Synthesis of Chiral Acyclic Pyrimidine Nucleoside Analogues from DHAP-Dependent Aldolases.

Dihydroxyacetone phosphate (DHAP)-dependent aldolases catalyze the aldol addition of DHAP to a variety of aldehydes and generate compounds with two stereocenters. This reaction is useful to synthesize chiral acyclic nucleosides, which constitute a well-known class of antiviral drugs currently used. In such compounds, the chirality of the aliphatic chain, which mimics the open pentose residue, is crucial for activity. In this work, three DHAP-dependent aldolases: fructose-1,6-biphosphate aldolase from rabbit muscle, rhanmulose-1-phosphate aldolase from Thermotoga maritima, and fuculose-1-phosphate aldolase from Escherichia coli, were used as biocatalysts. Aldehyde derivatives of thymine and cytosine were used as acceptor substrates, generating new acyclic nucleoside analogues containing two new stereocenters with conversion yields between 70% and 90%. Moreover, structural analyses by molecular docking were carried out to gain insights into the diasteromeric excess observed.

Glycerol-3-Phosphate Dehydrogenase: The K120 and K204 Side Chains Define an Oxyanion Hole at the Enzyme Active Site.

The cationic K120 and K204 side chains lie close to the C-2 carbonyl group of substrate dihydroxyacetone phosphate (DHAP) at the active site of glycerol-3-phosphate dehydrogenase (GPDH), and the K120 side chain is also positioned to form a hydrogen bond to the C-1 hydroxyl of DHAP. The kinetic parameters for unactivated and phosphite dianion-activated GPDH-catalyzed reduction of glycolaldehyde and acetaldehyde (AcA) show that the transition state for the former reaction is stabilized by ca 5 kcal/mole by interactions of the C-1 hydroxyl group with the protein catalyst. The K120A and K204A substitutions at wild-type GPDH result in similar decreases in kcat, but Km is only affected by the K120A substitution. These results are consistent with 3 kcal/mol stabilizing interactions between the K120 or K204 side chains and a negative charge at the C-2 oxygen at the transition state for hydride transfer from NADH to DHAP. This stabilization resembles that observed at oxyanion holes for other enzymes. There is no detectable rescue of the K204A variant by ethylammonium cation (EtNH3+), compared with the efficient rescue of the K120A variant. This is consistent with a difference in the accessibility of the variant enzyme active sites to exogenous EtNH3+. The K120A/K204A substitutions cause a (6 × 106)-fold increase in the promiscuity of wild-type hlGPDH for catalysis of the reduction of AcA compared to DHAP. This may reflect conservation of the active site for an ancestral alcohol dehydrogenase, whose relative activity for catalysis of reduction of AcA increases with substitutions that reduce the activity for reduction of the specific substrate DHAP.

Transient Formation of a Second Active Site Cavity during Quinolinic Acid Synthesis by NadA.

Quinolinate synthase, also called NadA, is a [4Fe-4S]-containing enzyme that uses what is probably the oldest pathway to generate quinolinic acid (QA), the universal precursor of the biologically essential cofactor nicotinamide adenine dinucleotide (NAD). Its synthesis comprises the condensation of dihydroxyacetone phosphate (DHAP) and iminoaspartate (IA), which involves dephosphorylation, isomerization, cyclization, and two dehydration steps. The convergence of the three homologous domains of NadA defines a narrow active site that contains a catalytically essential [4Fe-4S] cluster. A tunnel, which can be opened or closed depending on the nature (or absence) of the bound ligand, connects this cofactor to the protein surface. One outstanding riddle has been the observation that the so far characterized active site is too small to bind IA and DHAP simultaneously. Here, we have used site-directed mutagenesis, X-ray crystallography, functional analyses, and molecular dynamics simulations to propose a condensation mechanism that involves the transient formation of a second active site cavity to which one of the substrates can migrate before this reaction takes place.

Hydride Transfer Catalyzed by Glycerol Phosphate Dehydrogenase: Recruitment of an Acidic Amino Acid Side Chain to Rescue a Damaged Enzyme.

