Structural basis of substrate specificity in human glyoxylate reductase/hydroxypyruvate reductase

Human glyoxylate reductase/hydroxypyruvate reductase (GRHPR) is a D-2-hydroxy-acid dehydrogenase that plays a critical role in the removal of the metabolic by-product glyoxylate from within the liver. Deficiency of this enzyme is the underlying cause of primary hyperoxaluria type 2 (PH2) and leads to increased urinary oxalate levels, formation of kidney stones and renal failure. Here we describe the crystal structure of human GRHPR at 2.2 A resolution. There are four copies of GRHPR in the crystallographic asymmetric unit: in each homodimer, one subunit forms a ternary (enzyme+NADPH+reduced substrate) complex, and the other a binary (enzyme+NADPH) form. The spatial arrangement of the two enzyme domains is the same in binary and ternary forms. This first crystal structure of a true ternary complex of an enzyme from this family demonstrates the relationship of substrate and catalytic residues within the active site, confirming earlier proposals of the mode of substrate binding, stereospecificity and likely catalytic mechanism for these enzymes. GRHPR has an unusual substrate specificity, preferring glyoxylate and hydroxypyruvate, but not pyruvate. A tryptophan residue (Trp141) from the neighbouring subunit of the dimer is projected into the active site region and appears to contribute to the selectivity for hydroxypyruvate. This first crystal structure of a human GRHPR enzyme also explains the deleterious effects of naturally occurring missense mutations of this enzyme that lead to PH2.     

NAD+-specific D-arabinose dehydrogenase and its contribution to erythroascorbic acid production in Saccharomyces cerevisiae

Erythroascorbic acid (eAsA) is a five-carbon analog of ascorbic acid, and it is synthesized from D-arabinose by D-arabinose dehydrogenase (ARA) and D-arabinono-gamma-lactone oxidase. We found an NAD+-specific ARA activity which is operative under submillimolar level of d-arabinose in the extracts of Saccharomyces cerevisiae. The hypothetical protein encoded by YMR041c showed a significant homology to a l-galactose dehydrogenase which plays in plant ascorbic acid biosynthesis, and we named it as Ara2p. Recombinant Ara2p showed NAD+-specific ARA activity with Km=0.78 mM to d-arabinose, which is 200-fold lower than that for the conventional NADP+-specific ARA, Ara1p. Gene disruptant of ARA2 lost entire NAD+-specific ARA activity and the conspicuous increase in intracellular eAsA by exogenous d-arabinose feeding, while the double knockout mutant of ARA1 and ARA2 still retained measurable amount of eAsA. It demonstrates that Ara2p, not Ara1p, mainly contributes to the production of eAsA from d-arabinose in S. cerevisiae.     

Domain closure,substrate specificity and catalysis of D-lactate dehydrogenase from Lactobacillus bulgaricus

NAD-dependent Lactobacillus bulgaricus D-Lactate dehydrogenase (D-LDHb) catalyses the reversible conversion of pyruvate into D-lactate. Crystals of D-LDHb complexed with NADH were grown and X-ray data collected to 2.2 A. The structure of D-LDHb was solved by molecular replacement using the dimeric Lactobacillus helveticus D-LDH as a model and was refined to an R-factor of 20.7%. The two subunits of the enzyme display strong asymmetry due to different crystal environments. The opening angles of the two catalytic domains with respect to the core coenzyme binding domains differ by 16 degrees. Subunit A is in an "open" conformation typical for a dehydrogenase apo enzyme and subunit B is "closed". The NADH-binding site in subunit A is only 30% occupied, while in subunit B it is fully occupied and there is a sulphate ion in the substrate-binding pocket. A pyruvate molecule has been modelled in the active site and its orientation is in agreement with existing kinetic and structural data. On domain closure, a cluster of hydrophobic residues packs tightly around the methyl group of the modelled pyruvate molecule. At least three residues from this cluster govern the substrate specificity. Substrate binding itself contributes to the stabilisation of domain closure and activation of the enzyme. In pyruvate reduction, D-LDH can adapt another protonated residue, a lysine residue, to accomplish the role of the acid catalyst His296. Required lowering of the lysine pK(a) value is explained on the basis of the H296K mutant structure.     

