Antibacterial Target DXP Synthase Catalyzes the Cleavage of d-Xylulose 5-Phosphate: a Study of Ketose Phosphate Binding and Ketol Transfer Reaction.

The bacterial enzyme 1-deoxy-d-xylulose 5-phosphate synthase (DXPS) catalyzes the formation of DXP from pyruvate and d-glyceraldehyde 3-phosphate (d-GAP) in a thiamin diphosphate (ThDP)-dependent manner. In addition to its role in isoprenoid biosynthesis, DXP is required for ThDP and pyridoxal phosphate biosynthesis. Due to its function as a branch-point enzyme and its demonstrated substrate and catalytic promiscuity, we hypothesize that DXPS could be key for bacterial adaptation in the dynamic metabolic landscape during infection. Prior work in the Freel Meyers laboratory has illustrated that DXPS displays relaxed specificity toward donor and acceptor substrates and varies acceptor specificity according to the donor used. We have reported that DXPS forms dihydroxyethyl (DHE)ThDP from ketoacid or aldehyde donor substrates via decarboxylation and deprotonation, respectively. Here, we tested other DHE donors and found that DXPS cleaves d-xylulose 5-phosphate (X5P) at C2-C3, producing DHEThDP through a third mechanism involving d-GAP elimination. We interrogated DXPS-catalyzed reactions using X5P as a donor substrate and illustrated (1) production of a semi-stable enzyme-bound intermediate and (2) O2, H+, and d-erythrose 4-phosphate act as acceptor substrates, highlighting a new transketolase-like activity of DXPS. Furthermore, we examined X5P binding to DXPS and suggest that the d-GAP binding pocket plays a crucial role in X5P binding and turnover. Overall, this study reveals a ketose-cleavage reaction catalyzed by DXPS, highlighting the remarkable flexibility for donor substrate usage by DXPS compared to other C-C bond-forming enzymes.

Structural basis of light-induced redox regulation in the Calvin-Benson cycle in cyanobacteria.

Plants, algae, and cyanobacteria fix carbon dioxide to organic carbon with the Calvin-Benson (CB) cycle. Phosphoribulokinase (PRK) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) are essential CB-cycle enzymes that control substrate availability for the carboxylation enzyme Rubisco. PRK consumes ATP to produce the Rubisco substrate ribulose bisphosphate (RuBP). GAPDH catalyzes the reduction step of the CB cycle with NADPH to produce the sugar glyceraldehyde 3-phosphate (GAP), which is used for regeneration of RuBP and is the main exit point of the cycle. GAPDH and PRK are coregulated by the redox state of a conditionally disordered protein CP12, which forms a ternary complex with both enzymes. However, the structural basis of CB-cycle regulation by CP12 is unknown. Here, we show how CP12 modulates the activity of both GAPDH and PRK. Using thermophilic cyanobacterial homologs, we solve crystal structures of GAPDH with different cofactors and CP12 bound, and the ternary GAPDH-CP12-PRK complex by electron cryo-microscopy, we reveal that formation of the N-terminal disulfide preorders CP12 prior to binding the PRK active site, which is resolved in complex with CP12. We find that CP12 binding to GAPDH influences substrate accessibility of all GAPDH active sites in the binary and ternary inhibited complexes. Our structural and biochemical data explain how CP12 integrates responses from both redox state and nicotinamide dinucleotide availability to regulate carbon fixation.

Mind the GAP: Purification and characterization of urea resistant GAPDH during extreme dehydration.

The African clawed frog (Xenopus laevis) withstands prolonged periods of extreme whole-body dehydration that lead to impaired blood flow, global hypoxia, and ischemic stress. During dehydration, these frogs shift from oxidative metabolism to a reliance on anaerobic glycolysis. In this study, we purified the central glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to electrophoretic homogeneity and investigated structural, kinetic, subcellular localization, and post-translational modification properties between control and 30% dehydrated X. laevis liver. GAPDH from dehydrated liver displayed a 25.4% reduction in maximal velocity and a 55.7% increase in its affinity for GAP, as compared to enzyme from hydrated frogs. Under dehydration mimicking conditions (150 mM urea and 1% PEG), GAP affinity was reduced with a Km value 53.8% higher than controls. Frog dehydration also induced a significant increase in serine phosphorylation, methylation, acetylation, beta-N-acetylglucosamination, and cysteine nitrosylation, post-translational modifications (PTMs). These modifications were bioinformatically predicted and experimentally validated to govern protein stability, enzymatic activity, and nuclear translocation, which increased during dehydration. These dehydration-responsive protein modifications, however, did not appear to affect enzymatic thermostability as GAPDH melting temperatures remained unchanged when tested with differential scanning fluorimetry. PTMs could promote extreme urea resistance in dehydrated GAPDH since the enzyme from dehydrated animals had a urea I50 of 7.3 M, while the I50 from the hydrated enzyme was 5.3 M. The physiological consequences of these dehydration-induced molecular modifications of GAPDH likely suppress GADPH glycolytic functions during the reduced circulation and global hypoxia experienced in dehydrated X. laevis.

