D-lactate dehydrogenase is a member of the D-isomer-specific 2-hydroxyacid dehydrogenase family. Cloning, sequencing, and expression in Escherichia coli of the D-lactate dehydrogenase gene of Lactobacillus plantarum.

The gene encoding D-lactate dehydrogenase (D-lactate: NAD+ oxidoreductase, EC 1.1.1.28) of Lactobacillus plantarum has been sequenced, and expressed in Escherichia coli cells with an inducible expression plasmid, in which the 5'-noncoding region of the gene was replaced with the tac promoter. Comparison of the sequence of D-lactate dehydrogenase with L-lactate dehydrogenases, including the L. plantarum L-lactate dehydrogenase, showed no significant homology. In contrast, the D-lactate dehydrogenase is homologous to E. coli D-3-phosphoglycerate dehydrogenase and Lactobacillus casei D-2-hydroxyisocaproate dehydrogenase. This indicates that D-lactate dehydrogenase is a member of a new family of 2-hydroxyacid dehydrogenases recently proposed, being distinct from L-lactate dehydrogenase and L-malate dehydrogenase, and strongly suggests that the new family consists of D-isomer-stereospecific enzymes. In the reductive reaction, the enzyme showed a broad substrate specificity, although pyruvate was the most favorable of all 2-ketocarboxylic acids tested. In particular, hydroxypyruvate is effectively reduced by the enzyme, the reaction rate, and Km value being comparable to those in the case of pyruvate, indicating that the enzyme has not only D-lactate dehydrogenase activity but also D-glycerate dehydrogenase activity. The conserved residues in this family appear to be the residues involved in the substrate binding and the catalytic reaction, and thus to be targets for site-directed mutagenesis.     

A new D-2-hydroxyacid dehydrogenase with dual coenzyme-specificity from Haloferax mediterranei, sequence analysis and heterologous overexpression

A gene encoding a new D-2-hydroxyacid dehydrogenase (E.C. 1.1.1.) from the halophilic Archaeon Haloferax mediterranei has been sequenced, cloned and expressed in Escherichia coli cells with the inducible expression plasmid pET3a. The nucleotide sequence analysis showed an open reading frame of 927 bp which encodes a 308 amino acid protein. Multiple amino acid sequence alignments of the D-2-hydroxyacid dehydrogenase from H. mediterranei showed high homology with D-2-hydroxyacid dehydrogenases from different organisms and other enzymes of this family. Analysis of the amino acid sequence showed catalytic residues conserved in hydroxyacid dehydrogenases with d-stereospecificity. In the reductive reaction, the enzyme showed broad substrate specificity, although alpha-ketoisoleucine was the most favourable of all alpha-ketocarboxylic acids tested. Kinetic data revealed that this new D-2-hydroxyacid dehydrogenase from H. mediterranei exhibits dual coenzyme-specificity, using both NADPH and NADH as coenzymes. To date, all D-2-hydroxyacid dehydrogenases have been found to be NADH-dependent. Here, we report the first example of a D-2-hydroxyacid dehydrogenase with dual coenzyme-specificity.     

A new d-2-hydroxyacid dehydrogenase with dual coenzyme-specificity from Haloferax mediterranei, sequence analysis and heterologous overexpression

A gene encoding a new d-2-hydroxyacid dehydrogenase (E.C. 1.1.1.) from the halophilic Archaeon Haloferax mediterranei has been sequenced, cloned and expressed in Escherichia coli cells with the inducible expression plasmid pET3a. The nucleotide sequence analysis showed an open reading frame of 927 bp which encodes a 308 amino acid protein. Multiple amino acid sequence alignments of the D-2-hydroxyacid dehydrogenase from H. mediterranei showed high homology with D-2-hydroxyacid dehydrogenases from different organisms and other enzymes of this family. Analysis of the amino acid sequence showed catalytic residues conserved in hydroxyacid dehydrogenases with d-stereospecificity. In the reductive reaction, the enzyme showed broad substrate specificity, although α-ketoisoleucine was the most favourable of all α-ketocarboxylic acids tested. Kinetic data revealed that this new D-2-hydroxyacid dehydrogenase from H. mediterranei exhibits dual coenzyme-specificity, using both NADPH and NADH as coenzymes. To date, all D-2-hydroxyacid dehydrogenases have been found to be NADH-dependent. Here, we report the first example of a D-2-hydroxyacid dehydrogenase with dual coenzyme-specificity.     