K120 of glycerol 3-phosphate dehydrogenase (GPDH) lies close to the carbonyl group of the bound dihydroxyacetone phosphate (DHAP) dianion. pH rate (pH 4.6-9.0) profiles are reported for kcat and (kcat/Km)dianion for wild type and K120A GPDH-catalyzed reduction of DHAP by NADH, and for (kcat/KdKam) for activation of the variant-catalyzed reduction by CH3CH2NH3+, where Kam and Kd are apparent dissociation constants for CH3CH2NH3+ and DHAP, respectively. These profiles provide evidence that the K120 side chain cation, which is stabilized by an ion-pairing interaction with the D260 side chain, remains protonated between pH 4.6 and 9.0. The profiles for wild type and K120A variant GPDH show downward breaks at a similar pH value (7.6) that are attributed to protonation of the K204 side chain, which also lies close to the substrate carbonyl oxygen. The pH profiles for (kcat/Km)dianion and (kcat/KdKam) for the K120A variant show that the monoprotonated form of the variant is activated for catalysis by CH3CH2NH3+ but has no detectable activity, compared to the diprotonated variant, for unactivated reduction of DHAP. The pH profile for kcat shows that the monoprotonated K120A variant is active toward reduction of enzyme-bound DHAP, because of activation by a ligand-driven conformational change. Upward breaks in the pH profiles for kcat and (kcat/Km)dianion for K120A GPDH are attributed to protonation of D260. These breaks are consistent with the functional replacement of K120 by D260, and a plasticity in the catalytic roles of the active site side chains.

The Organization of Active Site Side Chains of Glycerol-3-phosphate Dehydrogenase Promotes Efficient Enzyme Catalysis and Rescue of Variant Enzymes.

A comparison of the values of kcat/Km for reduction of dihydroxyacetone phosphate (DHAP) by NADH catalyzed by wild type and K120A/R269A variant glycerol-3-phosphate dehydrogenase from human liver (hlGPDH) shows that the transition state for enzyme-catalyzed hydride transfer is stabilized by 12.0 kcal/mol by interactions with the cationic K120 and R269 side chains. The transition state for the K120A/R269A variant-catalyzed reduction of DHAP is stabilized by 1.0 and 3.8 kcal/mol for reactions in the presence of 1.0 M EtNH3+ and guanidinium cation (Gua+), respectively, and by 7.5 kcal/mol for reactions in the presence of a mixture of each cation at 1.0 M, so that the transition state stabilization by the ternary E·EtNH3+·Gua+ complex is 2.8 kcal/mol greater than the sum of stabilization by the respective binary complexes. This shows that there is cooperativity between the paired activators in transition state stabilization. The effective molarities (EMs) of ∼50 M determined for the K120A and R269A side chains are ≪106 M, the EM for entropically controlled reactions. The unusually efficient rescue of the activity of hlGPDH-catalyzed reactions by the HPi/Gua+ pair and by the Gua+/EtNH3+ activator pair is due to stabilizing interactions between the protein and the activator pieces that organize the K120 and R269 side chains at the active site. This "preorganization" of side chains promotes effective catalysis by hlGPDH and many other enzymes. The role of the highly conserved network of side chains, which include Q295, R269, N270, N205, T264, K204, D260, and K120, in catalysis is discussed.

One-Pot Multienzyme Synthesis of Rare Ketoses from Glycerol.

A facile approach is introduced here for the synthesis of rare ketoses from glycerol and d-/l-glyceraldehyde (d-/l-GA). The reactions were carried out in a one-pot multienzyme fashion in which the only carbon source is glycerol. In the enzymatic cascade, glycerol is phosphorylated and then oxidized at C2 to afford dihydroxyacetone phosphate (DHAP), the key donor for enzymatic aldol reaction. Meanwhile, the primary alcohol of glycerol is also oxidized to give the acceptor molecule GA in situ (d- or l-isomer could be formed stereospecifically with either alditol oxidase or horse liver alcohol dehydrogenase). Different DHAP-dependent aldolases were used to generate the aldol adducts (rare ketohexose phosphates) with various stereoconfigurations and diastereomeric ratios. It is worth noting that the enzyme that catalyzes the phosphorylation reaction in the first step could also help recycle the phosphate in the last step to provide free rare sugar molecules. This study provides a useful method for rare ketose synthesis on a 100 mg to g scale, starting from relatively inexpensive materials which solved the problem of supplying both glycerol 3-phosphate and GA in our previous work. It also demonstrates an example of green synthesis due to highly efficient carbon usage and recycling of cofactors.

Standard Gibbs energy of metabolic reactions: IV. Triosephosphate isomerase reaction.