Redirection of the central metabolism of Klebsiella pneumoniae towards dihydroxyacetone production.

BACKGROUND: Klebsiella pneumoniae is a bacterium that can be used as producer for numerous chemicals. Glycerol can be catabolised by K. pneumoniae and dihydroxyacetone is an intermediate of this catabolism pathway. Here dihydroxyacetone and glycerol were produced from glucose by this bacterium based a redirected glycerol catabolism pathway. RESULTS: tpiA, encoding triosephosphate isomerase, was knocked out to block the further catabolism of dihydroxyacetone phosphate in the glycolysis. After overexpression of a Corynebacterium glutamicum dihydroxyacetone phosphate dephosphorylase (hdpA), the engineered strain produced remarkable levels of dihydroxyacetone (7.0 g/L) and glycerol (2.5 g/L) from glucose. Further increase in product formation were obtained by knocking out gapA encoding an iosenzyme of glyceraldehyde 3-phosphate dehydrogenase. There are two dihydroxyacetone kinases in K. pneumoniae. They were both disrupted to prevent an inefficient reaction cycle between dihydroxyacetone phosphate and dihydroxyacetone, and the resulting strains had a distinct improvement in dihydroxyacetone and glycerol production. pH 6.0 and low air supplement were identified as the optimal conditions for dihydroxyacetone and glycerol production by K, pneumoniae ΔtpiA-ΔDHAK-hdpA. In fed batch fermentation 23.9 g/L of dihydroxyacetone and 10.8 g/L of glycerol were produced after 91 h of cultivation, with the total conversion ratio of 0.97 mol/mol glucose. CONCLUSIONS: This study provides a novel and highly efficient way of dihydroxyacetone and glycerol production from glucose.

The Antibiotic Fosfomycin Mimics the Effects of the Intermediate Metabolites Phosphoenolpyruvate and Glyceraldehyde-3-Phosphate on the Stenotrophomonas maltophilia Transcriptome.

Stenotrophomonas maltophilia is an opportunistic pathogen with an environmental origin, which presents a characteristically low susceptibility to antibiotics and is capable of acquiring increased levels of resistance to antimicrobials. Among these, fosfomycin resistance seems particularly intriguing; resistance to this antibiotic is generally due to the activity of fosfomycin-inactivating enzymes, or to defects in the expression or the activity of fosfomycin transporters. In contrast, we previously described that the cause of fosfomycin resistance in S. maltophilia was the inactivation of enzymes belonging to its central carbon metabolism. To go one step further, here we studied the effects of fosfomycin on the transcriptome of S. maltophilia compared to those of phosphoenolpyruvate-its structural homolog-and glyceraldehyde-3-phosphate-an intermediate metabolite of the mutated route in fosfomycin-resistant mutants. Our results show that transcriptomic changes present a large degree of overlap, including the activation of the cell-wall-stress stimulon. These results indicate that fosfomycin activity and resistance are interlinked with bacterial metabolism. Furthermore, we found that the studied compounds inhibit the expression of the smeYZ efflux pump, which confers intrinsic resistance to aminoglycosides. This is the first description of efflux pump inhibitors that can be used as antibiotic adjuvants to counteract antibiotic resistance in S. maltophilia.

The thermophilic biomass-degrading bacterium Caldicellulosiruptor bescii utilizes two enzymes to oxidize glyceraldehyde 3-phosphate during glycolysis.