A new family of 2-hydroxyacid dehydrogenases

The NADH-dependent hydroxypyruvate reductase from cucumber and the pdxB gene product of E. coli display significant homology to E. coli D-3-phosphoglycerate dehydrogenase. In contrast, these proteins do not display much similarity with other oxidoreductases or with other 2-hydroxyacid dehydrogenases in particular. On the basis of their relatedness and the structure of their substrates, these three enzymes constitute a new family of 2-hydroxyacid dehydrogenases distinct from malate and lactate dehydrogenase.     

Chirality of the hydrogen transfer to the coenzyme catalyzed by ribitol dehydrogenase from Klebsiella pneumoniae and D-mannitol 1-phosphate dehydrogenase from Escherichia coli.

The stereochemistry of the hydrogen transfer to NAD catalyzed by ribitol dehydrogenase (ribitol:NAD 2-oxidoreductase, EC 1.1.1.56) from Klebsiella pneumoniae and D-mannitol-1-phosphate dehydrogenase (D-mannitol-1-phosphate:NAD 2-oxidoreductase, EC 1.1.1.17) from Escherichia coli was investigated. [4-3H]NAD was enzymatically reduced with nonlabelled ribitol in the presence of ribitol dehydrogenase and with nonlabelled D-mannitol 1-phosphate and D-mannitol 1-phosphate dehydrogenase, respectively. In both cases the [4-3H]-NADH produced was isolated and the chirality at the C-4 position determined. It was found that after the transfer of hydride, the label was in both reactions exclusively confined to the (4R) position of the newly formed [4-3H]NADH. In order to explain these results, the hydrogen transferred from the nonlabelled substrates to [4-3H]NAD must have entered the (4S) position of the nicotinamide ring. These data indicate for both investigated inducible dehydrogenases a classification as B or (S) type enzymes. Ribitol also can be dehydrogenated by the constitutive A-type L-iditol dehydrogenase (L-iditol:NAD 5-oxidoreductase, EC 1.1.1.14) from sheep liver. When L-iditol dehydrogenase utilizes ribitol as hydrogen donor, the same A-type classification for this oxidoreductase, as expected, holds true. For the first time, opposite chirality of hydrogen transfer to NAD in one organic reaction--ribitol + NAD = D-ribu + NADH + H--is observed when two different dehydrogenases, the inducible ribitol dehydrogenase from K. pneumoniae and the constitutive L-iditol dehydrogenase from sheep liver, are used as enzymes. This result contradicts the previous generalization that the chirality of hydrogen transfer to the coenzyme for the same reaction is independent of the source of the catalyzing enzyme.     

Methanoarchaeal sulfolactate dehydrogenase: prototype of a new family of NADH-dependent enzymes

The crystal structure of the sulfolactate dehydrogenase from the hyperthermophilic and methanogenic archaeon Methanocaldococcus jannaschii was solved at 2.5 A resolution (PDB id. 1RFM). The asymmetric unit contains a tetramer of tight dimers. This structure, complexed with NADH, does not contain a cofactor-binding domain with 'Rossmann-fold' topology. Instead, the tertiary and quaternary structures indicate a novel fold. The NADH is bound in an extended conformation in each active site, in a manner that explains the pro-S specificity. Cofactor binding involves residues belonging to both subunits within the tight dimers, which are therefore the smallest enzymatically active units. The protein was found to be a homodimer in solution by size-exclusion chromatography, analytical ultracentrifugation and small-angle neutron scattering. Various compounds were tested as putative substrates. The results indicate the existence of a substrate discrimination mechanism, which involves electrostatic interactions. Based on sequence homology and phylogenetic analyses, several other enzymes were classified as belonging to this novel family of homologous (S)-2-hydroxyacid dehydrogenases.     

Combined coenzyme-substrate analogues of various dehydrogenases. Synthesis of (3S)- and (3R)-5-(3-carboxy-3-hydroxypropyl)nicotinamide adenine dinucleotide and their interaction with (S)- and (R)-lactate-specific dehydrogenases.