The glycolytic pathway is present in most organisms and represents a central part of the energy production mechanism in a cell. For a general understanding of glycolysis, the investigation from a thermodynamic point of view is essential and allows realising thermodynamic feasibility analyses under in vivo conditions. However, available literature standard Gibbs energies of reaction, ΔRg'0, are calculated using equilibrium-molality ratios Km', which might lead to a misinterpretation of the glycolytic pathway. It was the aim of this work to thermodynamically investigate the triosephosphate isomerase (TPI) reaction to provide new activity-based reaction data. In vitro equilibrium experiments were performed, and activity coefficients were predicted with the equation of state electrolyte PC-SAFT (ePC-SAFT). The combination of experimental concentrations and predicted activity coefficients yielded the thermodynamic equilibrium constant Ka and a new value for ΔRg'0(298.15 K, pH 7) = 7.1 ± 0.3 kJ mol­1. The availability of the new ΔRg'0 value allowed predicting influences of the reaction medium on the reaction equilibrium of the TPI reaction. In this work, influences of the initial substrate concentration, pH and Mg2+ concentration on the reaction equilibrium were investigated and a method is presented to predict these influences. The higher the substrate concentration and the higher the temperature, the stronger the reaction equilibrium is shifted on the product side. While the pH did not have a significant influence on the reaction equilibrium, Mg2+ yielded a shift of the reaction equilibrium to the substrate side. All these effects were predicted correctly with ePC-SAFT. Based on the ePC-SAFT predictions we concluded that a charge-reduction of the product by complexation of the product with Mg2+ was responsible for the strong influence of Mg2+ on the reaction equilibrium. Finally, the standard enthalpy of reaction of ΔRh'0(pH 7) = 18 ± 7 kJ mol­1 was determined with the equilibrium constants Ka at 298.15 K, 304.15 K and 310.15 K using the van 't Hoff equation.

Uncovering the Role of Key Active-Site Side Chains in Catalysis: An Extended Brønsted Relationship for Substrate Deprotonation Catalyzed by Wild-Type and Variants of Triosephosphate Isomerase.

We report results of detailed empirical valence bond simulations that model the effect of several amino acid substitutions on the thermodynamic (ΔG°) and kinetic activation (ΔG⧧) barriers to deprotonation of dihydroxyacetone phosphate (DHAP) and d-glyceraldehyde 3-phosphate (GAP) bound to wild-type triosephosphate isomerase (TIM), as well as to the K12G, E97A, E97D, E97Q, K12G/E97A, I170A, L230A, I170A/L230A, and P166A variants of this enzyme. The EVB simulations model the observed effect of the P166A mutation on protein structure. The E97A, E97Q, and E97D mutations of the conserved E97 side chain result in ≤1.0 kcal mol-1 decreases in the activation barrier for substrate deprotonation. The agreement between experimental and computed activation barriers is within ±1 kcal mol-1, with a strong linear correlation between ΔG⧧ and ΔG° for all 11 variants, with slopes ß = 0.73 (R2 = 0.994) and ß = 0.74 (R2 = 0.995) for the deprotonation of DHAP and GAP, respectively. These Brønsted-type correlations show that the amino acid side chains examined in this study function to reduce the standard-state Gibbs free energy of reaction for deprotonation of the weak α-carbonyl carbon acid substrate to form the enediolate phosphate reaction intermediate. TIM utilizes the cationic side chain of K12 to provide direct electrostatic stabilization of the enolate oxyanion, and the nonpolar side chains of P166, I170, and L230 are utilized for the construction of an active-site cavity that provides optimal stabilization of the enediolate phosphate intermediate relative to the carbon acid substrate.

An Unexpected Species Determined by X-ray Crystallography that May Represent an Intermediate in the Reaction Catalyzed by Quinolinate Synthase.

Quinolinic acid is a common intermediate in the biosynthesis of nicotinamide adenine dinucleotide and its derivatives in all organisms that synthesize the molecule de novo. In most prokaryotes, it is formed from the condensation of dihydroxyacetone phosphate (DHAP) and iminoaspartate (IA) by the action of quinolinate synthase (NadA). NadA contains a [4Fe-4S] cluster cofactor with a unique noncysteinyl-ligated iron ion (Fea), which is proposed to bind the hydroxyl group of an intermediate in its reaction to facilitate a dehydration step. However, direct evidence for this role in catalysis has yet to be provided, and the exact chemical mechanism that underlies this transformation remains elusive. Herein, we present a structure of NadA from Pyrococcus horikoshii (PhNadA) in complex with IA and show that a carboxylate group of the molecule is ligated to Fea of the iron-sulfur cluster, occupying the site to which DHAP has been proposed to bind during catalysis. When crystals of PhNadA in complex with IA are soaked briefly in DHAP before freezing, electron density for a new molecule is observed, which we suggest is related to an intermediate in the reaction. Similar, but slightly different, "intermediates" are observed when crystals of a PhNadA Glu198Gln variant are incubated with DHAP, oxaloacetate, and ammonium chloride, conditions under which IA is formed chemically. Continuous-wave and pulse electron paramagnetic resonance techniques are used to verify the binding mode of substrates and proposed intermediates in frozen solution.