Caldicellulosiruptor bescii is an extremely thermophilic, cellulolytic bacterium with a growth optimum at 78 °C and is the most thermophilic cellulose degrader known. It is an attractive target for biotechnological applications, but metabolic engineering will require an in-depth understanding of its primary pathways. A previous analysis of its genome uncovered evidence that C. bescii may have a completely uncharacterized aspect to its redox metabolism, involving a tungsten-containing oxidoreductase of unknown function. Herein, we purified and characterized this new member of the aldehyde ferredoxin oxidoreductase family of tungstoenzymes. We show that it is a heterodimeric glyceraldehyde-3-phosphate (GAP) ferredoxin oxidoreductase (GOR) present not only in all known Caldicellulosiruptor species, but also in 44 mostly anaerobic bacterial genera. GOR is phylogenetically distinct from the monomeric GAP-oxidizing enzyme found previously in several Archaea. We found that its large subunit (GOR-L) contains a single tungstopterin site and one iron-sulfur [4Fe-4S] cluster, that the small subunit (GOR-S) contains four [4Fe-4S] clusters, and that GOR uses ferredoxin as an electron acceptor. Deletion of either subunit resulted in a distinct growth phenotype on both C5 and C6 sugars, with an increased lag phase, but higher cell densities. Using metabolomics and kinetic analyses, we show that GOR functions in parallel with the conventional GAP dehydrogenase, providing an alternative ferredoxin-dependent glycolytic pathway. These two pathways likely facilitate the recycling of reduced redox carriers (NADH and ferredoxin) in response to environmental H2 concentrations. This metabolic flexibility has important implications for the future engineering of this and related species.

A Cytosolic Bypass and G6P Shunt in Plants Lacking Peroxisomal Hydroxypyruvate Reductase.

The oxygenation of ribulose 1,5-bisphosphate by Rubisco is the first step in photorespiration and reduces the efficiency of photosynthesis in C3 plants. Our recent data indicate that mutants in photorespiration have increased rates of photosynthetic cyclic electron flow around photosystem I. We investigated mutant lines lacking peroxisomal hydroxypyruvate reductase to determine if there are connections between 2-phosphoglycolate accumulation and cyclic electron flow in Arabidopsis (Arabidopsis thaliana). We found that 2-phosphoglycolate is a competitive inhibitor of triose phosphate isomerase, an enzyme in the Calvin-Benson cycle that converts glyceraldehyde 3-phosphate to dihydroxyacetone phosphate. This block in metabolism could be overcome if glyceraldehyde 3-phosphate is exported to the cytosol, where cytosolic triose phosphate isomerase could convert it to dihydroxyacetone phosphate. We found evidence that carbon is reimported as glucose-6-phosphate, forming a cytosolic bypass around the block of stromal triose phosphate isomerase. However, this also stimulates a glucose-6-phosphate shunt, which consumes ATP, which can be compensated by higher rates of cyclic electron flow.

Conformational dynamics of 1-deoxy-d-xylulose 5-phosphate synthase on ligand binding revealed by H/D exchange MS.

The enzyme 1-deoxy-d-xylulose 5-phosphate synthase (DXPS) is a key enzyme in the methylerythritol 4-phosphate pathway and is a target for the development of antibiotics, herbicides, and antimalarial drugs. DXPS catalyzes the formation of 1-deoxy-d-xylulose 5-phosphate (DXP), a branch point metabolite in isoprenoid biosynthesis, and is also used in the biosynthesis of thiamin (vitamin B1) and pyridoxal (vitamin B6). Previously, we found that DXPS is unique among the superfamily of thiamin diphosphate (ThDP)-dependent enzymes in stabilizing the predecarboxylation intermediate, C2-alpha-lactyl-thiamin diphosphate (LThDP), which has subsequent decarboxylation that is triggered by d-glyceraldehyde 3-phosphate (GAP). Herein, we applied hydrogen-deuterium (H/D) exchange MS (HDX-MS) of full-length Escherichia coli DXPS to provide a snapshot of the conformational dynamics of this enzyme, leading to the following conclusions. (i) The high sequence coverage of DXPS allowed us to monitor structural changes throughout the entire enzyme, including two segments (spanning residues 183-238 and 292-317) not observed by X-ray crystallography. (ii) Three regions of DXPS (spanning residues 42-58, 183-199, and 278-298) near the active center displayed both EX1 (monomolecular) and EX2 (bimolecuar) H/D exchange (HDX) kinetic behavior in both ligand-free and ligand-bound states. All other peptides behaved according to the common EX2 kinetic mechanism. (iii) The observation of conformational changes on DXPS provides support for the role of conformational dynamics in the DXPS mechanism: The closed conformation of DXPS is critical for stabilization of LThDP, whereas addition of GAP converts DXPS to the open conformation that coincides with decarboxylation of LThDP and DXP release.