Identification of the residue responsible for catalysing regeneration of activity in the inactive glutamate dehydrogenase mutant D165N

Previously a mutant of clostridial glutamate dehydrogenase with the catalytic Asp-165 replaced by Asn was shown to regain activity through spontaneous, specific deamidation of this residue. A double mutant D165N/K125A has now been constructed, in which the catalytic Lys is also replaced. This was successfully over-expressed and according to several criteria appears to be correctly folded. The double mutant was incubated for 35 days under conditions where D165N reactivates. LC-MS analysis of tryptic digests of timed samples showed no significant deamidation. This confirms that the reactivation of D165N is a consequence of the catalytic chemistry of the enzyme's active site.     

X-ray structures of Aerococcus viridans lactate oxidase and its complex with D-lactate at pH 4.5 show an alpha-hydroxyacid oxidation mechanism

l-Lactate oxidase (LOX) belongs to a family of flavin mononucleotide (FMN)-dependent α-hydroxy acid-oxidizing enzymes. Previously, the crystal structure of LOX (pH 8.0) from Aerococcus viridans was solved, revealing that the active site residues are located around the FMN. Here, we solved the crystal structures of the same enzyme at pH 4.5 and its complex with d-lactate at pH 4.5, in an attempt to analyze the intermediate steps. In the complex structure, the d-lactate resides in the substrate-binding site, but interestingly, an active site base, His265, flips far away from the d-lactate, as compared with its conformation in the unbound state at pH 8.0. This movement probably results from the protonation of His265 during the crystallization at pH 4.5, because the same flip is observed in the structure of the unbound state at pH 4.5. Thus, the present structure appears to mimic an intermediate after His265 abstracts a proton from the substrate. The flip of His265 triggers a large structural rearrangement, creating a new hydrogen bonding network between His265-Asp174-Lys221 and, furthermore, brings molecular oxygen in between d-lactate and His265. This mimic of the ternary complex intermediate enzyme-substrate-O₂ could explain the reductive half-reaction mechanism to release pyruvate through hydride transfer. In the mechanism of the subsequent oxidative half-reaction, His265 flips back, pushing molecular oxygen into the substrate-binding site as the second substrate, and the reverse reaction takes place to produce hydrogen peroxide. During the reaction, the flip-flop action of His265 has a dual role as an active base/acid to define the major chemical steps. Our proposed reaction mechanism appears to be a common mechanistic strategy for this family of enzymes.     

Molecular and functional characterization of D-3-phosphoglycerate dehydrogenase in the serine biosynthetic pathway of the hyperthermophilic archaeon Sulfolobus tokodaii

A gene (ST1218) encoding a d-3-phosphoglycerate dehydrogenase (PGDH; EC 1.1.1.95) homolog was found in the genome of Sulfolobus tokodaii strain 7 by screening a database of enzymes likely to contribute to l-serine biosynthesis in hyperthermophilic archaea. After expressing the gene in Escherichia coli, the PGDH activity of the recombinant enzyme was assessed. Homogeneous PGDH was obtained using conventional chromatography steps, though during the purification an unexpected decline in enzyme activity was observed if the enzyme was stored in plastic tubes, but not in glass ones. The purified enzyme was a homodimer with a subunit molecular mass of about 35 kDa and was highly thermostable. It preferably acted as an NAD-dependent d-3-phosphoglycerate (3PGA) dehydrogenase. Although NADP had no activity as the electron acceptor, both NADPH and NADH acted as electron donors. Kinetic analyses indicated that the enzyme reaction proceeds via a Theorell-Chance Bi-Bi mechanism. Unlike E. coli PGDH, the S. tokodaii enzyme was not inhibited by l-serine. In addition, both the NAD-dependent 3PGA oxidation and the reverse reaction were enhanced by phosphate and sulfate ions, while NADPH-dependent 3-phosphohydroxypyruvate (PHP) reduction was inhibited. Thus S. tokodaii PGDH appears to be subject to a novel regulatory mechanism not seen elsewhere. A database analysis showed that ST1218 gene forms a cluster with ST1217 gene, and a functional analysis of the ST1217 product expressed in E. coli revealed that it possesses l-glutamate-PHP aminotransferase activity. Taken together, our findings represent the first example of a phosphorylated serine pathway in a hyperthermophilic archaeon.     