Synthesis of Branched-Chain Sugars with a DHAP-Dependent Aldolase: Ketones are Electrophile Substrates of Rhamnulose-1-phosphate Aldolases.

Dihydroxyacetone phosphate (DHAP)-dependent rhamnulose aldolases display an unprecedented versatility for ketones as electrophile substrates. We selected and characterized a rhamnulose aldolase from Bacteroides thetaiotaomicron (RhuABthet) to provide a proof of concept. DHAP was added as a nucleophile to several α-hydroxylated ketones used as electrophiles. This aldol addition was stereoselective and produced branched-chain monosaccharide adducts with a tertiary alcohol moiety. Several aldols were readily obtained in good to excellent yields (from 76 to 95 %). These results contradict the general view that aldehydes are the only electrophile substrates for DHAP-dependent aldolases and provide a new C-C bond-forming enzyme for stereoselective synthesis of tertiary alcohols.

Enzyme Architecture: Modeling the Operation of a Hydrophobic Clamp in Catalysis by Triosephosphate Isomerase.

Triosephosphate isomerase (TIM) is a proficient catalyst of the reversible isomerization of dihydroxyacetone phosphate (DHAP) to d-glyceraldehyde phosphate (GAP), via general base catalysis by E165. Historically, this enzyme has been an extremely important model system for understanding the fundamentals of biological catalysis. TIM is activated through an energetically demanding conformational change, which helps position the side chains of two key hydrophobic residues (I170 and L230), over the carboxylate side chain of E165. This is critical both for creating a hydrophobic pocket for the catalytic base and for maintaining correct active site architecture. Truncation of these residues to alanine causes significant falloffs in TIM's catalytic activity, but experiments have failed to provide a full description of the action of this clamp in promoting substrate deprotonation. We perform here detailed empirical valence bond calculations of the TIM-catalyzed deprotonation of DHAP and GAP by both wild-type TIM and its I170A, L230A, and I170A/L230A mutants, obtaining exceptional quantitative agreement with experiment. Our calculations provide a linear free energy relationship, with slope 0.8, between the activation barriers and Gibbs free energies for these TIM-catalyzed reactions. We conclude that these clamping side chains minimize the Gibbs free energy for substrate deprotonation, and that the effects on reaction driving force are largely expressed at the transition state for proton transfer. Our combined analysis of previous experimental and current computational results allows us to provide an overview of the breakdown of ground-state and transition state effects in enzyme catalysis in unprecedented detail, providing a molecular description of the operation of a hydrophobic clamp in triosephosphate isomerase.

Crystal structure of fuculose aldolase from the Antarctic psychrophilic yeast Glaciozyma antarctica PI12.

Fuculose-1-phosphate aldolase (FucA) catalyses the reversible cleavage of L-fuculose 1-phosphate to dihydroxyacetone phosphate (DHAP) and L-lactaldehyde. This enzyme from mesophiles and thermophiles has been extensively studied; however, there is no report on this enzyme from a psychrophile. In this study, the gene encoding FucA from Glaciozyma antarctica PI12 (GaFucA) was cloned and the enzyme was overexpressed in Escherichia coli, purified and crystallized. The tetrameric structure of GaFucA was determined to 1.34 Šresolution. The overall architecture of GaFucA and its catalytically essential histidine triad are highly conserved among other fuculose aldolases. Comparisons of structural features between GaFucA and its mesophilic and thermophilic homologues revealed that the enzyme has typical psychrophilic attributes, indicated by the presence of a high number of nonpolar residues at the surface and a lower number of arginine residues.

Crystal Structures of the Iron-Sulfur Cluster-Dependent Quinolinate Synthase in Complex with Dihydroxyacetone Phosphate, Iminoaspartate Analogues, and Quinolinate.

The quinolinate synthase of prokaryotes and photosynthetic eukaryotes, NadA, contains a [4Fe-4S] cluster with unknown function. We report crystal structures of Pyrococcus horikoshii NadA in complex with dihydroxyacetone phosphate (DHAP), iminoaspartate analogues, and quinolinate. DHAP adopts a nearly planar conformation and chelates the [4Fe-4S] cluster via its keto and hydroxyl groups. The active site architecture suggests that the cluster acts as a Lewis acid in enediolate formation, like zinc in class II aldolases. The DHAP and putative iminoaspartate structures suggest a model for a condensed intermediate. The ensemble of structures suggests a two-state system, which may be exploited in early steps.

