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Enzymes_Two - Biology


Section overview

Enzymes are biological catalysts that accelerate chemical reactions by lowering the activation energy. Enzymes are proteins consisting of one or more polypeptide chains. Enzymes have an active site that provides a unique chemical environment made up of certain amino acid R groups (residues). This unique environment is well suited to convert particular chemical reactants for that enzyme, called substrates, into unstable intermediates, called transition states. Enzymes and substrates are thought to bind with an induced fit, which means that enzymes and substrates undergo slight conformational adjustments upon substrate contact, leading to binding. Enzymes bind to substrates and catalyze reactions in four different ways: bringing substrates together in an optimal orientation, compromising the bond structures of substrates so that bonds can be more easily broken, providing optimal environmental conditions for a reaction to occur, or participating directly in their chemical reaction by forming transient covalent bonds with the substrates.

Enzyme action must be regulated so that, in a given cell at a given time, the desired reactions are being catalyzed and the undesired reactions are not. Enzymes are regulated by cellular conditions, such as temperature and pH. They are also regulated through their location within a cell, sometimes being compartmentalized so that they can only catalyze reactions under certain circumstances. Inhibition and activation of enzymes via other molecules are other important ways that enzymes are regulated. Inhibitors can act competitively, noncompetitively, or allosterically; noncompetitive inhibitors are usually allosteric. Activators can also enhance the function of enzymes allosterically. The most common method by which cells regulate the enzymes in metabolic pathways is through feedback inhibition. During feedback inhibition, the products of a metabolic pathway serve as inhibitors (usually allosteric) of one or more of the enzymes (usually the first committed enzyme of the pathway) involved in the pathway that produces them.

Enzymes

A substance that helps a chemical reaction to occur is a catalyst, and the special molecules that catalyze biochemical reactions are called enzymes. Almost all enzymes are proteins, made up of chains of amino acids, and they perform the critical task of lowering the activation energies of chemical reactions inside the cell. Enzymes do this by binding to the reactant molecules and holding them in such a way as to make the chemical bond-breaking and bond-forming processes take place more readily. It is important to remember that enzymes don’t change the ∆G of a reaction. In other words, they don’t change whether a reaction is exergonic (spontaneous) or endergonic (not spontaneous). This is because they don’t change the free energy of the reactants or products. They only reduce the activation energy required to reach the transition state.

Figure 1. Enzymes lower the activation energy of the reaction but do not change the free energy of the reaction. Here, the solid line in the graph shows the energy required for reactants to turn into products without a catalyst. The dotted line shows the energy required using a catalyst. This figure should say Gibbs Free Energy on the Y-axis and instead of noting deltaH should have deltaG. Attribution: Marc T. Facciotti (own work)

Enzyme active site and substrate specificity

The chemical reactants to which an enzyme binds are the enzyme’s substrates. There may be one or more substrates, depending on the particular chemical reaction. In some reactions, a single-reactant substrate is broken down into multiple products. In others, two substrates may come together to create one larger molecule. Two reactants might also enter a reaction, both become modified, and leave the reaction as two products. The location within the enzyme where the substrate binds is called the enzyme’s active site. The active site is where the “action” happens, so to speak. Since enzymes are proteins, there is a unique combination of amino acid residues (also called side chains, or R groups) within the active site. Each amino acid side chain is characterized by different properties. Amino acids can be classified as large or small, weakly acidic or basic, hydrophilic or hydrophobic, positively or negatively charged, or neutral. The unique combination of amino acids (their positions, sequences, structures, and properties) creates a very specific chemical environment within the active site. This specific environment is suited to bind, albeit briefly, to a specific chemical substrate (or substrates). Due to this jigsaw-puzzle-like match between an enzyme and its substrates (which adapts to find the best fit between the transition state and the active site), enzymes are known for their specificity. The “best fit” between an enzyme and its substrates results from their respective shapes and the chemical complementarity of the functional groups on each binding partner.

Figure 2. This is an enzyme with two different substrates bound in the active site. The enzymes are represented as blobs, except for the active site, which shows the three R-groups of each of the three amino acids located in the active site. These R groups are interacting with the substrates through hydrogen bonding (represented as dashed lines).

At this point in the class, you should be familiar with all the types of bonds as well as the chemical characteristics of all the functional groups. For example, the R group of R180 in the enzyme depicted above is the amino acid Arginine (abbreviated as R) and has an R group that consists of several amino functional groups. Amino functional groups contain a nitrogen (N) and hydrogen (H) atoms. Nitrogen is more electronegative than hydrogen, so the covalent bond between N-H is a polar covalent bond. The hydrogen atoms in this bond will have a positive dipole moment, and the nitrogen atom will have a negative dipole moment. This allows amino groups to form hydrogen bonds with other polar compounds. Likewise, the backbone carbonyl oxygens of valine (V) 81 and glycine (G) 121 the backbone amino hydrogen of V81 are depicted engaged in hydrogen bonds with the small molecule substrate.

Exercise

Look to see which atoms in Figure 2 (above) are involved in the hydrogen bonds between the amino acid R groups and the substrate. You will need to be able to identify these on your own; hydrogen bonds may not be drawn in for you on the test.

If you changed the pH of the solution that this enzyme is located in, would the enzyme still be able to form hydrogen bonds with the substrate?

Which substrate (the left or right one) do you think is more stable in the active site? Why? How?

Figure 3. This is a depiction of an enzyme active site. Only the amino acids in the active site are drawn. The substrate is sitting directly in the center.
Source: created by Marc T. Facciotti (original work)

Exercise

First, identify the type of macromolecule in Figure 3. Second, draw in and label the appropriate interactions between the R groups and the substrate. Explain how these interactions might change if the pH of the solution changed.

Structural instability of enzymes

The fact that active sites are so well suited to provide specific environmental conditions also means that they are subject to influences by the local environment. It is true that increasing the environmental temperature generally increases reaction rates, enzyme-catalyzed or otherwise. However, increasing or decreasing the temperature outside of an optimal range can affect chemical bonds within the active site in such a way that they are less well suited to bind substrates. High temperatures will eventually cause enzymes, like other biological molecules, to denature, a process that changes the natural properties of a substance. Likewise, the pH of the local environment can also affect enzyme function. Active site amino acid residues have their own acidic or basic properties that are optimal for catalysis. These residues are sensitive to changes in pH that can impair the way substrate molecules bind. Enzymes are suited to function best within a certain pH range, and, as with temperature, extreme pH values (acidic or basic) of the environment can cause enzymes to denature.

Figure 4. Enzymes have an optimal pH. The pH at which the enzyme is most active will be the pH where the active site R groups are protonated/deprotonated such that the substrate can enter the active site and the initial step in the reaction can begin. Some enzymes require a very low pH (acidic) to be completely active. In the human body, these enzymes are most likely located in the lower stomach, or located in lysosomes (a cellular organelle used to digest large compounds inside the cell).
Source: http://biowiki.ucdavis.edu/Biochemis..._pH_Inhibition

The process where enzymes denature usually starts with the unwinding of the tertiary structure through destabilization of the bonds holding the tertiary structure together. Hydrogen bonds, ionic bonds, and covalent bonds (disulfide bridges and peptide bonds) can all be disrupted by large changes in temperate and pH. Using the chart of enzyme activity and temperature below, make an energy story for the red enzyme. Explain what might be happening from 37 °C to 95 °C.

Figure 5. Enzymes have an optimal temperature. The temperature at which the enzyme is most active will usually be the temperature where the structure of the enzyme is stable or uncompromised. Some enzymes require a specific temperature to remain active and not denature. Source: http://academic.brooklyn.cuny.edu/bi...ge/enz_act.htm

Induced fit and enzyme function

For many years, scientists thought that enzyme-substrate binding took place in a simple “lock-and-key” fashion. This model asserted that the enzyme and substrate fit together perfectly in one instantaneous step. However, current research supports a more refined view called induced fit. The induced-fit model expands upon the lock-and-key model by describing a more dynamic interaction between enzyme and substrate. As the enzyme and substrate come together, their interaction causes a mild shift in the enzyme’s structure that confirms a more productive binding arrangement between the enzyme and the transition state of the substrate. This energetically favorable binding maximizes the enzyme’s ability to catalyze its reaction.

When an enzyme binds its substrate, an enzyme-substrate complex is formed. This complex lowers the activation energy of the reaction and promotes its rapid progression in one of many ways. On a basic level, enzymes promote chemical reactions that involve more than one substrate by bringing the substrates together in an optimal orientation. The appropriate region (atoms and bonds) of one molecule is juxtaposed to the appropriate region of the other molecule with which it must react. Another way in which enzymes promote the reaction of their substrates is by creating an energetically favorable environment within the active site for the reaction to occur. Certain chemical reactions might proceed best in a slightly acidic or nonpolar environment. The chemical properties that emerge from the particular arrangement of amino acid residues within an active site create the energetically favorable environment for an enzyme’s specific substrates to react.

The activation energy required for many reactions includes the energy involved in slightly contorting chemical bonds so that they can more easily react. Enzymatic action can aid this process. The enzyme-substrate complex can lower the activation energy by contorting substrate molecules in such a way as to facilitate bond breaking. Finally, enzymes can also lower activation energies by taking part in the chemical reaction itself. The amino acid residues can provide certain ions or chemical groups that actually form covalent bonds with substrate molecules as a necessary step of the reaction process. In these cases, it is important to remember that the enzyme will always return to its original state at the completion of the reaction. One of the hallmark properties of enzymes is that they remain ultimately unchanged by the reactions they catalyze. After an enzyme is done catalyzing a reaction, it releases its product(s).

Figure 6. According to the induced-fit model, both enzyme and substrate undergo dynamic conformational changes upon binding. The enzyme contorts the substrate into its transition state, thereby increasing the rate of the reaction.