A novel glycoside hydrolase family 105: the structure of family 105 unsaturated rhamnogalacturonyl hydrolase complexed with a disaccharide in comparison with family 88 enzyme complexed with the disaccharide

YteR, a hypothetical protein with unknown functions, is derived from Bacillus subtilis strain 168 and has an overall structure similar to that of bacterial unsaturated glucuronyl hydrolase (UGL), although it exhibits little amino acid sequence identity with UGL. UGL releases unsaturated glucuronic acid from glycosaminoglycan treated with glycosaminoglycan lyases. The amino acid sequence of YteR shows a significant homology (26% identity) with the hypothetical protein YesR also from B. subtilis strain 168. To clarify the intrinsic functions of YteR and YesR, both proteins were overexpressed in Escherichia coli, purified, and characterized. Based on their gene arrangements in genome and enzyme properties, YteR and YesR were found to constitute a novel enzyme activity, "unsaturated rhamnogalacturonyl hydrolase," classified as new glycoside hydrolase family 105. This enzyme acts specifically on unsaturated rhamnogalacturonan (RG) obtained from RG type-I treated with RG lyases and releases an unsaturated galacturonic acid. The crystal structure of YteR complexed with unsaturated chondroitin disaccharide (UGL substrate) was obtained and compared to the structure of UGL complexed with the same disaccharide. The UGL substrate is sterically hindered with the active pocket of YteR. The protruding loop of YteR prevents the UGL substrate from being bound effectively. The most likely candidate catalytic residues for general acid/base are Asp143 in YteR and Asp135 in YesR. This is supported by three-dimensional structural and site-directed mutagenesis studies. These findings provide molecular insights into novel enzyme catalysis and sequential reaction mechanisms involved in RG-I depolymerization by bacteria.     

Inducibility of Rat Liver Drug-Metabolizing Enzymes as an Index of Food-Screeing for Safety Assessment

D-Lactate dehydrogenase (D-LDH) from Pediococcus pentosaceus ATCC 25745 was found to produce D-3-phenyllactic acid from phenylpyruvate. The optimum pH and temperature for enzyme activity were pH 5.5 and 45 °C. The Michaelis-Menten constant (K _m), turnover number (k _cat), and catalytic efficiency (k _cat?K _m) values for the substrate phenylpyruvate were estimated to be 1.73 mmol/L, 173 s^?1, and 100 (mmol/L)^?1 s^?1 respectively.     

Effect of Dietary l-Glutamine on the Hepatotoxic Action of d-Galactosamine in Rats

The protective effect of dietary l-glutamine against the hepatotoxic action of d-galactosamine (GalN) was investigated by model experiments with rats. Rats fed with 20% casein diets containing 10% free amino acids were injected with GalN, and the serum aspartate aminotransferase, alanine aminotransferase and lactate dehydrogenase activities and the hepatic glycogen content were assayed 20 hours after the injection. These enzyme activities in the group fed with the 10% l-glutamine diet for 8 days were lower than those in the groups fed with the control, 10% l-glutamic acid and 10% l-alanine diets for 8 days. The more prolonged the feeding period with the 10% l-glutamine diet was, the more the serum activity levels of such enzymes were decreased. Although neomycin also lowered these enzyme activities, its simultaneous ingestion with neomycin did not show any additive or synergistic effect. The hepatic glycogen content in the 10% glutamine group still remained high after the GalN treatment. It is therefore assumed that the effectiveness of glutamine intake would have been mediated by glycogen metabolism rather than by uridine metabolism.     