Two diastereomeric nicotinamide adenine dinucleotide (NAD+) derivatives were synthesized in which the substrates of (S)-and (R)-lactate-specific dehydrogenases are covalently attached via a methylene spacer at position 5 of the nicotinamide ring. The corresponding nicotinamide derivatives were obtained stereospecifically by enzymatic reduction of 5-(2-oxalylethyl)nicotinamide. (3S)-5-(3-Carboxy-3-hydroxypropyl)-NAD+ undergoes and intramolecular hydride transfer in the presence of pig heart lactate dehydrogenase, forming the corresponding coenzyme-substrate analogue composed of pyruvate and NADH. No cross-reaction products resulting from an intermolecular reaction are observed. Two (R)-lactate specific dehydrogenases, however, do not catalyze a similar reaction in either one of the two diastereomers. A possible arrangement of the substrates in the active centers of these enzymes is proposed. 5-Methyl-NAD+ and 5-methyl-NADH are active coenzymes of pig heart lactate dehydrogenase in contrast to reports in the literature. (S)-Lactate binds to this enzyme in the absence of coenzyme, exhibiting a dissociation constant of 11 mM.     

Properties and primary structure of the L-malate dehydrogenase from the extremely thermophilic archaebacterium Methanothermus fervidus

L-Malate dehydrogenase from the extremely thermophilic mathanogen Methanothermus fervidus was isolated and its phenotypic properties were characterized. The primary structure of the protein was deducted from the coding gene. The enzyme is a homomeric dimer with a molecular mass of 70 kDa, possesses low specificity for NAD+ or NADP+ and catalyzes preferentially the reduction of oxalacetate. The temperature dependence of the activity as depicted in the Arrhenius and van't Hoff plots shows discontinuities near 52 degrees C, as was found for glyceraldehyde-3-phosphate dehydrogenase from the same organism. With respect to the primary structure, the archaebacterial L-malate dehydrogenase deviates strikingly from the eubacterial and eukaryotic enzymes. The sequence similarity is even lower than that between the L-malate dehydrogenases and L-lactate dehydrogenases of eubacteria and eukaryotes. The phylogenetic meaning of this relationship is discussed.     

Action of surfactants on porcine heart malate dehydrogenase isoenzymes and a simple method for the differential assay of these isoenzymes

The cationic surfactant, cetyl (hexadecyl) trimethylammonium bromide (CTAB), completely inactivates porcine heart cytoplasmic malate dehydrogenase (L-malate: NAD⁺ oxidoreductase, EC 1.1.1.37) at concentrations (of surfactant) which do not affect the activity of the mitochondrial isoenzyme. These concentrations are close to, or higher than, the critical micelle concentration of CTAB. An increase in the ionic strength of the medium significantly retards the CTAB-induced inactivation of the cytoplasmic enzyme. The enzyme is also markedly protected against CTAB inactivation by NADH; L-malate on its own has no effect but a combination of NADH and L-malate affords greater protection than NADH alone. The CTAB inactivation is not reversed by dilution of the surfactant. The highly selective action of CTAB on the two malate dehydrogenases, which correlates well with their electrostatic charges, has been exploited for a simple and reliable differential assay of these isoenzymes. The anionic surfactant, sodium dodecyl sulphate (SDS), at concentrations well below the critical micelle concentration, inactivates both isoenzymes, but the mitochondrial enzyme is significantly more sensitive than its cytoplasmic counterpart. There is thus some correlation, though not as strong as with CTAB, between SDS inactivation and the charges of the two malat dehydrogenases. An increase in ionic strength has opposite effects on the two isoenzymes: the mitochondrial enzyme becomes more resistant and the cytoplasmic enzyme less so. Both isoenzymes are rendered more resistant to SDS by the inclusion of NADH. Inactivation of the enzymes caused by short exposure to SDS is largely reversed by dilution of the detergent, but longer exposure leads to progressive irreversible loss of activity. NADH very effectively protects the isoenzymes against irreversible inactivation. It is likely that a reversible phase of inactivation precedes an irreversible phase and that in the former phase SDS acts competitively with NADH. Both malate dehydrogenases possess considerable resistance to the nonionic detergent, Triton X-100.     

Biochemical and structural studies of uncharacterized protein PA0743 from Pseudomonas aeruginosa revealed NAD+-dependent L-serine dehydrogenase