Creating an energy story for the reaction above

Using Figure 6, answer the questions posed in the energy story.
1. What are the reactants? What are the products?
2. What work was accomplished by the enzyme?
3. What state is the energy in initially? What state is the energy transformed into in the final state? This one might be tricky still, but try to identify where the energy is in the initial state and the final state.

Enzyme regulation

Why regulate enzymes?

Cellular needs and conditions vary from cell to cell and change within individual cells over time. The required enzymes and energetic demands of stomach cells are different from those of fat storage cells, skin cells, blood cells, and nerve cells. Furthermore, a digestive cell works much harder to process and break down nutrients during the time that closely follows a meal compared with many hours after a meal. As these cellular demands and conditions vary, so do the needed amounts and functionality of different enzymes.

Regulation of enzymes by molecules

Enzymes can be regulated in ways that either promote or reduce their activity. There are many different kinds of molecules that inhibit or promote enzyme function, and various mechanisms exist for doing so. In some cases of enzyme inhibition, for example, an inhibitor molecule is similar enough to a substrate that it can bind to the active site and simply block the substrate from binding. When this happens, the enzyme is inhibited through competitive inhibition, because an inhibitor molecule competes with the substrate for active site binding. On the other hand, in noncompetitive inhibition, an inhibitor molecule binds to the enzyme in a location other than an allosteric site and still manages to block substrate binding to the active site.

Figure 7. Competitive and noncompetitive inhibition affect the rate of reaction differently. Competitive inhibitors affect the initial rate but do not affect the maximal rate, whereas noncompetitive inhibitors affect the maximal rate.

Some inhibitor molecules bind to enzymes in a location where their binding induces a conformational change that reduces the affinity of the enzyme for its substrate. This type of inhibition is called allosteric inhibition. Most allosterically regulated enzymes are made up of more than one polypeptide, meaning that they have more than one protein subunit. When an allosteric inhibitor binds to an enzyme, all active sites on the protein subunits are changed slightly such that they bind their substrates with less efficiency. There are allosteric activators as well as inhibitors. Allosteric activators bind to locations on an enzyme away from the active site, inducing a conformational change that increases the affinity of the enzyme’s active site(s) for its substrate(s).

Figure 8. Allosteric inhibitors modify the active site of the enzyme so that substrate binding is reduced or prevented. In contrast, allosteric activators modify the active site of the enzyme so that the affinity for the substrate increases.

Video link

Check out this short (one-minute) video on competitive vs. noncompetitive enzymatic inhibition. Also, take a look at this video (1.2 minutes) on feedback inhibition.

Many enzymes don’t work optimally, or even at all, unless bound to other specific non-protein helper molecules, either temporarily through ionic or hydrogen bonds or permanently through stronger covalent bonds. Two types of helper molecules are cofactors and coenzymes. Binding to these molecules promotes optimal conformation and function for their respective enzymes. Cofactors are inorganic ions such as iron(II) (Fe2+) and magnesium(II) (Mg2+). One example of an enzyme that requires a metal ion as a cofactor is the enzyme that builds DNA molecules, DNA polymerase, which requires a bound zinc(II) ion (Zn2+) to function. Coenzymes are organic helper molecules, with a basic atomic structure made up of carbon and hydrogen, that are required for enzyme action. The most common sources of coenzymes are dietary vitamins. Some vitamins are precursors to coenzymes, and others act directly as coenzymes. Vitamin C is a coenzyme for multiple enzymes that take part in building the important connective tissue component, collagen. An important step in the breakdown of glucose to yield energy is catalysis by a multi-enzyme complex called pyruvate dehydrogenase. Pyruvate dehydrogenase is a complex of several enzymes that actually requires one cofactor (a magnesium ion) and five different organic coenzymes to catalyze its specific chemical reaction. Therefore, enzyme function is, in part, regulated by an abundance of various cofactors and coenzymes, which are supplied primarily by the diets of most organisms.

Enzyme compartmentalization

In eukaryotic cells, molecules such as enzymes are usually compartmentalized into different organelles. This allows for yet another level of regulation of enzyme activity. Enzymes required only for certain cellular processes can be housed separately along with their substrates, allowing for more efficient chemical reactions. Examples of this sort of enzyme regulation based on location and proximity include the enzymes involved in the latter stages of cellular respiration, which take place exclusively in the mitochondria, and the enzymes involved in the digestion of cellular debris and foreign materials, located within lysosomes.

Additional links

Khan Academy

The following links will take you to a series of videos on kinetics. The first link contains four videos on reaction rates, and the second link contains nine videos related to the relationship between reaction rates and concentration. These videos are supplemental and are provided to give you an outside resource to further explore enzyme kenetics.

  • Introduction to enzyme kinetics
  • Reaction mechanism

Enzymes

A substance that helps a chemical reaction to occur is called a catalyst, and the molecules that catalyze biochemical reactions are called enzymes. Most enzymes are proteins and perform the critical task of lowering the activation energies of chemical reactions inside the cell. Most of the reactions critical to a living cell happen too slowly at normal temperatures to be of any use to the cell. Without enzymes to speed up these reactions, life could not persist. Enzymes do this by binding to the reactant molecules and holding them in such a way as to make the chemical bond-breaking and -forming processes take place more easily. It is important to remember that enzymes do not change whether a reaction is exergonic (spontaneous) or endergonic. This is because they do not change the free energy of the reactants or products. They only reduce the activation energy required for the reaction to go forward (Figure 1). In addition, an enzyme itself is unchanged by the reaction it catalyzes. Once one reaction has been catalyzed, the enzyme is able to participate in other reactions.

Figure 1 Enzymes lower the activation energy of the reaction but do not change the free energy of the reaction.

The chemical reactants to which an enzyme binds are called the enzyme’s substrates. There may be one or more substrates, depending on the particular chemical reaction. In some reactions, a single reactant substrate is broken down into multiple products. In others, two substrates may come together to create one larger molecule. Two reactants might also enter a reaction and both become modified, but they leave the reaction as two products. The location within the enzyme where the substrate binds is called the enzyme’s active site. The active site is where the “action” happens. Since enzymes are proteins, there is a unique combination of amino acid side chains within the active site. Each side chain is characterized by different properties. They can be large or small, weakly acidic or basic, hydrophilic or hydrophobic, positively or negatively charged, or neutral. The unique combination of side chains creates a very specific chemical environment within the active site. This specific environment is suited to bind to one specific chemical substrate (or substrates).

Active sites are subject to influences of the local environment. Increasing the environmental temperature generally increases reaction rates, enzyme-catalyzed or otherwise. However, temperatures outside of an optimal range reduce the rate at which an enzyme catalyzes a reaction. Hot temperatures will eventually cause enzymes to denature, an irreversible change in the three-dimensional shape and therefore the function of the enzyme (Figure 8). Enzymes are also suited to function best within a certain pH and salt concentration range, and, as with temperature, extreme pH, and salt concentrations can cause enzymes to denature.

Figure 2 Heat applied to an egg during cooking irreversibly denatures the proteins. (credit: “K-Wall”/Flickr)

Typically, enzymes function optimally in the environment where they are typically found and used. For example, the enzyme amylase is found in saliva, where it functions to break down starch (a polysaccharide – carbohydrate chain) into smaller sugars. Note that in this example, amylase is the enzyme, starch is the substrate, and smaller sugars are the product. The pH of saliva is typically between 6.2 and 7.6, with roughly 6.7 being the average. The optimum pH of amylase is between 6.7 and 7.0, which is close to neutral (Figure 3). The optimum temperature for amylase is close to 37ºC (which is human body temperature).

Figure 3 The effect of pH and temperature on the activity of an enzyme. Amylase is shown in blue in both graphs. (top) Amylase (blue) has an optimum pH of about 7. The green enzyme, which has an optimum pH of about 2.3, might function in the stomach where it is very acidic. (bottom) Amylase (blue) has an optimum temperature of about 37 degrees C. The orange enzyme, which has an optimum temperature of about 15 degrees C (about 60F) might function in a plant found outdoors.

For many years, scientists thought that enzyme-substrate binding took place in a simple “lock and key” fashion. This model asserted that the enzyme and substrate fit together perfectly in one instantaneous step. However, current research supports a model called induced fit (Figure 9). The induced-fit model expands on the lock-and-key model by describing a more dynamic binding between enzyme and substrate. As the enzyme and substrate come together, their interaction causes a mild shift in the enzyme’s structure that forms an ideal binding arrangement between enzyme and substrate.

When an enzyme binds its substrate, an enzyme-substrate complex is formed. This complex lowers the activation energy of the reaction and promotes its rapid progression in one of multiple possible ways.

  • On a basic level, enzymes promote chemical reactions that involve more than one substrate by bringing the substrates together in an optimal orientation for reaction.
  • Enzymes promote the reaction of their substrates is by creating an optimal environment within the active site for the reaction to occur. The chemical properties that emerge from the particular arrangement of amino acid R groups (side chains) within an active site create the perfect environment for an enzyme’s specific substrates to react.
  • The enzyme-substrate complex can also lower activation energy by compromising the bond structure so that it is easier to break.
  • Finally, enzymes can also lower activation energies by taking part in the chemical reaction itself. In these cases, it is important to remember that the enzyme will always return to its original state by the completion of the reaction.

One of the hallmark properties of enzymes is that they remain ultimately unchanged by the reactions they catalyze. After an enzyme has catalyzed a reaction, it releases its product(s) and can catalyze a new reaction.

Figure 9 The induced-fit model is an adjustment to the lock-and-key model and explains how enzymes and substrates undergo dynamic modifications during the transition state to increase the affinity of the substrate for the active site.