Structure of Quinolinate Synthase from Pyrococcus horikoshii in the Presence of Its Product, Quinolinic Acid.

Quinolinic acid (QA) is a common intermediate in the biosynthesis of nicotinamide adenine dinucleotide (NAD(+)) and its derivatives in all organisms that synthesize the molecule de novo. In most prokaryotes, it is formed from the condensation of dihydroxyacetone phosphate (DHAP) and aspartate-enamine by the action of quinolinate synthase (NadA). NadA contains a [4Fe-4S] cluster cofactor with a unique, non-cysteinyl-ligated, iron ion (Fea), which is proposed to bind the hydroxyl group of a postulated intermediate in the last step of the reaction to facilitate a dehydration. However, direct evidence for this role in catalysis has yet to be provided. Herein, we present the structure of NadA in the presence of the product of its reaction, QA. We find that N1 and the C7 carboxylate group of QA ligate to Fea in a bidentate fashion, which is confirmed by Hyperfine Sublevel Correlation (HYSCORE) spectroscopy. This binding mode would place the C5 hydroxyl group of the postulated final intermediate distal to Fea and virtually incapable of coordinating to it. The structure shows that three strictly conserved amino acids, Glu198, Tyr109, and Tyr23, are in close proximity to the bound product. Substitution of these amino acids with Gln, Phe, and Phe, respectively, leads to complete loss of activity.

Structure and Stability of the Dimeric Triosephosphate Isomerase from the Thermophilic Archaeon Thermoplasma acidophilum.

Thermoplasma acidophilum is a thermophilic archaeon that uses both non-phosphorylative Entner-Doudoroff (ED) pathway and Embden-Meyerhof-Parnas (EMP) pathway for glucose degradation. While triosephosphate isomerase (TPI), a well-known glycolytic enzyme, is not involved in the ED pathway in T. acidophilum, it has been considered to play an important role in the EMP pathway. Here, we report crystal structures of apo- and glycerol-3-phosphate-bound TPI from T. acidophilum (TaTPI). TaTPI adopts the canonical TIM-barrel fold with eight α-helices and parallel eight ß-strands. Although TaTPI shares ~30% sequence identity to other TPIs from thermophilic species that adopt tetrameric conformation for enzymatic activity in their harsh physiological environments, TaTPI exists as a dimer in solution. We confirmed the dimeric conformation of TaTPI by analytical ultracentrifugation and size-exclusion chromatography. Helix 5 as well as helix 4 of thermostable tetrameric TPIs have been known to play crucial roles in oligomerization, forming a hydrophobic interface. However, TaTPI contains unique charged-amino acid residues in the helix 5 and adopts dimer conformation. TaTPI exhibits the apparent Td value of 74.6°C and maintains its overall structure with some changes in the secondary structure contents at extremely acidic conditions (pH 1-2). Based on our structural and biophysical analyses of TaTPI, more compact structure of the protomer with reduced length of loops and certain patches on the surface could account for the robust nature of Thermoplasma acidophilum TPI.

Casuarine stereoisomers from achiral substrates: chemoenzymatic synthesis and inhibitory properties.

A straightforward chemoenzymatic synthesis of four uncovered casuarine stereoisomers is described. The strategy consists of L-fuculose-1-phosphate aldolase F131A-variant-catalyzed aldol addition of dihydroxyacetone phosphate to aldehyde derivatives of 1,4-dideoxy-1,4-imino-D-arabinitol (DAB) and its enantiomer (LAB) and subsequent one-pot catalytic deprotection-reductive amination. DAB and LAB were obtained from dihydroxyacetone and aminoethanol using D-fructose-6-phosphate aldolase and L-rhamnulose-1-phosphate aldolase catalysts, respectively. The new ent-3-epi-casuarine is a strong inhibitor of α-d-glucosidase from rice and of rat intestinal sucrase.

Enzyme architecture: the effect of replacement and deletion mutations of loop 6 on catalysis by triosephosphate isomerase.