The β-hydroxyacid dehydrogenases form a large family of ubiquitous enzymes that catalyze oxidation of various β-hydroxy acid substrates to corresponding semialdehydes. Several known enzymes include β-hydroxyisobutyrate dehydrogenase, 6-phosphogluconate dehydrogenase, 2-(hydroxymethyl)glutarate dehydrogenase, and phenylserine dehydrogenase, but the vast majority of β-hydroxyacid dehydrogenases remain uncharacterized. Here, we demonstrate that the predicted β-hydroxyisobutyrate dehydrogenase PA0743 from Pseudomonas aeruginosa catalyzes an NAD⁺-dependent oxidation of l-serine and methyl-l-serine but exhibits low activity against β-hydroxyisobutyrate. Two crystal structures of PA0743 were solved at 2.2–2.3-Å resolution and revealed an N-terminal Rossmann fold domain connected by a long α-helix to the C-terminal all-α domain. The PA0743 apostructure showed the presence of additional density modeled as HEPES bound in the interdomain cleft close to the predicted catalytic Lys-171, revealing the molecular details of the PA0743 substrate-binding site. The structure of the PA0743-NAD⁺ complex demonstrated that the opposite side of the enzyme active site accommodates the cofactor, which is also bound near Lys-171. Site-directed mutagenesis of PA0743 emphasized the critical role of four amino acid residues in catalysis including the primary catalytic residue Lys-171. Our results provide further insight into the molecular mechanisms of substrate selectivity and activity of β-hydroxyacid dehydrogenases.     

2-Keto-4-methylthiobutyrate, an intermediate in the methionine salvage pathway, is a good substrate for CtBP1

In the present work, we have studied the kinetic properties of the catalytic domain of CtBP1, a co-repressor belonging to the d-2-hydroxyacid dehydrogenase family and known to reduce pyruvate in the presence of NADH. CtBP1 acted on a variety of alpha-keto acids, for which it displayed biphasic curves with inhibition at elevated concentrations, as observed with other dehydrogenases of the same family. Based on catalytic efficiencies, the best substrate was 2-keto-4-methylthiobutyrate, an intermediate of the methionine salvage pathway. It was about 20-fold better than 2-ketoisocaproate and glyoxylate, and 80-fold better than pyruvate. From these data we conclude that 2-keto-4-methylthiobutyrate may be an important regulator of CtBP activity, possibly linking gene repression to the activity of the methionine salvage and spermine synthesis pathways.     

Structural characterization of a D-isomer specific 2-hydroxyacid dehydrogenase from Lactobacillus delbrueckii ssp. bulgaricus

Hydroxyacid dehydrogenases, responsible for the stereospecific conversion of 2-keto acids to 2-hydroxyacids in lactic acid producing bacteria, have a range of biotechnology applications including antibiotic synthesis, flavor development in dairy products and the production of valuable synthons. The genome of Lactobacillus delbrueckii ssp. bulgaricus, a member of the heterogeneous group of lactic acid bacteria, encodes multiple hydroxyacid dehydrogenases whose structural and functional properties remain poorly characterized. Here, we report the apo and coenzyme NAD⁺ complexed crystal structures of the L. bulgaricus D-isomer specific 2-hydroxyacid dehydrogenase, D2-HDH. Comparison with closely related members of the NAD-dependent dehydrogenase family reveals that whilst the D2-HDH core fold is structurally conserved, the substrate-binding site has a number of non-canonical features that may influence substrate selection and thus dictate the physiological function of the enzyme.     

Amino acid transport in membrane vesicles of obligately anaerobic Veillonella alcalescens.

Membrane vesicles of Veillonella alcalescens, grown in the presence of L-lactate and KNO-3, actively transport amino acids under anaerobic conditions in the presence of several electron donors and the electron acceptor nitrate. The highest initial rates of uptake are obtained with L-lactate, followed by reduced nicotinamide adenine dinucleotide, glycerol-1-phosphate, formate, and L-malate.. The membrane vesicles contain the dehydrogenases for these electron donors, and these enzymes are coupled with nitrate reductase. In membrane vesicles from cells, grown in the presence of nitrate, the dehydrogenases are not coupled with fumarate reducatase, and anaerobic transport of amino acids does not occur with fumarate as electron acceptor. Under aerobic conditions none of the physiological electron donors can energize transport. However, a high rate of uptake is observed with the electron donor system ascorbate-phenazine metho-sulfate. This electron donor system also effectively energizes transport under anaerobicconditions in the presence of the electron acceptor nitrate.     

Structure and function of L-lactate dehydrogenases from thermophilic and mesophilic bacteria. I) Isolation and characterization of lactate dehydrogenases from thermophilic and mesophilic bacilli.