It would seem ideal to have a scenario in which all of an organism’s enzymes existed in abundant supply and functioned optimally under all cellular conditions, in all cells, at all times. However, a variety of mechanisms ensures that this does not happen. Cellular needs and conditions constantly vary from cell to cell, and change within individual cells over time. The required enzymes of stomach cells differ from those of fat storage cells, skin cells, blood cells, and nerve cells. Furthermore, a digestive organ cell works much harder to process and break down nutrients during the time that closely follows a meal compared with many hours after a meal. As these cellular demands and conditions vary, so must the amounts and functionality of different enzymes.

Since the rates of biochemical reactions are controlled by activation energy, and enzymes lower and determine activation energies for chemical reactions, the relative amounts and functioning of the variety of enzymes within a cell ultimately determine which reactions will proceed and at what rates. This determination is tightly controlled in cells. In certain cellular environments, enzyme activity is partly controlled by environmental factors like pH, temperature, salt concentration, and, in some cases, cofactors or coenzymes.

Enzymes can also be regulated in ways that either promote or reduce enzyme activity. There are many kinds of molecules that inhibit or promote enzyme function, and various mechanisms by which they do so. In some cases of enzyme inhibition, an inhibitor molecule is similar enough to a substrate that it can bind to the active site and simply block the substrate from binding. When this happens, the enzyme is inhibited through competitive inhibition, because an inhibitor molecule competes with the substrate for binding to the active site.

On the other hand, in noncompetitive inhibition, an inhibitor molecule binds to the enzyme in a location other than the active site, called an allosteric site, but still manages to block substrate binding to the active site. Some inhibitor molecules bind to enzymes in a location where their binding induces a conformational change that reduces the affinity of the enzyme for its substrate. This type of inhibition is called allosteric inhibition (Figure 10). Most allosterically regulated enzymes are made up of more than one polypeptide, meaning that they have more than one protein subunit. When an allosteric inhibitor binds to a region on an enzyme, all active sites on the protein subunits are changed slightly such that they bind their substrates with less efficiency. There are allosteric activators as well as inhibitors. Allosteric activators bind to locations on an enzyme away from the active site, inducing a conformational change that increases the affinity of the enzyme’s active site(s) for its substrate(s) (Figure 10).

Figure 10 Allosteric inhibition works by indirectly inducing a conformational change to the active site such that the substrate no longer fits. In contrast, in allosteric activation, the activator molecule modifies the shape of the active site to allow a better fit of the substrate.

Many enzymes do not work optimally, or even at all, unless bound to other specific non-protein helper molecules. They may bond either temporarily through ionic or hydrogen bonds, or permanently through stronger covalent bonds. Binding to these molecules promotes optimal shape and function of their respective enzymes. Two examples of these types of helper molecules are cofactors and coenzymes. Cofactors are inorganic ions such as ions of iron and magnesium. Coenzymes are organic helper molecules, those with a basic atomic structure made up of carbon and hydrogen. Like enzymes, these molecules participate in reactions without being changed themselves and are ultimately recycled and reused. Vitamins are the source of coenzymes. Some vitamins are the precursors of coenzymes and others act directly as coenzymes. Vitamin C is a direct coenzyme for multiple enzymes that take part in building the important connective tissue, collagen. Therefore, enzyme function is, in part, regulated by the abundance of various cofactors and coenzymes, which may be supplied by an organism’s diet or, in some cases, produced by the organism.


Enzymes_Two - Biology

As unnecessary activity within a cell can be wasteful or harmful, enzymes are regulated by four primary means: proteolytic cleavage (irreversible covalent modification), reversible covalent modification, control proteins, and allosteric interactions.

Enzymes select which reactions take place within a cell and, therefore, must regulate them. Catalytic activity is desirable only when needed, so enzymes are regulated by four primary means: proteolytic cleavage (irreversible covalent modification), reversible covalent modification, control proteins, and allosteric interactions.

Proteolytic cleavage (irreversible covalent modification): Many enzymes are released into their environment in an active form called a zymogen (or proenzyme). When specific peptide bonds on zymogens are cleaved, the zymogens become irreversibly activated. Activation of zymogens may be instigated by other enzymes, or by a change in the environment.

Reversible covalent modification: Some enzymes are activated or deactivated by phosphorylation or the addition of some other modifier (e.g. AMP). The removal of all modifiers is almost always done by hydrolysis.

Control proteins: Control proteins are protein subunits that associate with certain enzymes to activate or inhibit their activity (e.g. calmodulin, G-proteins).

Allosteric interactions: Allosteric regulation is the modification of an enzyme’s configuration through the binding of an activator or inhibitor at a specific binding site of the enzyme called the allosteric site. In allosteric inhibition, inhibitor molecules bind to an enzyme at the allosteric site. Their binding induces a conformational change that reduces the affinity of the enzyme’s active site for its substrate. The binding of this allosteric inhibitor changes the conformation of the enzyme and its active site, so the substrate is not able to bind. This prevents the enzyme from lowering the activation energy of the reaction, and the reaction rate is reduced. Allosteric activators can increase reaction rates. They bind to an allosteric site which induces a conformational change that increases the affinity of the enzyme’s active site for its substrate. This increases the reaction rate.

Practice Questions

Khan Academy

MCAT Official Prep (AAMC)

Biology Question Pack, Vol 2. Passage 15 Question 98

• Enzymes select which reactions take place within a cell and, therefore, must regulate them. Catalytic activity is desirable only when needed, so enzymes are regulated by four primary means: proteolytic cleavage (irreversible covalent modification), reversible covalent modification, control proteins, and allosteric interactions.

• Proteolytic cleavage (irreversible covalent modification) is when specific peptide bonds on zymogens are cleaved the zymogens become irreversibly activated.

• Reversible covalent modification is when some enzymes are activated or deactivated by phosphorylation or the addition of some other modifier.

• Control proteins are protein subunits that associate with certain enzymes to activate or inhibit their activity.

• Allosteric regulation is the modification of an enzyme’s configuration through the binding of an activator or inhibitor at a specific binding site of the enzyme.

zymogen: enzymes released into their environment in an active form.

phosphorylation: a biochemical process that involves the addition of phosphate to an organic compound.

catalytic activity: defined as the rate constant for the slow step

hydrolysis: the chemical breakdown of a compound due to reaction with water

allosteric site: a site other than the active site on an enzyme


Structural biology of starch-degrading enzymes and their regulation

Self-stabilisation of amylose in the active site of sugar beet GH31 α-glucosidase.

Substrate complex of the debranching barley limit dextrinase.

Carbohydrate surface binding sites (SBSs) in isoamylase.

Structural details of catalytic steps in plant disproportionating enzyme.

Regulatory protein–protein interactions of barley limit dextrinase.

Starch is a major energy source for all domains of life. Recent advances in structures of starch-degrading enzymes encompass the substrate complex of starch debranching enzyme, the function of surface binding sites in plant isoamylase, details on individual steps in the mechanism of plant disproportionating enzyme and a self-stabilised conformation of amylose accommodated in the active site of plant α-glucosidase. Important inhibitor complexes include a flavonol glycoside, montbretin A, binding at the active site of human pancreatic α-amylase and barley limit dextrinase inhibitor binding to the debranching enzyme, limit dextrinase using a new binding mode for cereal protein inhibitors.


Do-it-yourself enzymes

Enzymes have long captivated biologists and chemists by performing difficult reactions rapidly and selectively in aqueous environments. The designs of enzymes catalyzing a retro-aldol reaction and a Kemp elimination demonstrate that complex, biochemically novel reactions can be achieved using cutting-edge computational methods.

Each new advance in the field of protein design raises our expectations for future designs targets that seemed impossible only ten years ago are now attainable with better computational tools. New protein folds have been built from scratch, such as a right-handed four-helix coiled coil 1 and a novel α/β protein 2 . Biosensors with a wide range of ligand specificities 3 and high-affinity peptides that target transmembrane protein surfaces 4 have also been designed computationally. Protein design challenges our fundamental grasp of molecular determinants of structure, stability and function. As such, catalysis has been one of the most difficult features of proteins to recapitulate. Catalysis requires an amino acid sequence that binds the substrate, stabilizes transition state intermediates, releases the product and restores active site residues to facilitate turnover, all while maintaining the fold and stability of the remainder of the protein. Previous computational designs have tackled many of these obstacles. A 'protozyme' was constructed by introducing single histidine mutations to thioredoxin to catalyze hydrolysis of p-nitrophenol acetate 5 . Carving a substrate binding channel into a de novo–designed helical bundle with a diiron cofactor allowed it to catalyze two-electron oxidation of a 4-aminophenol substrate with multiple turnovers 6 . Now, Jiang et al. 7 and Rothlisberger et al. 8 have raised the bar for enzyme design several notches.


Immunological aspects and pathogenicity

Given the scale of the global tuberculosis burden, vaccination is not only a priority but remains the only realistic public health intervention that is likely to affect both the incidence and the prevalence of the disease 29 . Several areas of vaccine development are promising, including DNA vaccination, use of secreted or surface-exposed proteins as immunogens, recombinant forms of BCG and rational attenuation of M. tuberculosis 29 . All of these avenues of research will benefit from the genome sequence as its availability will stimulate more focused approaches. Genes encoding ∼ 90 lipoproteins were identified, some of which are enzymes or components of transport systems, and a similar number of genes encoding preproteins (with type I signal peptides) that are probably exported by the Sec-dependent pathway. M. tuberculosis seems to have two copies of secA. The potent T-cell antigen Esat-6 (ref. 30), which is probably secreted in a Sec-independent manner, is encoded by a member of a multigene family. Examination of the genetic context reveals several similarly organized operons that include genes encoding large ATP-hydrolysing membrane proteins that might act as transporters. One of the surprises of the genome project was the discovery of two extensive families of novel glycine-rich proteins, which may be of immunological significance as they are predicted to be abundant and potentially polymorphic antigens.