Engineering Pseudomonas putida for isoprenoid production by manipulating endogenous and shunt pathways supplying precursors.

BACKGROUND: The soil bacterium Pseudomonas putida is a promising platform for the production of industrially valuable natural compounds. In the case of isoprenoids, the availability of biosynthetic precursors is a major limiting factor. In P. putida and most other bacteria, these precursors are produced from pyruvate and glyceraldehyde 3-phosphate by the methylerythritol 4-phosphate (MEP) pathway, whereas other bacteria synthesize the same precursors from acetyl-CoA using the unrelated mevalonate (MVA) pathway. RESULTS: Here we explored different strategies to increase the supply of isoprenoid precursors in P. putida cells using lycopene as a read-out. Because we were not aiming at producing high isoprenoid titers but were primarily interested in finding ways to enhance the metabolic flux to isoprenoids, we engineered the well-characterized P. putida strain KT2440 to produce low but detectable levels of lycopene under conditions in which MEP pathway steps were not saturated. Then, we compared lycopene production in cells expressing the Myxococcus xanthus MVA pathway genes or endogenous MEP pathway genes (dxs, dxr, idi) under the control of IPTG-induced and stress-regulated promoters. We also tested a shunt pathway producing isoprenoid precursors from ribulose 5-phosphate using a mutant version of the Escherichia coli ribB gene. CONCLUSIONS: The most successful combination led to a 50-fold increase in lycopene levels, indicating that P. putida can be successfully engineered to substantially increase the supply of metabolic substrates for the production of industrially valuable isoprenoids.

The effect of specific proline residues on the kinetic stability of the triosephosphate isomerases of two trypanosomes.

The effect of specific residues on the kinetic stability of two closely related triosephosphate isomerases (from Trypanosoma cruzi, TcTIM and Trypanosoma brucei, TbTIM) has been studied. Based on a comparison of their ß-turn occurrence, we engineered two chimerical enzymes where their super secondary ß-loop-α motifs 2 ((ßα)2 ) were swapped. Differential scanning calorimetry (DSC) experiments showed that the (ßα)2 motif of TcTIM inserted into TbTIM (2Tc) increases the kinetic stability. On the other hand, the presence of the (ßα)2 motif of TbTIM inserted into TcTIM (2Tb) gave a chimerical protein difficult to purify in soluble form and with a significantly reduced kinetic stability. The comparison of the contact maps of the (ßα)2 of TbTIM and TcTIM showed differences in the contact pattern of residues 43 and 49. In TcTIM these residues are prolines, located at the N-terminal of loop-2 and the C-terminal of α-helix-2. Twelve mutants were engineered involving residues 43 and 49 to study the effect over the unfolding activation energy barrier (EA ). A systematic analysis of DSC data showed a large decrease on the EA of TcTIM (ΔEA ranging from 468 to 678 kJ/mol) when the single and double proline mutations are present. The relevance of Pro43 to the kinetic stability is also revealed by mutation S43P, which increased the free energy of the transition state of TbTIM by 17.7 kJ/mol. Overall, the results indicate that protein kinetic stability can be severely affected by punctual mutations, disturbing the complex network of interactions that, in concerted action, determine protein stability. Proteins 2017; 85:571-579. © 2016 Wiley Periodicals, Inc.

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.

Phosphoglycerate kinase acts as a futile cycle at high temperature.

In (hyper)thermophilic organisms metabolic processes have to be adapted to function optimally at high temperature. We compared the gluconeogenic conversion of 3-phosphoglycerate via 1,3-bisphosphoglycerate to glyceraldehyde-3-phosphate at 30 °C and at 70 °C. At 30 °C it was possible to produce 1,3-bisphosphoglycerate from 3-phosphoglycerate with phosphoglycerate kinase, but at 70 °C, 1,3-bisphosphoglycerate was dephosphorylated rapidly to 3-phosphoglycerate, effectively turning the phosphoglycerate kinase into a futile cycle. When phosphoglycerate kinase was incubated together with glyceraldehyde 3-phosphate dehydrogenase it was possible to convert 3-phosphoglycerate to glyceraldehyde 3-phosphate, both at 30 °C and at 70 °C, however, at 70 °C only low concentrations of product were observed due to thermal instability of glyceraldehyde 3-phosphate. Thus, thermolabile intermediates challenge central metabolic reactions and require special adaptation strategies for life at high temperature.