Crystal structure and functional characterization of a D-stereospecific amino acid amidase from Ochrobactrum anthropi SV3, a new member of the penicillin-recognizing proteins

D-amino acid amidase (DAA) from Ochrobactrum anthropi SV3, which catalyzes the stereospecific hydrolysis of D-amino acid amides to yield the D-amino acid and ammonia, has attracted increasing attention as a catalyst for the stereospecific production of D-amino acids. In order to clarify the structure-function relationships of DAA, the crystal structures of native DAA, and of the D-phenylalanine/DAA complex, were determined at 2.1 and at 2.4 A resolution, respectively. Both crystals contain six subunits (A-F) in the asymmetric unit. The fold of DAA is similar to that of the penicillin-recognizing proteins, especially D-alanyl-D-alanine-carboxypeptidase from Streptomyces R61, and class C beta-lactamase from Enterobacter cloacae strain GC1. The catalytic residues of DAA and the nucleophilic water molecule for deacylation were assigned based on these structures. DAA has a flexible Omega-loop, similar to class C beta-lactamase. DAA forms a pseudo acyl-enzyme intermediate between Ser60 O(gamma) and the carbonyl moiety of d-phenylalanine in subunits A, B, C, D, and E, but not in subunit F. The difference between subunit F and the other subunits (A, B, C, D and E) might be attributed to the order/disorder structure of the Omega-loop: the structure of this loop cannot assigned in subunit F. Deacylation of subunit F may be facilitated by the relative movement of deprotonated His307 toward Tyr149. His307 N(epsilon2) extracts the proton from Tyr149 O(eta), then Tyr149 O(eta) attacks a nucleophilic water molecule as a general base. Gln214 on the Omega-loop is essential for forming a network of water molecules that contains the nucleophilic water needed for deacylation. Although peptidase activity is found in almost all penicillin-recognizing proteins, DAA lacks peptidase activity. The lack of transpeptidase and carboxypeptidase activities may be attributed to steric hindrance of the substrate-binding pocket by a loop comprised of residues 278-290 and the Omega-loop.     

Cathepsin L Plays an Important Role in the Lysosomal Degradation of L-Lactate Dehydrogenase

A cystatin α-sensitive cysteine proteinase that plays an important role in the lysosomal inactivation and degradation of L-lactate dehydrogenase (LDH) was purified by column chromatography from an ammonium sulfate precipitate of lysosome extract prepared from rat livers. It was eluted with marked delay from cathepsins B and H in a Sephacryl S-200 column by its specific interaction with the gel, and then effectively separated from cathepsins B and H and other proteins. It was eluted with 0.5 M NaCl after washing with 0.2 M NaCl in a CM-Sephadex column, indicating that it showed the same elution behavior as cathepsin L from the CM-Sephadex column. It had activity to hydrolyze z-Phe-Arg-NH-Mec, a synthetic substrate for cysteine proteinases, including cathepsins B and L. The N-terminal sequences of the final preparation of LDH-inactivating enzyme were identical with those of rat cathepsin L. Inactivation and degradation of LDH by the final preparation were observed and effectively inhibited by a low level of cystatin α as well as a general cysteine proteinase inhibitor, leupeptin or (L-3-trans-carboxyoxirane-2-carbonyl)-L-leucine (3-methylbutyl)amide (E-64-c). From these results, it is concluded that cathepsin L plays a critical role in the lysosomal degradation of native LDH.     

Characterization of human D-amino acid oxidase

D-Amino acid oxidase (DAAO) has been proposed to be involved in the oxidation of D-serine, an allosteric activator of the NMDA-type glutamate receptor in the brain, and to be associated with the onset of schizophrenia. The recombinant human DAAO was expressed in Escherichia coli and was isolated as an active homodimeric flavoenzyme. It shows the properties of the dehydrogenase-oxidase class of flavoproteins, possesses a low kinetic efficiency, and follows a ternary complex (sequential) kinetic mechanism. In contrast to the other known DAAOs, the human enzyme is a stable homodimer even in the apoprotein form and weakly binds the cofactor in the free form.     

Asp-196-->Ala mutant of Leuconostoc mesenteroides sucrose phosphorylase exhibits altered stereochemical course and kinetic mechanism of glucosyl transfer to and from phosphate

Mutagenesis of Asp-196 into Ala yielded an inactive variant of Leuconostoc mesenteroides sucrose phosphorylase (D196A). External azide partly complemented the catalytic defect in D196A with a second-order rate constant of 0.031 M-1 s-1 (pH 5, 30 degrees C) while formate, acetate and halides could not restore activity. The mutant utilized azide to convert alpha-D-glucose 1-phosphate into beta-D-glucose 1-azide, reflecting a change in stereochemical course of glucosyl transfer from alpha-retaining in wild-type to inverting in D196A. Phosphorolysis of beta-D-glucose 1-azide by D196A occurred through a ternary complex kinetic mechanism, in marked contrast to the wild-type whose reactions feature a common glucosyl enzyme intermediate and Ping-Pong kinetics. Therefore, Asp-196 is identified unambiguously as the catalytic nucleophile of sucrose phosphorylase, and its substitution by Ala forces the reaction to proceed via single nucleophilic displacement. D196A is not detectably active as alpha-glucosynthase.     