The PE and PPE multigene families. About 10% of the coding capacity of the genome is devoted to two large unrelated families of acidic, glycine-rich proteins, the PE and PPE families, whose genes are clustered ( Figs 1 , 2 (PDF File: 890K)) and are often based on multiple copies of the polymorphic repetitive sequences referred to as PGRSs, and major polymorphic tandem repeats (MPTRs), respectively 31 , 32 . The names PE and PPE derive from the motifs Pro–Glu (PE) and Pro–Pro–Glu (PPE) found near the N terminus in most cases 33 . The 99 members of the PE protein family all have a highly conserved N-terminal domain of ∼ 110 amino-acid residues that is predicted to have a globular structure, followed by a C-terminal segment that varies in size, sequence and repeat copy number ( Fig. 5). Phylogenetic analysis separated the PE family into several subfamilies. The largest of these is the highly repetitive PGRS class, which contains 61 members members of the other subfamilies, share very limited sequence similarity in their C-terminal domains (Fig. 5). The predicted molecular weights of the PE proteins vary considerably as a few members contain only the N-terminal domain, whereas most have C-terminal extensions ranging in size from 100 to 1,400 residues. The PGRS proteins have a high glycine content (up to 50%), which is the result of multiple tandem repetitions of Gly–Gly–Ala or Gly–Gly–Asn motifs, or variations thereof.

a, Classification of the PE and PPE protein families. b, Sequence variation between M. tuberculosis H37Rv and M. bovis BCG-Pasteur in the PE-PGRS encoded by open reading frame (ORF) Rv0746.

The 68 members of the PPE protein family (Fig. 5) also have a conserved N-terminal domain that comprises ∼ 180 amino-acid residues, followed by C-terminal segments that vary markedly in sequence and length. These proteins fall into at least three groups, one of which constitutes the MPTR class characterized by the presence of multiple, tandem copies of the motif Asn–X–Gly–X–Gly–Asn–X–Gly. The second subgroup contains a characteristic, well-conserved motif around position 350, whereas the third contains proteins that are unrelated except for the presence of the common 180-residue PPE domain.

The subcellular location of the PE and PPE proteins is unknown and in only one case, that of a lipase (Rv3097), has a function been demonstrated. On examination of the protein database from the extensively sequenced M. leprae 15 , no PGRS- or MPTR-related polypeptides were detected but a few proteins belonging to the non-MPTR subgroup of the PPE family were found. These proteins include one of the major antigens recognized by leprosy patients, the serine-rich antigen 34 . Although it is too early to attribute biological functions to the PE and PPE families, it is tempting to speculate that they could be of immunological importance. Two interesting possibilities spring to mind. First, they could represent the principal source of antigenic variation in what is otherwise a genetically and antigenically homogeneous bacterium. Second, these glycine-rich proteins might interfere with immune responses by inhibiting antigen processing.

Several observations and results support the possibility of antigenic variation associated with both the PE and the PPE family proteins. The PGRS member Rv1759 is a fibronectin-binding protein of relative molecular mass 55,000 (ref. 35) that elicits a variable antibody response, indicating either that individuals mount different immune responses or that this PGRS protein may vary between strains of M. tuberculosis. The latter possibility is supported by restriction fragment length polymorphisms for various PGRS and MPTR sequences in clinical isolates 33 . Direct support for genetic variation within both the PE and the PPE families was obtained by comparative DNA sequence analysis (Fig. 5). The gene for the PE–PGRS protein Rv0746 of BCG differs from that in H37Rv by the deletion of 29 codons and the insertion of 46 codons. Similar variation was seen in the gene for the PPE protein Rv0442 (data not shown). As these differences were all associated with repetitive sequences they could have resulted from intergenic or intragenic recombinational events or, more probably, from strand slippage during replication 32 . These mechanisms are known to generate antigenic variability in other bacterial pathogens 36 .

There are several parallels between the PGRS proteins and the Epstein–Barr virus nuclear antigens (EBNAs). Members of both polypeptide families are glycine-rich, contain extensive Gly–Ala repeats, and exhibit variation in the length of the repeat region between different isolates. The Gly–Ala repeat region of EBNA1 functions as a cis-acting inhibitor of the ubiquitin/proteasome antigen-processing pathway that generates peptides presented in the context of major histocompatibility complex (MHC) class I molecules 37 , 38 . MHC class I knockout mice are very susceptible to M. tuberculosis , underlining the importance of a cytotoxic T-cell response in protection against disease 3 , 39 . Given the many potential effects of the PPE and PE proteins, it is important that further studies are performed to understand their activity. If extensive antigenic variability or reduced antigen presentation were indeed found, this would be significant for vaccine design and for understanding protective immunity in tuberculosis, and might even explain the varied responses seen in different BCG vaccination programmes 40 .

Pathogenicity. Despite intensive research efforts, there is little information about the molecular basis of mycobacterial virulence 41 . However, this situation should now change as the genome sequence will accelerate the study of pathogenesis as never before, because other bacterial factors that may contribute to virulence are becoming apparent. Before the completion of the genome sequence, only three virulence factors had been described 41 : catalase-peroxidase, which protects against reactive oxygen species produced by the phagocyte mce, which encodes macrophage-colonizing factor 42 and a sigma factor gene, sigA (aka rpoV ), mutations in which can lead to attenuation 41 . In addition to these single-gene virulence factors, the mycobacterial cell wall 4 is also important in pathology, but the complex nature of its biosynthesis makes it difficult to identify critical genes whose inactivation would lead to attenuation.

On inspection of the genome sequence, it was apparent that four copies of mce were present and that these were all situated in operons, comprising eight genes, organized in exactly the same manner. In each case, the genes preceding mce code for integral membrane proteins, whereas mce and the following five genes are all predicted to encode proteins with signal sequences or hydrophobic stretches at the N terminus. These sets of proteins, about which little is known, may well be secreted or surface-exposed this is consistent with the proposed role of Mce in invasion of host cells 42 . Furthermore, a homologue of smpB, which has been implicated in intracellular survival of Salmonella typhimurium, has also been identified 43 . Among the other secreted proteins identified from the genome sequence that could act as virulence factors are a series of phospholipases C, lipases and esterases, which might attack cellular or vacuolar membranes, as well as several proteases. One of these phospholipases acts as a contact-dependent haemolysin (N. Stoker, personal communication). The presence of storage proteins in the bacillus, such as the haemoglobin-like oxygen captors described above, points to its ability to stockpile essential growth factors, allowing it to persist in the nutrient-limited environment of the phagosome. In this regard, the ferritin-like proteins, encoded by bfrA and bfrB, may be important in intracellular survival asthe capacity to acquire enough iron in the vacuole is very limited.


Absorption

General principles

Absorption refers to the transfer of compounds from the gut lumen across the gut wall to the body tissues, including the lymph or blood of vertebrates and hemolymph of arthropods. At the cellular level, organic compounds can be absorbed from the gut lumen by paracellular and transcellular routes. Paracellular transport refers to movement between cells of the gut epithelium, while the transcellular route involves transport across the apical cell membrane of gut epithelial cells, transit across the cell (for some molecules with metabolic transformations in the cell), and then export at the basolateral membrane. We distinguish the term �sorption” (transport from gut lumen to body tissues by either the paracellular or transcellular route) from “uptake,” which refers to the transport from the gut lumen across the apical membrane of the gut epithelial cell (one step in transcellular transport).

This section considers absorption of organic compounds, particularly products of digestion: monosaccharides, the digestive breakdown products of complex carbohydrates peptide and amino acid products of protein digestion and lipids, SCFAs (generated by hydrolysis of triglycerides), and SCFAs (products of fermentative breakdown of complex carbohydrates by gut microbes). With the exception of SCFAs, these products are absorbed principally distal to the gastric region of the alimentary tract, for example, small intestine of vertebrates and midgut of insects. The absorptive cells are columnar epithelial cells called enterocytes. Exceptionally, SCFAs produced by the microbiota in the hindgut (e.g., mammalian colon and cecum) are absorbed across the hindgut wall by cells that are variously known as enterocytes, colonic enterocytes, or colonocytes.

In this section, two aspects of nutrient absorption are addressed: the modes of transport of the major classes of organic solutes and variation in nutrient absorption among animal taxa, in relation to nutritional habits and phylogeny and its mechanistic basis. Diet-related determinants of absorption in individual animals are addressed in Section “Matches of GI system biochemistry (enzymes, transporters) to changes in diet composition.”

Transcellular transport of organic solutes

Carrier-mediated transport

Most organic compounds absorbed across animal guts are polar, and their transport is predominantly or exclusively carrier-mediated, that is, mediated by membrane-bound transporters and displaying the twin characteristics of saturation kinetics and competitive inhibition. Two forms of carrier-mediated transport are recognized: facilitated diffusion, which is energy-independent and mediates transport down the electrochemical potential gradient and active transport, which is concentrative and dependent, directly or indirectly, on cellular energy. Simple diffusion, that is, down the concentration gradient and involving neither a carrier nor cellular energy, is an additional mode of absorption that is especially important for small, nonpolar molecules.

Absorption of carbohydrates

Monosaccharides cross the apical and basolateral membranes of gut epithelial cells by carrier-mediated mechanisms. The key glucose transporters in mammals and birds (184) are a Na+/glucose cotransporter SGLT1 (a member of the Na+/solute symporter family) and the facilitative transporter GLUT2, which transports glucose, fructose, mannose, and galactose with low affinity and N-acetyl-glucosamine with high affinity (444). Fructose is transported principally via the facilitative transporter GLUT5 (126). These transporters are expressed predominantly in the small intestine.