Active-site remodelling in the bifunctional fructose-1,6-bisphosphate aldolase/phosphatase.

Fructose-1,6-bisphosphate (FBP) aldolase/phosphatase is a bifunctional, thermostable enzyme that catalyses two subsequent steps in gluconeogenesis in most archaea and in deeply branching bacterial lineages. It mediates the aldol condensation of heat-labile dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (GAP) to FBP, as well as the subsequent, irreversible hydrolysis of the product to yield the stable fructose-6-phosphate (F6P) and inorganic phosphate; no reaction intermediates are released. Here we present a series of structural snapshots of the reaction that reveal a substantial remodelling of the active site through the movement of loop regions that create different catalytic functionalities at the same location. We have solved the three-dimensional structures of FBP aldolase/phosphatase from thermophilic Thermoproteus neutrophilus in a ligand-free state as well as in complex with the substrates DHAP and FBP and the product F6P to resolutions up to 1.3 Å. In conjunction with mutagenesis data, this pinpoints the residues required for the two reaction steps and shows that the sequential binding of additional Mg(2+) cations reversibly facilitates the reaction. FBP aldolase/phosphatase is an ancestral gluconeogenic enzyme optimized for high ambient temperatures, and our work resolves how consecutive structural rearrangements reorganize the catalytic centre of the protein to carry out two canonical reactions in a very non-canonical type of bifunctionality.

Structural basis for the bifunctionality of fructose-1,6-bisphosphate aldolase/phosphatase.

Enzymes catalyse specific reactions and are essential for maintaining life. Although some are referred to as being bifunctional, they consist of either two distinct catalytic domains or a single domain that displays promiscuous substrate specificity. Thus, one enzyme active site is generally responsible for one biochemical reaction. In contrast to this conventional concept, archaeal fructose-1,6-bisphosphate (FBP) aldolase/phosphatase (FBPA/P) consists of a single catalytic domain, but catalyses two chemically distinct reactions of gluconeogenesis: (1) the reversible aldol condensation of dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (GA3P) to FBP; (2) the dephosphorylation of FBP to fructose-6-phosphate (F6P). Thus, FBPA/P is fundamentally different from ordinary enzymes whose active sites are responsible for a specific reaction. However, the molecular mechanism by which FBPA/P achieves its unusual bifunctionality remains unknown. Here we report the crystal structure of FBPA/P at 1.5-Å resolution in the aldolase form, where a critical lysine residue forms a Schiff base with DHAP. A structural comparison of the aldolase form with a previously determined phosphatase form revealed a dramatic conformational change in the active site, demonstrating that FBPA/P metamorphoses its active-site architecture to exhibit dual activities. Thus, our findings expand the conventional concept that one enzyme catalyses one biochemical reaction.

The Peculiar Glycolytic Pathway in Hyperthermophylic Archaea: Understanding Its Whims by Experimentation In Silico.

Mathematical models are key to systems biology where they typically describe the topology and dynamics of biological networks, listing biochemical entities and their relationships with one another. Some (hyper)thermophilic Archaea contain an enzyme, called non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN), which catalyzes the direct oxidation of glyceraldehyde-3-phosphate to 3-phosphoglycerate omitting adenosine 5'-triphosphate (ATP) formation by substrate-level-phosphorylation via phosphoglycerate kinase. In this study we formulate three hypotheses that could explain functionally why GAPN exists in these Archaea, and then construct and use mathematical models to test these three hypotheses. We used kinetic parameters of enzymes of Sulfolobus solfataricus (S. solfataricus) which is a thermo-acidophilic archaeon that grows optimally between 60 and 90 °C and between pH 2 and 4. For comparison, we used a model of Saccharomyces cerevisiae (S. cerevisiae), an organism that can live at moderate temperatures. We find that both the first hypothesis, i.e., that the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plus phosphoglycerate kinase (PGK) route (the alternative to GAPN) is thermodynamically too much uphill and the third hypothesis, i.e., that GAPDH plus PGK are required to carry the flux in the gluconeogenic direction, are correct. The second hypothesis, i.e., that the GAPDH plus PGK route delivers less than the 1 ATP per pyruvate that is delivered by the GAPN route, is only correct when GAPDH reaction has a high rate and 1,3-bis-phosphoglycerate (BPG) spontaneously degrades to 3PG at a high rate.