A structural basis for substrate selectivity and stereoselectivity in octopine dehydrogenase from Pecten maximus

Octopine dehydrogenase [N²-(d-1-carboxyethyl)-l-arginine:NAD⁺ oxidoreductase] (OcDH) from the adductor muscle of the great scallop Pecten maximus catalyzes the reductive condensation of l-arginine and pyruvate to octopine during escape swimming. This enzyme, which is a prototype of opine dehydrogenases (OpDHs), oxidizes glycolytically born NADH to NAD⁺, thus sustaining anaerobic ATP provision during short periods of strenuous muscular activity. In contrast to some other OpDHs, OcDH uses only l-arginine as the amino acid substrate. Here, we report the crystal structures of OcDH in complex with NADH and the binary complexes NADH/l-arginine and NADH/pyruvate, providing detailed information about the principles of substrate recognition, ligand binding and the reaction mechanism. OcDH binds its substrates through a combination of electrostatic forces and size selection, which guarantees that OcDH catalysis proceeds with substrate selectivity and stereoselectivity, giving rise to a second chiral center and exploiting a “molecular ruler” mechanism.     

Crystal structures of human glycerol 3-phosphate dehydrogenase 1 (GPD1)

Homo sapiens L-alpha-glycerol-3-phosphate dehydrogenase 1 (GPD1) catalyzes the reversible biological conversion of dihydroxyacetone (DHAP) to glycerol-3-phosphate. The GPD1 protein was expressed in Escherichia coli, and purified as a fusion protein with glutathione S-transferase. Here we report the apoenzyme structure of GPD1 determined by multiwavelength anomalous diffraction phasing, and other complex structures with small molecules (NAD+ and DHAP) by the molecular replacement method. This enzyme structure is organized into two distinct domains, the N-terminal eight-stranded beta-sheet sandwich domain and the C-terminal helical substrate-binding domain. An electrophilic catalytic mechanism by the epsilon-NH3+ group of Lys204 is proposed on the basis of the structural analyses. In addition, the inhibitory effects of zinc and sulfate on GPDHs are assayed and discussed.     

Kinetic resolution of 2-hydroxybutanoate racemic mixtures by NAD-independent L-lactate dehydrogenase

Optically active d-2-hydroxybutanoate is an important building block intermediate for medicines and biodegradable poly(2-hydroxybutanoate). Kinetic resolution of racemic 2-hydroxybutanoate may be a green and desirable alternative for d-2-hydroxybutanoate production. In this work, d-2-hydroxybutanoate at a high concentration (0.197 M) and a high enantiomeric excess (99.1%) was produced by an NAD-independent l-lactate dehydrogenase (l-iLDH) containing biocatalyst. 2-Oxobutanoate, another important intermediate, was co-produced at a high concentration (0.193 M). Using a simple ion exchange process with the macroporous anion exchange resin D301, d-2-hydroxybutanoate was separated from the biotransformation system with a high recovery of 84.7%.     

Ascorbic acid synthesis due to L-gulono-1,4-lactone oxidase expression enhances NO production in endothelial cells