The expression of SGLT1 in the intestine is restricted to the apical membrane of enterocytes. Its capacity to take up glucose from very low concentrations in the intestinal lumen is driven by the downhill gradient of Na + ions maintained by the Na + /K + -ATPase on the basolateral membrane ( Fig. 9 ) (206). Once in the cell, the glucose is widely accepted to be transported down its concentration gradient across the basolateral membrane into the circulation by GLUT2. Under conditions of high luminal glucose content, however, GLUT2 in rodents is inserted into the apical membrane, where it mediates the high flux of glucose into the enterocyte (254). Some data suggest that sugar-induced translocation of GLUT2 may not occur universally in mammals (18, 330), and further research is required to establish the distribution of this effect with respect to phylogeny and diet.

Transport of glucose and fructose across the mammalian enterocyte by SGLT1, GLUT2, and GLUT5. The insertion of GLUT2 into the apical membrane is mediated by the detection of luminal glucose by the TIR2/3 receptors and Ca 2+ signaling, as described in text.

The mechanism by which GLUT2 is inserted into the apical enterocyte membrane is understood in outline (253). Under high glucose conditions, the inward flux of Na + ions via SGLT1 results in depolarization of the membrane and Ca 2+ influx, which, in turn, causes a large-scale reorganization of the cytoskeleton, facilitating access of proteins to the apical membrane. In parallel, high concentrations of luminal glucose and fructose activate the TIR2/3 receptor on the apical membrane, resulting in trafficking of phospholipase (PLC)㬢 and protein kinase C (PKC)βII to the apical membrane. Diacylglycerol generated by PLC㬢, together with the high Ca 2+ , activates PKCβII, permitting the insertion of GLUT2 into the apical membrane and the resultant high capacity uptake of glucose and fructose. This process occurs very rapidly.

In the mouse, the responsiveness of GLUT2 insertion to luminal sugars varies among sugars, being triggered much less efficiently by glucose and complex sugars than by fructose, sucrose, and a mixture of glucose and fructose (193) mice fed on a high-fructose diet have been reported to bear GLUT2 permanently on the apical membrane of enterocytes (434). Artificial sweeteners, such as sucralose, dramatically increase GLUT2 insertion and the resultant uptake of glucose, such that the sugar is absorbed efficiently from lower concentrations in the presence of the artificial sweetener than in its absence (302). The implications of these rodent studies for human nutrition are not yet fully resolved.

Phylogenetic analysis assigns the mammalian GLUT2 to a clade that includes three further mammalian GLUTs (GLUT1, 3, and 4) and invertebrate, but no nonmetazoan, GLUTs, suggesting that this group of transporters may have evolved in the basal metazoans or immediate ancestors of animals (472). There is also evidence that SGLT1 and GLUT transporters contribute to intestinal glucose absorption in nonmammalian vertebrates, including fish (72, 269). The molecular basis of sugar uptake across the gut wall has not, however, been investigated widely in the invertebrates. Among insects, glucose transport across the midgut of the hymenopteran parasite Aphidius ervi is mediated by a SGLT1-like transporter on the apical membrane, together with a GLUT2-like transporter on both the apical and basolateral membranes of the enterocytes and a second passive transporter similar to GLUT-5 is implicated in fructose uptake (58). There is also persuasive molecular and physiological evidence for the involvement of SGLT and GLUT transporters in glucose absorption from the midgut of the pyrrochorid bug Dysdercus peruvianus, with K + , not Na + , as the likely counterion of SGLT (28). This condition is not, however, universal among insects. For example, genome annotation of the pea aphid Acyrthosiphon pisum revealed no Na + /solute symporter with plausible specificity for sugars, but 29 candidate sugar transporters in the MFS family, equivalent to GLUT (368). These included an abundantly expressed gene ApSt3, a hexose uniporter with specificity for glucose and fructose in the distal midgut. Aphids may not, however, be typical of insects because their diet of plant phloem sap is sugar rich, and a concentration gradient from gut lumen to epithelial cell and hemocoel is maintained by the excess sugar in the gut lumen (127).

Pathways for amino acid and peptide absorption

The products of protein digestion taken up by enterocytes of the mammalian intestine are free amino acids, dipeptides, and tripeptides. Free amino acids are taken up from the small intestine of mammals by multiple carriers with overlapping specificities, with the result that most individual amino acids are transported by more than one transporter. By contrast, peptides are taken up by a single transporter with very low selectivity, as considered at the end of this section.

The amino acid transporters are classified by their activity (specificity and kinetics) into multiple systems, and by sequence homology into solute carrier (SLC) families. The SLC nomenclature was devised by the Human Genome Organization for transporters in the human genome (with all members of each family having 㸠%�% amino acid sequence homology), and is widely used for other animals. The principal transporters mediating amino acid transport in the human intestine are summarized in Table 3 .

Table 3

Amino Acid Transport Systems in the Mammalian Intestine [Data From Table 1 of Reference (41)]

System a Solute carrier
(SLC) group
Amino acids
transported c
Transport propertiesLocation
Neutral amino acids (AA 0 )
B 0 B 0 AT1SLC6A19All AA 0 AA 0 /Na + symportApical
ASCASCT2SLC1A5A, S, C, T, QAA 0 antiporter (no net AA 0 uptake of
L4F2hc/LAT2 2 SLC3A2/SLC7A8All AA 0 except PNa + independent transportBasolateral
TTATISLC16A19F, Y, W
Cationic amino acids (AA + )
b 0,+ rBAT/b 0,+ AT b SLC3A1/SLC7A9R, K, O, cystineAA + or cystine (uptake)/AA 0 (efflux) antiportApical
y + L4F2hc/y + LAT1 b SLC3A2/SLC7A7K, RAA + (efflux)/AA 0 antiportBasolateral
4F2hc/y + LAT2 b SLC3A2/SLC7A6K, R, C
Anionic amino acids (AA − )
X − AGEAAT3SLC1A1E, DAA − /3Na + symportApical
Proline and glycine
IMINOIMINOSLC6A20P, HO-PP/Na + symportApical
PATPAT1SLC36A1P, G, AP or G/H + symport d

Studies on human, rodent and rabbit suggest that the amino acid transporters in the mammalian small intestine can be assigned to four groups, mediating the transport of neutral, cationic, anionic, and imino acids, respectively (41). Uptake across the apical membrane is mediated by: Na + -coupled transporters, for example, the B 0 transporter with broad specificity for neutral amino acids and found in all parts of the small intestine proton-motive force, as in the uptake of proline and glycine by the transporter PAT and amino acid exchange, for example, uptake of cationic amino acids and cystine linked to efflux of neutral amino acids by b 0,+ system. Transport across the basolateral membrane is also mediated by amino acid exchange, for example, y + L for efflux of cationic amino acids, or by facilitative diffusion, for example, transporters of the L and T system for efflux of neutral and aromatic amino acids, respectively.

The rich classical literature on the kinetics of amino acid transport across the intestinal epithelium of various nonmammalian vertebrates and invertebrates is summarized by (246) and (341), and there is increasing interest in analysis from a molecular perspective [e.g., for birds, see reference (184)]. The midgut amino acid transporters that have been studied in insects belong principally to the Na + -coupled symporter family SLC6. As in mammals, multiple transporters are expressed, with overlapping specificities for amino acids. Some are very specific, for example, NAT6 and NAT8 in the distal midgut of mosquito Anopheles gambiae transport just aromatic amino acids (318, 319). Other SLC6 transporters have a very broad range. Notably, the neutral amino acid transporter in Drosophila (DmNAT6) can mediate the transport of most amino acids apart from lysine, arginine, aspartate, and glutamate and, remarkably, it can also take up D-isomers of several amino acids (321). This capability can be linked to the abundance of D-amino acids in the cell walls of bacteria, which are an important component of the natural diet of Drosophila species. DmNAT6 is an active transporter, capable of mediating uptake against the concentration gradient.

Exceptionally, amino acid transport in the midgut of larval Lepidoptera is coupled to K + ions, and not Na + ions (158, 340). This trait is believed to be linked to the high K + /low Na + conditions in the gut of these insects, which eat plants with high ratios of K + /Na + . Multiple transporters are involved with a range of specificities, including two neutral amino acid transporters in Manduca sexta (KAAT1 and CAATCH1), both members of the SL6 family (71, 145) with distinctive amino acid selectivities (322). The cotransport of the K + ions and amino acid into enterocytes is coupled to the ATPase-dependent extrusion of K + ions from adjacent goblet cells. The coupled functions of electrogenic K + transport and K + /amino acid uptake are mediated by different cells, presumably because the high emf generated by the goblet cells could compromise the function of the SL6 and other transporters.

Amino acid transporters are also expressed in the apical membrane of the insect hindgut epithelium, where they mediate the uptake of amino acids in the primary urine produced in the Malpighian tubules. For example, glycine, serine, alanine, and threonine are actively resorbed into the cells of the rectal pads of the locust by a Na + cotransporter of the SLC6 family (430). Proline is also taken up, and is a major respiratory substrate of rectal cells (76).

Returning to mammals, a single proton-oligopeptide transporter, PEPT1 (member of SLC15A family) mediates the uptake of peptides across the apical membrane ( Fig. 10 ). It can transport thousands of di- and tripeptides with low affinity and high capacity, but neither free amino acids nor tetrapeptides (106). This property is intelligible from the structural features of the binding pocket of the protein, which can accommodate compounds with oppositely charged head groups (carboxyl and amino groups) separated by a carbon backbone of 0.55 to 0.63 nm (compatible with di-/tripeptides) and a capacity to accommodate a great variety of size and charge in the side groups (125). The acid load of the enterocyte imposed by H + influx associated with PEPT1-mediated peptide/H + symport is relieved by Na + /H + exchange at the apical membrane (170). (Early reports that peptide transport is Na + -linked are erroneous.) Neutral and most cationic peptides are cotransported with one proton, while anionic peptides require two protons (228). Peptides taken up into the enterocyte are hydrolyzed by a diversity of cytoplasmic peptidases ( Fig. 10 ), and the resultant amino acids are exported via transporters on the basolateral membrane ( Table 3 ).