Structure-Function Studies of Hydrophobic Residues That Clamp a Basic Glutamate Side Chain during Catalysis by Triosephosphate Isomerase.

Kinetic parameters are reported for the reactions of whole substrates (kcat/Km, M(-1) s(-1)) (R)-glyceraldehyde 3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP) and for the substrate pieces [(kcat/Km)E·HPi/Kd, M(-2) s(-1)] glycolaldehyde (GA) and phosphite dianion (HPi) catalyzed by the I172A/L232A mutant of triosephosphate isomerase from Trypanosoma brucei brucei (TbbTIM). A comparison with the corresponding parameters for wild-type, I172A, and L232A TbbTIM-catalyzed reactions shows that the effect of I172A and L232A mutations on ΔG(⧧) for the wild-type TbbTIM-catalyzed reactions of the substrate pieces is nearly the same as the effect of the same mutations on TbbTIM previously mutated at the second side chain. This provides strong evidence that mutation of the first hydrophobic side chain does not affect the functioning of the second side chain in catalysis of the reactions of the substrate pieces. By contrast, the effects of I172A and L232A mutations on ΔG(⧧) for wild-type TbbTIM-catalyzed reactions of the whole substrate are different from the effect of the same mutations on TbbTIM previously mutated at the second side chain. This is due to the change in the rate-determining step that determines the barrier to the isomerization reaction. X-ray crystal structures are reported for I172A, L232A, and I172A/L232A TIMs and for the complexes of these mutants to the intermediate analogue phosphoglycolate (PGA). The structures of the PGA complexes with wild-type and mutant enzymes are nearly superimposable, except that the space opened by replacement of the hydrophobic side chain is occupied by a water molecule that lies ∼3.5 Å from the basic side chain of Glu167. The new water at I172A mutant TbbTIM provides a simple rationalization for the increase in the activation barrier ΔG(⧧) observed for mutant enzyme-catalyzed reactions of the whole substrate and substrate pieces. By contrast, the new water at the L232A mutant does not predict the decrease in ΔG(⧧) observed for the mutant enzyme-catalyzed reactions of the substrate piece GA.

Crystal structures capture three states in the catalytic cycle of a pyridoxal phosphate (PLP) synthase.

PLP synthase (PLPS) is a remarkable single-enzyme biosynthetic pathway that produces pyridoxal 5'-phosphate (PLP) from glutamine, ribose 5-phosphate, and glyceraldehyde 3-phosphate. The intact enzyme includes 12 synthase and 12 glutaminase subunits. PLP synthesis occurs in the synthase active site by a complicated mechanism involving at least two covalent intermediates at a catalytic lysine. The first intermediate forms with ribose 5-phosphate. The glutaminase subunit is a glutamine amidotransferase that hydrolyzes glutamine and channels ammonia to the synthase active site. Ammonia attack on the first covalent intermediate forms the second intermediate. Glyceraldehyde 3-phosphate reacts with the second intermediate to form PLP. To investigate the mechanism of the synthase subunit, crystal structures were obtained for three intermediate states of the Geobacillus stearothermophilus intact PLPS or its synthase subunit. The structures capture the synthase active site at three distinct steps in its complicated catalytic cycle, provide insights into the elusive mechanism, and illustrate the coordinated motions within the synthase subunit that separate the catalytic states. In the intact PLPS with a Michaelis-like intermediate in the glutaminase active site, the first covalent intermediate of the synthase is fully sequestered within the enzyme by the ordering of a generally disordered 20-residue C-terminal tail. Following addition of ammonia, the synthase active site opens and admits the Lys-149 side chain, which participates in formation of the second intermediate and PLP. Roles are identified for conserved Asp-24 in the formation of the first intermediate and for conserved Arg-147 in the conversion of the first to the second intermediate.

Mechanistic binding insights for 1-deoxy-D-Xylulose-5-Phosphate synthase, the enzyme catalyzing the first reaction of isoprenoid biosynthesis in the malaria-causing protists, Plasmodium falciparum and Plasmodium vivax.