As a primary antioxidant, ascorbic acid (AA) provides beneficial effects for vascular health mitigating oxidative stress and endothelial dysfunction. However, the association of intracellular AA with NO production occurring inside the endothelial cells remains unclear. In the present study, we addressed this issue by increasing intracellular AA directly through de novo synthesis. To restore AA synthesis pathway, bovine aortic endothelial cells were transfected with the plasmid vector encoding L-gulono-1,4-lactone oxidase (GULO, EC 1.1.3.8), the missing enzyme converting L-gulono-1,4-lactone (GUL) to AA. Functional expression of GULO was verified by Western blotting and in vitro enzyme activity assay. GULO expression alone did not lead to AA synthesis but the supply of GUL resulted in a marked increase of intracellular AA. When the cells were stimulated with calcium ionophore, A23187, NO production was more active in the GULO-expressing cells supplied with GUL, in comparison with the cells without GULO expression or without GUL supply, indicating that intracellular AA regulated NO production. Enhancement of NO production by intracellular AA was further verified in aortic endothelial cells obtained from eNOS knockout mice that were cotransfected with eNOS and GULO constructs. GULO-dependent AA synthesis also elevated intracellular tetrahydrobiopterin content, implicating that this essential cofactor of endothelial nitric oxide synthase (eNOS) might mediate the AA effect. The present study strongly suggests that intracellular AA plays critical roles in vascular physiology through enhancing endothelial NO production.     

Mechanisms of the protective effect of L-alanine to D-galactosamine-induced hepatocellular injury: comparative studies of L-alanine and pyruvate

The addition of L-alanine reduced lactate dehydrogenase leakage from primary cultured rat hepatocytes treated with galactosamine (D-gal), while D-alanine and other amino acids did not. However, the mechanisms have not yet been entirely clarified. In this study, we used various inhibitors of metabolism, i.e., aminooxyacetate, oligomycin, and quinolinic acid, to examine the relation between this protective effect and the metabolism of L-alanine. Quinolinic acid (10 mM) did not affect the hepatoprotective effect of L-alanine, while oligomycin (0.1 mug/ml) and aminooxyacetate (1 mM) eliminated the hepatoprotective effect of L-alanine. L-Alanine also increased the albumin secretion by cultured hepatocytes treated with D-gal, while pyruvate had little effect. It was revealed that the intracellular content of pyruvate did not increase as a result of addition of L-alanine. These results are consistent with the hypothesis that L-alanine metabolism is important for hepatoprotection, but pyruvate cannot be used as a substitute for L-alanine.     

l-Lactate generates hydrogen peroxide in purified rat liver mitochondria due to the putative l-lactate oxidase localized in the intermembrane space

In order to ascertain whether and how mitochondria can produce hydrogen peroxide (H₂O₂) as a result of l-lactate addition, we monitored H₂O₂ generation in rat liver mitochondria and in submitochondrial fractions free of peroxisomal and cytosolic contamination. We found that H₂O₂ is produced independently on the respiratory chain with 1:1 stoichiometry with pyruvate, due to a putative flavine-dependent l-lactate oxidase restricted to the intermembrane space. The l-lactate oxidase reaction shows a hyperbolic dependence on l-lactate concentration and is inhibited by NAD⁺ in a competitive manner, being the enzyme different from the l-lactate dehydrogenase isoenzymes as shown by their pH profiles.     

Three-dimensional structures of apo- and holo-L-alanine dehydrogenase from Mycobacterium tuberculosis reveal conformational changes upon coenzyme binding

l-Alanine dehydrogenase from Mycobacterium tuberculosis catalyzes the NADH-dependent reversible conversion of pyruvate and ammonia to l-alanine. Expression of the gene coding for this enzyme is up-regulated in the persistent phase of the organism, and alanine dehydrogenase is therefore a potential target for pathogen control by antibacterial compounds. We have determined the crystal structures of the apo- and holo-forms of the enzyme to 2.3 and 2.0 Å resolution, respectively. The enzyme forms a hexamer of identical subunits, with the NAD-binding domains building up the core of the molecule and the substrate-binding domains located at the apical positions of the hexamer. Coenzyme binding stabilizes a closed conformation where the substrate-binding domains are rotated by about 16° toward the dinucleotide-binding domains, compared to the open structure of the apo-enzyme. In the structure of the abortive ternary complex with NAD+ and pyruvate, the substrates are suitably positioned for hydride transfer between the nicotinamide ring and the C2 carbon atom of the substrate. The approach of the nucleophiles water and ammonia to pyruvate or the reaction intermediate iminopyruvate, respectively, is, however, only possible through conformational changes that make the substrate binding site more accessible. The crystal structures identified the conserved active-site residues His96 and Asp270 as potential acid/base catalysts in the reaction. Amino acid replacements of these residues by site-directed mutagenesis led to inactive mutants, further emphasizing their essential roles in the enzymatic reaction mechanism.