Peptide absorption. Uptake of di- and tripeptides across the apical membrane of enterocytes is mediated by PEPT1/H + symport, with the H + transport coupled to the Na + /H + antiporter NHE3. The peptides are hydrolyzed by multiple cytosolic hydrolases, and the resultant amino acids are exported via the basolateral membrane by multiple transporters (see Table 3 ). The efflux of unhydrolyzed peptides across the basolateral membrane is mediated by peptide transporters that have not been identified at molecular level.

Low-affinity/high-capacity peptide transporters expressed in the alimentary tract have been characterized functionally in nonmammalian vertebrates, notably the chicken (184), zebrafish (454), and other fish (455), and in Caenorhabditis elegans (317) and Drosophila (382). The peptide transporter family to which the mammalian PEPT1 protein belongs is ancient, with the defining peptide transporter motif (PTR) motif evident in proteins of bacteria, fungi, plants, and animals (107). Analysis of basal animal groups is required to establish the evolutionary origin(s) of gut-borne peptide transporter(s) in metazoans.

Of central importance is the relative importance of peptide and amino acid uptake in the protein nutrition of the animal. Humans with mutational defects in amino acid uptake systems do not suffer from essential amino acid deficiencies, for example, abolition of cystine uptake caused by defect in b 0,+ system (condition known as cystinuria), and aromatic amino acid uptake by defect in B 0 system (Hartnup disease) and this suggests that PEPT1-mediated uptake of peptides can be substantial, sufficient to meet the dietary requirements for these essential amino acids (106). The significance of PEPT1 for the protein nutrition of other animals remains to be established.

Transcellular pathways for lipid absorption

In vertebrates, the absorption of lipid hydrolysis products and sterols is dependent on their incorporation into micelles formed in the lumen of the small intestine. Micelles are 4 to 8 nm diameter aggregations of the hydrophobic lipid products with bile acids, which act as amphipathic detergents and mediate the passage of the lipid products across the aqueous boundary layer to the apical membrane of intestinal enterocytes. A proportion of the micelle-associated molecules pass across the apical membrane by simple diffusion, according to the concentration and permeability coefficient of each compound, but carrier-mediated transport is also involved.

The dominant lipids in most diets are triacylglycerols (TAGs), accompanied by small amounts of various polar and nonpolar lipids, including phospholipids, sterols, and the fat-soluble vitamins A and E. The products of lipid digestion include free FAs, glycerol, monoglycerides, and lysophospholipids. Following uptake by diffusion and via transporters, these products are transported to the endoplasmic reticulum, where they are used to synthesize diacylglycerols (DAGs), TAGs, phospholipids, cholesterol esters, etc. They are then packaged with lipoproteins to form chylomicrons, which are passed through the Golgi apparatus for exocytosis. In mammals, the chylomicrons are delivered to the lymphatic vessels. The mechanism of chylomicron assembly is reviewed by reference (227).

Of particular note are the transporters mediating sterol flux across the apical membrane of enterocytes. In mammals, a steep diffusion gradient across the apical membrane is generated by acyl-CoA:cholesterol acyltransferase (ACAT2)-mediated esterification of cholesterol in the enterocyte ( Fig. 11 ), and it used to be assumed𠅎rroneously—that cholesterol is taken up exclusively by simple diffusion. There is now overwhelming physiological and molecular evidence for carrier-mediated uptake and also efflux across the apical membrane ( Fig. 11 ). The key transporter mediating cholesterol uptake is Niemann Pick C1-like 1 (NPC1L1) protein, identified initially as the transporter sensitive to ezetimibe, a highly specific and potent inhibitor of intestinal cholesterol absorption (6, 111, 234). However, overexpression of NPC1L1 in nonenterocyte cells has not yielded cholesterol transport activity, suggesting that additional proteins may be required to reconstitute a fully functional cholesterol transporter. NPC1L1 has 50% amino acid homology to the NPC1 protein, which functions in intracellular cholesterol trafficking and is defective in the Niemann Pick type C cholesterol storage disease (70). Importantly, cholesterol is also exported across the apical membrane, via the ATP-binding cassette (ABC) transporters ABCG5 and ABCG8 (24). ABC transporters generally have 12 transmembrane domains, but each of ABCG5 and ABCG8 has just six transmembrane domains transport activity is mediated by the heterodimer, comprising a 12-transmembrane protein complex (194). Cholesterol molecules that are not esterified in the endoplasmic reticulum are eliminated from the enterocyte to the intestinal lumen and voided via the feces.

Absorption of cholesterol in mammalian intestine. Cholesterol presented in micelles to the apical membranes of enterocytes is taken up by Niemann-Pick C1-like-1 (NPC1L1) transporter, and esterified by acyl-CoA:cholesterol acyltransferase (ACAT2), an enzyme in the endoplasmic reticulum membrane. These esterified products are incorporated into apolipoprotein (apo)B48-containing chylomicrons in a microsomal triglyceride transport protein-dependent manner. After further processing, the chylomicrons are released from the basolateral membrane by exocytosis. Nonesterified sterol is eliminated into the gut lumen via ATP-binding cassette (ABC) transporters ABCG5 and ABCG8.

Nevertheless, ABCG5/G8 does not function exclusively in relation to cholesterol. Mammals feeding on fungal or plant material need to process the dominant sterols in these foods: ergosterol and phytosterols, respectively. These sterols have the tetracyclic ring structure and side chain at C17, as in cholesterol, but the side chain in phytosterols is alkylated at C-24 (e.g., with ethyl substituent in sitosterol), and some phytosterols (e.g., stigmasterol) also have double bonds in the side chain. They are taken up by NCP1L1 into enterocytes, but they are not esterified by ACAT2 and are eliminated via ABCG5/G8. Wang (2007) has described ABCG5/G8 as “the gatekeeper to avoid high plant sterols in plasma." This role is illustrated vividly by patients with mutations in ABCG5/G8, resulting in elevated absorption and plasma levels of sitosterol, a condition known as sitosterolemia. In healthy individuals, dietary phytosterols reduce serum cholesterol levels, probably through their more efficient incorporation than cholesterol into micelles, resulting in reduced cholesterol uptake (223) this is why sitosterol is sold as a functional food. A dietary supply of cholesterol is not required by mammals, which can synthesize sterols de novo.

Among invertebrates, most research on lipid absorption has concerned insects. The products of insect lipid digestion are absorbed principally across the midgut epithelium, although absorption in the foregut, e.g. the crop of the cockroach Periplaneta americana, can also occur (63, 447). Lipid absorption in insects differs from vertebrates in several important respects. (i) Although, as in vertebrates, the products of lipid hydrolysis are packaged into micelles, the amphipathic molecules of insect micelles are fatty acid-amino acid, lysophospholipid, and glycolipid complexes (442), and not bile acids (which insects lack). (ii) The lipids synthesized in all insect enterocytes studied to date are dominated by DAGs, not TAGs and sterols appear to be absorbed without esterification in the enterocyte (442). (iii) The functional equivalent to chylomicrons in insects is the high-density lipoprotein, lipophorin, which mediates the transport of DAGs exported from enterocytes (9). Unlike chylomicrons, lipophorin is not synthesized in enterocytes it is localized in the hemolymph (blood), where it acts as a shuttle delivering lipids to the fat body and other organs. Lipophorin has been implicated in the transport of hydrocarbons, carotenoids, sterols, and phosopholipids, as well as DAGs. (iv) The role of transporters in the absorption of lipidic compounds in insects is poorly studied, although a NPC-like transporter, NPC1b, has been demonstrated to mediate sterol uptake from the midgut of Drosophila (456), and a fatty acid transporter on the apical membrane has been invoked (63).

The products of lipid digestion in the gut of the spider Polybetes phythagoricus are taken up by cells of the midgut diverticulum, where they are processed to TAGs and phospholipids and exported via two distinct carriers: a high-density lipoprotein (equivalent to the insect lipophorin) and a very high density lipoprotein that also contains hemocyanin (275).

Pathways for absorption of short chain fatty acids

This class of lipid-related molecules is distinctive from other lipids in two important respects. First, they have lower hydrophobicity than long-chain fatty acids. Consequently, SCFAs permeate membranes more slowly by simple diffusion, and cellular transport mechanisms are especially important for SCFA absorption. Second, they are waste products of fermentative respiration of resident bacteria in nongastric, anoxic regions of the alimentary tract (not products of animal digestion), with the implication that they are produced and absorbed across the hindgut (and pregastric fermentation chambers of some animals, see Section �sic designs of digestive tracts”), not midgut, small intestine etc. For example, in humans, acetate, propionate, and butyrate are produced in the ratio 3:1:1 and contribute up to 10% of respiratory fuel butyrate is particularly important, as the primary carbon source for colonocytes (156). Topics not considered here are the role of SCFAs in the regulation of fluid and electrolyte movement of the vertebrate gut, reviewed by reference (32), and importance of butyrate in the regulation of colonic cell proliferation and differentiation [see review of reference (198)].

SCFAs are transported across the colon wall of mammals by a combination of simple diffusion and carrier-mediated processes. The SCFA transporter(s) have yet to be identified definitively. Studies with colonic epithelial tissue and luminal perfusion experiments point to SCFA/HCO3 − exchangers, with evidence for saturation kinetics and competitive inhibition by acetate, butyrate, and propionate, but not lactate (203, 204, 312, 378). However, the transport proteins responsible for SCFA/HCO3 − exchange have yet to be identified, raising the possibility that SCFA is coupled to HCO3 − via multiple transporters, for example, SCFA/H + cotransport and Cl − /HCO3 − exchange (99). SCFAs are transported by the H + /monocarboxylate transporter MCT1 in several colonic cancer cell lines, including Caco-2 cells, (282) and by a Na + -dependent SCFA transporter, SLCA8, cloned from the human intestine (324), but the relevance of these transporters to SCFA transport in the colon and cecum of healthy mammals in vivo is uncertain.