We have successfully truncated and recombinantly-expressed 1-deoxy-D-xylulose-5-phosphate synthase (DXS) from both Plasmodium vivax and Plasmodium falciparum. We elucidated the order of substrate binding for both of these ThDP-dependent enzymes using steady-state kinetic analyses, dead-end inhibition, and intrinsic tryptophan fluorescence titrations. Both enzymes adhere to a random sequential mechanism with respect to binding of both substrates: pyruvate and D-glyceraldehyde-3-phosphate. These findings are in contrast to other ThDP-dependent enzymes, which exhibit classical ordered and/or ping-pong kinetic mechanisms. A better understanding of the kinetic mechanism for these two Plasmodial enzymes could aid in the development of novel DXS-specific inhibitors that might prove useful in treatment of malaria.

Metabolic pathway of 3,6-anhydro-D-galactose in carrageenan-degrading microorganisms.

Complete hydrolysis of κ-carrageenan produces two sugars, D-galactose and 3,6-anhydro-D-galactose (D-AnG). At present, however, we do not know how carrageenan-degrading microorganisms metabolize D-AnG. In this study, we investigated the metabolic pathway of D-AnG degradation by comparative genomic analysis of Cellulophaga lytica LIM-21, Pseudoalteromonas atlantica T6c, and Epulopiscium sp. N.t. morphotype B, which represent the classes Flavobacteria, Gammaproteobacteria, and Clostridia, respectively. In this bioinformatic analysis, we found candidate common genes that were believed to be involved in D-AnG metabolism. We then experimentally confirmed the enzymatic function of each gene product in the D-AnG cluster. In all three microorganisms, D-AnG metabolizing genes were clustered and organized in operon-like arrangements, which we named as the dan operon (3,6-d-anhydro-galactose). Combining bioinformatic analysis and experimental data, we showed that D-AnG is metabolized to pyruvate and D-glyceraldehyde-3-phosphate via four enzyme-catalyzed reactions in the following route: 3,6-anhydro-D-galactose â†’ 3,6-anhydro-D-galactonate â†’ 2-keto-3-deoxy-D-galactonate (D-KDGal) â†’ 2-keto-3-deoxy-6-phospho-D-galactonate â†’ pyruvate + D-glyceraldehyde-3-phosphate. The pathway of D-AnG degradation is composed of two parts: transformation of D-AnG to D-KDGal using two D-AnG specific enzymes and breakdown of D-KDGal to two glycolysis intermediates using two DeLey-Doudoroff pathway enzymes. To our knowledge, this is the first report on the metabolic pathway of D-AnG degradation.

Mechanism of the Enzymatic Synthesis of 4-(Hydroxymethyl)-2- furancarboxaldehyde-phosphate (4-HFC-P) from Glyceraldehyde-3-phosphate Catalyzed by 4-HFC-P Synthase.

A single enzyme, 4-(hydroxymethyl)-2-furancarboxaldehyde-phosphate synthase (MfnB), from the methanogen Methanocaldococcus jannaschii catalyzed at least 10 separate chemical reactions in converting two molecules of glyceraldehyde-3-P (GA-3-P) to 4-(hydroxymethyl)-2-furancarboxaldehyde-P (4-HFC-P), the first discrete intermediate in the biosynthetic pathway to the furan moiety of the coenzyme methanofuran. Here we describe the biochemical characterization of the recombinantly expressed MfnB to understand its catalytic mechanism. Site-directed mutagenesis showed that the strictly conserved residues (Asp25, Lys27, Lys85, and Asp151) around the active site are all essential for enzyme catalysis. Matrix-assisted laser desorption/ionization analysis of peptide fragments of MfnB incubated with GA-3-P followed by NaBH4 reduction and trypsin digestion identified a peptide with a mass/charge ratio of 1668.8 m/z present only in the D25N, D151N, and K155R mutants, which is consistent with Lys27 having increased by a mass of 58 m/z, indicating that Lys27 forms a Schiff base with a methylglyoxal-like intermediate. In addition, incubation of MfnB with GA-3-P in the presence of deuterated water or incubation of MfnB with C-2 deuterated GA-3-P showed essentially no deuterium incorporated into the 4-HFC-P. Combined with structural analysis and molecular docking, we predict the potential binding sites for two GA-3P molecules in the active site. On the basis of our observations, a possible catalytic mechanism of MfnB is proposed in this study. A phosphate elimination reaction and a triose phosphate isomerase-like reaction occur at the GA-3-P binding site I and II, respectively, prior to the aldol condensation between the enzyme-bound enol form of methylglyoxal and dihydroxyacetone phosphate (DHAP), after which the catalytic cycle is completed by a cyclization and two dehydration reactions assisted by several general acids/bases at the same active site.