Two mutations of the phosphodianion gripper loop in chicken muscle triosephosphate isomerase (cTIM) were examined: (1) the loop deletion mutant (LDM) formed by removal of residues 170-173 [Pompliano, D. L., et al. (1990) Biochemistry 29, 3186-3194] and (2) the loop 6 replacement mutant (L6RM), in which the N-terminal hinge sequence of TIM from eukaryotes, 166-PXW-168 (X = L or V), is replaced by the sequence from archaea, 166-PPE-168. The X-ray crystal structure of the L6RM shows a large displacement of the side chain of E168 from that for W168 in wild-type cTIM. Solution nuclear magnetic resonance data show that the L6RM results in significant chemical shift changes in loop 6 and surrounding regions, and that the binding of glycerol 3-phosphate (G3P) results in chemical shift changes for nuclei at the active site of the L6RM that are smaller than those of wild-type cTIM. Interactions with loop 6 of the L6RM stabilize the enediolate intermediate toward the elimination reaction catalyzed by the LDM. The LDM and L6RM result in 800000- and 23000-fold decreases, respectively, in kcat/Km for isomerization of GAP. Saturation of the LDM, but not the L6RM, by substrate and inhibitor phosphoglycolate is detected by steady-state kinetic analyses. We propose, on the basis of a comparison of X-ray crystal structures for wild-type TIM and the L6RM, that ligands bind weakly to the L6RM because a large fraction of the ligand binding energy is utilized to overcome destabilizing electrostatic interactions between the side chains of E168 and E129 that are predicted to develop in the loop-closed enzyme. Similar normalized yields of DHAP, d-DHAP, and d-GAP are formed in LDM- and L6RM-catalyzed reactions of GAP in D2O. The smaller normalized 12-13% yield of DHAP and d-DHAP observed for the mutant cTIM-catalyzed reactions compared with the 79% yield of these products for wild-type cTIM suggests that these mutations impair the transfer of a proton from O-2 to O-1 at the initial enediolate phosphate intermediate. No products are detected for the LDM-catalyzed isomerization reactions in D2O of [1-(13)C]GA and HPi, but the L6RM-catalyzed reaction in the presence of 0.020 M dianion gives a 2% yield of the isomerization product [2-(13)C,2-(2)H]GA.

Aldolase-catalyzed synthesis of conformationally constrained iminocyclitols: preparation of polyhydroxylated benzopyrrolizidines and cyclohexapyrrolizidines.

A straightforward chemo-enzymatic synthesis of new polyhydroxylated benzopyrrolizidines and cyclohexapyrrolizidines is developed. The two-step strategy consists of l-fuculose-1-phosphate aldolase variant F131A-catalyzed aldol addition of dihydroxyacetone phosphate to rac-N-benzyloxycarbonylindoline-2-carbaldehyde as well as (2S*,3aS*,7aS*)- and (2S*,3aR*,7aR*)-N-benzyloxycarbonyloctahydroindole-2-carbaldehydes and a subsequent one-step catalytic deprotection-reductive amination.

DHAP-dependent aldolases from (hyper)thermophiles: biochemistry and applications.

Generating new carbon-carbon (C-C) bonds in an enantioselective way is one of the big challenges in organic synthesis. Aldolases are a natural tool for stereoselective C-C bond formation in a green and sustainable way. This review will focus on thermophilic aldolases in general and on dihydroxyacetone phosphate-dependent aldolases in particular. Biochemical properties and applications for synthesis of rare sugars and carbohydrates will be discussed.

Transmitting the allosteric signal in methylglyoxal synthase.

The homohexameric enzyme methylglyoxal synthase (MGS) converts dihydroxyacetone phosphate (DHAP) to methylglyoxal and phosphate. This enzyme is allosterically inhibited by phosphate. The allosteric signal induced by phosphate in MGS from Thermus sp. GH5 (TMGS) has been tracked by site-directed mutagenesis, from the binding site of phosphate to the pathways that transmit the signal, and finally to the active site which is the receiver of the signal. In TMGS, Ser-55 distinguishes the inhibitory phosphate from the phosphoryl group of the substrate, DHAP, and transmits the allosteric signal through Pro-82, Arg-97 and Val-101 to the active site. Furthermore, the addition of a C-terminal tail to TMGS reinforces the allosteric signal by introducing a new salt bridge between Asp-10 and an Arg in this tail. Lastly, the active site amino acid, Gly-56, is shown to be involved in both allostery and phosphate elimination step from DHAP by TMGS. Interestingly, some of the mutations also trigger homotropic allostery, supporting the hypothesis that allostery is an intrinsic property of all dynamic proteins. The details of the TMGS allosteric network discussed in this study can serve as a model system for understanding the enigmatic allosteric mechanism of other proteins.