The fate of SCFAs in the gut epithelium has been studied particularly in the rumen. A proportion of the SCFAs taken up is metabolized to lactate and ketonic acids (including acetoacetate and 3-hydroxybutyrate) these products are transported from the basolateral membrane of epithelial cells, probably via MCT1, to the blood. The intraepithelial metabolism of SCFAs contributes to the high-energy demands of these cells. Additional advantages are the maintenance of the concentration gradient between the lumen of the rumen and epithelial cell contents, so promoting sustained SCFA uptake, and the greater solubility of the products (lactate etc.) than SCFAs, and therefore, facilitating transport in the blood to other organs.

Paracellular transport of organic molecules

Paracellular transport across the gut is constrained by tight junctions at the apical end of the lateral membrane of all cells in the epithelium. Tight junctions have selective permeability, discriminating among solutes by charge and size. Two pathways across the tight junction have been identified in various epithelial cell types, including gut epithelia: a high-capacity pore pathway, permeable to small uncharged molecules and ions (π.8 nm diam.) and a leak pathway mediating low capacity flux of larger, uncharged molecules. Caco-2 cells display a third pathway that allows the passage of molecules up to 0.13 nm diameter, suggesting an additional route in the mammalian gut intestine (448). Although the contribution of the various tight junction proteins to the restriction of movement between epithelial cells is not fully understood, there is growing evidence that: (i) the claudins (a family of membrane proteins spanning the tight junction) play a crucial role in the pore pathway, with individual family members forming cation- or anion-selective pores (ii) two further tight junction proteins, occludin and zona occludens-1, are important in the leak pathway and (iii) various intracellular and extracellular signals mediate cross-talk between the two pathways, resulting in dynamic regulation of flux of different classes of compounds by the paracellular route. For an excellent review on the molecular determinants of the function and plasticity of tight junctions, the reader is referred to (398).

For humans and biomedical rodent models, the paracellular pathway makes a negligible contribution to absorption of many solutes. Despite the growing evidence for dynamic selective permeability of tight junctions, the predominance of transcellular transport has been attributed to the superior selectivity of transcellular transport via carrier-mediated transporters on the apical membrane of enterocytes, thereby protecting the animal from many toxins or otherwise deleterious compounds breaching the gut wall.

Nevertheless, there is substantial evidence for extensive paracellular transport of solutes in flying birds and fruit bats. Particular insight into the mode of sugar transport comes from parallel analysis of absorption of L-glucose (the stereoisomer that does not interact with the glucose transporters and is transported exclusively by paracellular route), and D-glucose or 3-O-methyl-d-glucose (3OMD-glucose), a nonmetabolizable analogue of D-glucose that can be transported into cells. Karasov and colleagues measured total absorption (mediated and passive) of D-glucose or 3OMD-glucose and passive absorption of L-glucose in intact animals by a standard pharmacokinetic methodology, for example, references (78, 244, 278, 280). In experiments conducted on avian species, the fractional absorption of D-glucose and 3OMD-glucose did not differ significantly and L-glucose was found to account for the majority (range 50 to > 90%) of glucose absorption (79, 238, 316) ( Fig. 12 ). In analogous studies in rats (443), dogs (277), and humans (154) L-glucose, and hence passive absorption, is quantitatively much less important, confirming the likely phylogenetic difference between birds and mammals in the importance of paracellular transport.

Paracellular absorption of glucose in the American robin (Turdus migratorius) investigated by pharmacokinetic methodology, using D-glucose, L-glucose (the glucose stereoisomer that is not be transported across the intestinal membrane), and 3-O-methyl- d -glucose (3OMD-glucose, a nonmetabolizable but actively transported analogue of D-glucose). (A) The dose-corrected plasma concentration of [ 3 H]L-glucose as a function of time since American robins were injected (unfilled symbols) or gavaged (filled symbols) with the probe solution containing L-glucose. The areas under the curves (AUCs) are used to calculate fractional absorption, f, which averaged 87 ± 3%. (B) Time course of absorption of [ 3 H]L-glucose, and [ 14 C]D-glucose and 3OMD-glucose. Over early time points, the amounts of L-glucose absorbed was 50% to 70% of the amounts of D-glucose absorbed, which was interpreted to mean that the majority of glucose was absorbed by the paracellular pathway. Adapted from Figures 1 and ​ and2 2 from reference (316), with permission.

Intestinal paracellular absorption in nonflying mammals and birds appears to be qualitatively similar in regards to molecular size selectivity, as characterized using a series of nonelectrolyte water-soluble probes that differ in molecular dimension (80, 199) and in charge selectivity as characterized using relatively inert charged peptides (81, 205). Quantitatively, paracellular absorption is at least twice greater in small birds (< 400g) than in nonflying mammals ( Fig. 13A ), with the difference declining with increasing body size (278).

(A) Fractional absorption of water soluble carbohydrates by intact birds (triangles, solid line) and nonflying eutherian mammals (circles, dashed line). Arabinose, rhamnose, cellobiose, and lactulose are inert, nonactively transported compounds whereas 3-O-methyl- d -glucose is not metabolized but is transported actively as well as passively absorbed. Fractional absorption of the passively absorbed probes declined with increasing molecule size and differed significantly between the two taxa, although the difference diminished with increasing molecule size. In contrast, absorption of 3-Omethyl- d -glucose did not differ significantly between the taxa. The interpretation is that species in both groups absorb most glucose, but that birds relied more on the passive, paracellular route. Figure 4A adapted, with permission, from reference (243). (B) Small intestine nominal (smoothbore tube) surface area in omnivorous birds and mammals (same symbols and lines as in A). There was no significant difference in slope between birds and nonflying mammals (n = 46 species and 41 species in birds and mammals, respectively). When the lines were fit to the common slope of 0.73, the calculated proportionality coefficients (intercept at unity) were significantly lower for birds than for mammals. Hence, small intestine nominal surface area in birds is 36% lower than that in nonflying mammals. Figure 4B adapted from reference (75).

The difference in paracellular absorption between birds and nonflying mammals is not simply explained by mediated absorption in birds of the carbohydrate probes that are presumed to be absorbed passively. In studies using radiolabeled L-glucose and L-arabinose, their uptake by intestine in vitro was not significantly inhibited by high concentrations (50� mmol/L) of unlabeled L-glucose, L-arabinose, L-rhamnose, or D-glucose (280), which makes it unlikely that their absorption is carrier mediated. Nor is the difference in paracellular absorption between birds and nonflying mammals explained by longer retention of digesta in the gut of the former relative to the latter. Avian species typically have shorter mean retention time of digesta than do similar sized nonflying mammalian species (315). Because birds typically achieve higher paracellular absorption with less intestinal length and surface area than do similar sized nonflying mammals, there apparently are differences in intestinal permeability per unit intestinal tissue. This was confirmed in a comparison of pigeons and laboratory rats. Under similar recirculating duodenal perfusion conditions, anesthetized rats, and pigeons absorbed D-glucose at a comparable rate but pigeons had significantly greater (Ϣ× higher) absorption of inert carbohydrate probes (280). The difference in paracellular solute absorption between mammals and birds cannot be linked to differences in solvent drag because it is so difficult (155) to distinguish between water absorbed by the paracellular route versus aquaporins, which occur in intestine of both mammals and birds (229).

Enhanced paracellular absorption may have evolved as a compensation for smaller intestinal size in birds compared with nonflying mammals ( Fig. 13B ). In a phylogenetically informed allometric analysis, flying birds had shorter intestines and about 36% less nominal small intestine surface area (area of a smooth bore tube) as compared with nonflying mammals (279). Small intestine volume, a direct function of tube length and area, and consequently the potential mass of digesta carried, was relatively smaller in birds, by 32%. The difference in intestinal surface area between birds and nonflying mammals did not depend on diet in the analysis. (Diet did have a significant effect on gut size, but the effect was on cecal and large intestine size.) Another advantage of paracellular absorption is that it is an energetically cheap way to match absorption rate to substrate concentration in the diet and lumen.

If there has indeed been natural selection for smaller intestinal size in fliers, and increased paracellular absorption as a compensation, then one might expect to find the same patterns found in flying birds versus nonflying mammals in a comparison within mammals between fliers (i.e., bats) and nonfliers. Preliminary evidence suggests that this is the case (75), but more extensive sampling is necessary.

Dietary and phylogenetic correlates of transporter activity

Generally, in vertebrates, the more carnivorous the species, the lower its rate of intestinal mediated glucose absorption (246). This pattern, first described in a survey of more than 40 species drawn from the major vertebrate classes (245), is apparent also in comparative studies within fish (51) and birds (247). Based on phlorizin-binding studies in a limited number of species, it appeared that species differences in tissue-specific glucose uptake may largely reflect species differences in the number of copies of the main apical membrane glucose transporter SGLT1, although it is possible that differences in turnover time of the transporter can also contribute (150).

There was no marked pattern of higher intestinal transport activity for amino acids among the more carnivorous vertebrate species (245, 246). Likewise for digestive enzymes, it seems typical to find significant positive relationships between carbohydrases and dietary carbohydrate but not between proteases/peptidases and dietary protein, at least for fish (179), and in birds (261). This is perhaps expected because all animals, regardless of diet, need protein and so there should not be strong selection for very low protein processing capability in animals. In addition, it has been argued (214) that it would be advantageous for herbivores with relatively rapid gut throughput to have compensatorily higher biochemical capacity to process proteins and recover them rather than excrete them.


Enzymes_Two - Biology

Figure 1. Enzymes lower the activation energy of the reaction but do not change the free energy of the reaction.

A substance that helps a chemical reaction to occur is called a catalyst, and the molecules that catalyze biochemical reactions are called enzymes. Most enzymes are proteins and perform the critical task of lowering the activation energies of chemical reactions inside the cell. Most of the reactions critical to a living cell happen too slowly at normal temperatures to be of any use to the cell. Without enzymes to speed up these reactions, life could not persist. Enzymes do this by binding to the reactant molecules and holding them in such a way as to make the chemical bond-breaking and -forming processes take place more easily. It is important to remember that enzymes do not change whether a reaction is exergonic (spontaneous) or endergonic. This is because they do not change the free energy of the reactants or products. They only reduce the activation energy required for the reaction to go forward (Figure 1). In addition, an enzyme itself is unchanged by the reaction it catalyzes. Once one reaction has been catalyzed, the enzyme is able to participate in other reactions.

The chemical reactants to which an enzyme binds are called the enzyme’s substrates. There may be one or more substrates, depending on the particular chemical reaction. In some reactions, a single reactant substrate is broken down into multiple products. In others, two substrates may come together to create one larger molecule. Two reactants might also enter a reaction and both become modified, but they leave the reaction as two products. The location within the enzyme where the substrate binds is called the enzyme’s active site. The active site is where the “action” happens. Since enzymes are proteins, there is a unique combination of amino acid side chains within the active site. Each side chain is characterized by different properties. They can be large or small, weakly acidic or basic, hydrophilic or hydrophobic, positively or negatively charged, or neutral. The unique combination of side chains creates a very specific chemical environment within the active site. This specific environment is suited to bind to one specific chemical substrate (or substrates).

Active sites are subject to influences of the local environment. Increasing the environmental temperature generally increases reaction rates, enzyme-catalyzed or otherwise. However, temperatures outside of an optimal range reduce the rate at which an enzyme catalyzes a reaction. Hot temperatures will eventually cause enzymes to denature, an irreversible change in the three-dimensional shape and therefore the function of the enzyme. Enzymes are also suited to function best within a certain pH and salt concentration range, and, as with temperature, extreme pH, and salt concentrations can cause enzymes to denature.

For many years, scientists thought that enzyme-substrate binding took place in a simple “lock and key” fashion. This model asserted that the enzyme and substrate fit together perfectly in one instantaneous step. However, current research supports a model called induced fit (Figure 2). The induced-fit model expands on the lock-and-key model by describing a more dynamic binding between enzyme and substrate. As the enzyme and substrate come together, their interaction causes a mild shift in the enzyme’s structure that forms an ideal binding arrangement between enzyme and substrate.

When an enzyme binds its substrate, an enzyme-substrate complex is formed. This complex lowers the activation energy of the reaction and promotes its rapid progression in one of multiple possible ways. On a basic level, enzymes promote chemical reactions that involve more than one substrate by bringing the substrates together in an optimal orientation for reaction. Another way in which enzymes promote the reaction of their substrates is by creating an optimal environment within the active site for the reaction to occur.

Figure 2. The induced-fit model is an adjustment to the lock-and-key model and explains how enzymes and substrates undergo dynamic modifications during the transition state to increase the affinity of the substrate for the active site.

Careers in Action: Pharmaceutical Drug Developer

Figure 3. Have you ever wondered how pharmaceutical drugs are developed? (credit: Deborah Austin)

Enzymes are key components of metabolic pathways. Understanding how enzymes work and how they can be regulated are key principles behind the development of many of the pharmaceutical drugs on the market today. Biologists working in this field collaborate with other scientists to design drugs.

Consider statins for example—statins is the name given to one class of drugs that can reduce cholesterol levels. These compounds are inhibitors of the enzyme HMG-CoA reductase, which is the enzyme that synthesizes cholesterol from lipids in the body. By inhibiting this enzyme, the level of cholesterol synthesized in the body can be reduced. Similarly, acetaminophen, popularly marketed under the brand name Tylenol, is an inhibitor of the enzyme cyclooxygenase. While it is used to provide relief from fever and inflammation (pain), its mechanism of action is still not completely understood.

How are drugs discovered? One of the biggest challenges in drug discovery is identifying a drug target. A drug target is a molecule that is literally the target of the drug. In the case of statins, HMG-CoA reductase is the drug target. Drug targets are identified through painstaking research in the laboratory. Identifying the target alone is not enough scientists also need to know how the target acts inside the cell and which reactions go awry in the case of disease. Once the target and the pathway are identified, then the actual process of drug design begins. In this stage, chemists and biologists work together to design and synthesize molecules that can block or activate a particular reaction. However, this is only the beginning: If and when a drug prototype is successful in performing its function, then it is subjected to many tests from in vitro experiments to clinical trials before it can get approval from the U.S. Food and Drug Administration to be on the market.

Many enzymes do not work optimally, or even at all, unless bound to other specific non-protein helper molecules. They may bond either temporarily through ionic or hydrogen bonds, or permanently through stronger covalent bonds. Binding to these molecules promotes optimal shape and function of their respective enzymes. Two examples of these types of helper molecules are cofactors and coenzymes. Cofactors are inorganic ions such as ions of iron and magnesium. Coenzymes are organic helper molecules, those with a basic atomic structure made up of carbon and hydrogen. Like enzymes, these molecules participate in reactions without being changed themselves and are ultimately recycled and reused. Vitamins are the source of coenzymes. Some vitamins are the precursors of coenzymes and others act directly as coenzymes. Vitamin C is a direct coenzyme for multiple enzymes that take part in building the important connective tissue, collagen. Therefore, enzyme function is, in part, regulated by the abundance of various cofactors and coenzymes, which may be supplied by an organism’s diet or, in some cases, produced by the organism.


Collaborative Biosynthesis of a Class of Bioactive Azaphilones by Two Separate Gene Clusters Containing Four PKS/NRPSs with Transcriptional Crosstalk in Fungi

Shandong Provincial Key Laboratory of Synthetic Biology, Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, No. 189 Songling Road, Qingdao, 266101 China

These authors contributed equally to this work.

Shandong Provincial Key Laboratory of Synthetic Biology, Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, No. 189 Songling Road, Qingdao, 266101 China

These authors contributed equally to this work.

Shandong Provincial Key Laboratory of Synthetic Biology, Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, No. 189 Songling Road, Qingdao, 266101 China

College of Bioscience and Bioengineering, Jiangxi Agricultural University, No. 1101 Zhimin Road, Nanchang, 330045 China

These authors contributed equally to this work.

Shandong Provincial Key Laboratory of Synthetic Biology, Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, No. 189 Songling Road, Qingdao, 266101 China

Shandong Provincial Key Laboratory of Synthetic Biology, Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, No. 189 Songling Road, Qingdao, 266101 China

Marine Biology and Biotechnology Laboratory, Qingdao National Laboratory for Marine Science and Technology, No. 1 Wenhai Road, Aoshanwei, Qingdao, 266101 China

Shandong Provincial Key Laboratory of Synthetic Biology, Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, No. 189 Songling Road, Qingdao, 266101 China

These authors contributed equally to this work.

Shandong Provincial Key Laboratory of Synthetic Biology, Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, No. 189 Songling Road, Qingdao, 266101 China

These authors contributed equally to this work.

Shandong Provincial Key Laboratory of Synthetic Biology, Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, No. 189 Songling Road, Qingdao, 266101 China

College of Bioscience and Bioengineering, Jiangxi Agricultural University, No. 1101 Zhimin Road, Nanchang, 330045 China

These authors contributed equally to this work.

Shandong Provincial Key Laboratory of Synthetic Biology, Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, No. 189 Songling Road, Qingdao, 266101 China

Shandong Provincial Key Laboratory of Synthetic Biology, Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, No. 189 Songling Road, Qingdao, 266101 China

Marine Biology and Biotechnology Laboratory, Qingdao National Laboratory for Marine Science and Technology, No. 1 Wenhai Road, Aoshanwei, Qingdao, 266101 China

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Abstract

Azaphilones are a family of fungal polyketide metabolites with diverse chemical structures and biological activities with a highly oxygenated pyranoquinone bicyclic core. Here, a class of azaphilones possessing a 6/6/6/6 tetracyclic ring system was identified in Aspergillus terreus, and exhibited potential anticancer activities. The gene deletions and biochemical investigations demonstrated that these azaphilones were collaboratively synthesized by two separate clusters containing four core-enzymes, two nonreducing PKSs, one highly reducing PKS, and one NRPS-like. More interestingly, we found that the biosynthesis is coordinately regulated by a crosstalk mechanism between these two gene clusters based on three transcriptional factors. This is a meaningful mechanism of fungal secondary metabolism, which allows fungi to synthesize more complex compounds and gain new physiological functions. The results provide a new insight into fungal natural product biosynthesis.

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What Is the Main Function of Enzymes?

Enzymes are naturally occurring proteins that are found in the bodies of certain living things, including humans and other animals, and that cause chemical changes such as breaking down food in the stomach. Within the human body, enzymes can be found in bodily fluids, such as blood, saliva, the gastric juices or the stomach and fluids in the intestines. In general, enzymes serve as catalysts for biological functions, including natural, involuntary bodily functions, such as blood clotting.

Enzymes have three main characteristics. First, they increase the rate of a natural chemical reaction. Secondly, they typically only react with one specific substrate or reactant, and thirdly, enzyme activity is regulated and controlled within the cell through several different means, including regulation by inhibitors and activators. It is possible to group enzymes into different categories, including oxidases, transferases, hydrolases, lyaes, isomerases and ligases. In naming enzymes, the "-ase" suffix is often appended to the name of the substrate molecule upon which which the enzyme reacts. For example, the enzyme sucrase catalyzes the transformation of the sugar sucrose in to glucose and fructose. In this case, the "sucr-" suffix represents the molecule upon which the sucrase enzyme reacts. Not all enzymes are named according to this convention.