Lactate dehydrogenases from thermophilic bacilli (Bacillus stearothermophilus, Bacillus caldotenax) and from mesophilic bacilli (Bacillus X1, Bacillus subtilis) have been isolated by a two-step purification procedure. Only one type (LDH-P4) composed of four identical subunits (Mr 34 000 or 36 000) was found in each bacillus. The tetrameric enzymes were characterized with respect to thermostability, pH and temperature dependence of the pyruvate reduction and the L-lactate oxidation, substrate specificity, saturation kinetics (Km values of pyruvate, lactate, NAD, NADH), pyruvate and oxamate inhibition, and activation by fructose bisphosphate. The thermophilic and mesophilic enzymes differ characteristically in these parameters. Preliminary structural data (amino acid composition, comparative N-terminal sequence analysis) show the expected close phylogenetic relationship (high degree of sequence homology), but also typical differences between thermophilic and mesophilic dehydrogenases, a suitable basis for further comparative studies.     

Archaebacterial malate dehydrogenases. The enzymes from the thermoacidophilic organisms Sulfolobus acidocaldarius and Thermoplasma acidophilum show A-side stereospecificity for NAD+.

The stereoselective transfer of hydrogen from NADH to oxaloacetate catalysed by malate dehydrogenases (EC 1.1.1.37) from the thermoacidophilic archaebacteria Sulfolobus acidocaldarius and Thermoplasma acidophilum was studied by the p.m.r. method described by Zhou & Wong [(1981) J. Biochem. Biophys. Methods 4, 329-338]. Both enzymes are A-side (pro-R) stereospecific for NADH.     

Recognition site for the side chain of 2-ketoacid substrate in d-lactate dehydrogenase

Replacement of Tyr52 with Val or Ala in Lactobacillus pentosus d-lactate dehydrogenase induced high activity and preference for large aliphatic 2-ketoacids and phenylpyruvate. On the other hand, replacements with Arg, Thr or Asp severely reduced the enzyme activity, and the Tyr52Arg enzyme, the only one that exhibited significant enzyme activity, showed a similar substrate preference to the Tyr52Val and Tyr52Ala enzymes. Replacement of Phe299 with Gly or Ser greatly reduced the enzyme activity with less marked change in the substrate preference. Except for the Phe299Ser enzyme, these mutant enzymes with low catalytic activity consistently stimulated NADH oxidation in the absence of 2-ketoacid substrates. However, the double mutant enzymes, Tyr52Arg/Phe299Gly and Tyr52Thr/Phe299Ser, did not exhibit synergically decreased enzyme activity or the substrate-independent NADH oxidation, but rather increased activities toward certain 2-ketoacid substrates. These results indicate that the coordinative combination of amino acid residues at two positions is pivotal in both the functional recognition of the 2-ketoacid side chain and the protection of the bound NADH molecule from the solvent. Multiplicity in such combinations appears to provide d-LDH-related 2-hydroxyacid dehydrogenases with a great variety of catalytic and physiological functions.     

NADP+-Preferring d-Lactate Dehydrogenase from Sporolactobacillus inulinus

Hydroxy acid dehydrogenases, including l- and d-lactate dehydrogenases (L-LDH and D-LDH), are responsible for the stereospecific conversion of 2-keto acids to 2-hydroxyacids and extensively used in a wide range of biotechnological applications. A common feature of LDHs is their high specificity for NAD⁺ as a cofactor. An LDH that could effectively use NADPH as a coenzyme could be an alternative enzymatic system for regeneration of the oxidized, phosphorylated cofactor. In this study, a d-lactate dehydrogenase from a Sporolactobacillus inulinus strain was found to use both NADH and NADPH with high efficiencies and with a preference for NADPH as its coenzyme, which is different from the coenzyme utilization of all previously reported LDHs. The biochemical properties of the D-LDH enzyme were determined by X-ray crystal structural characterization and in vivo and in vitro enzymatic activity analyses. The residue Asn¹⁷⁴ was demonstrated to be critical for NADPH utilization. Characterization of the biochemical properties of this enzyme will contribute to understanding of the catalytic mechanism and provide referential information for shifting the coenzyme utilization specificity of 2-hydroxyacid dehydrogenases.     

Mechanism of transfer of reduced nicotinamide adenine dinucleotide among dehydrogenases

The pathway for the transfer of NADH from one dehydrogenase (E1) to another dehydrogenase (E2) has been investigated by studying the E2-catalyzed reduction of S2 by NADH. The experimental conditions are that the concentration of E1 exceeds that of NADH, which in turn is very much greater than E2; hence, the concentration of free (aqueous) NADH is exceedingly low. The rate of reduction of S2 will hence be very slow if unliganded aqueous NADH is required for the E2-catalyzed reaction. Our results with eight dehydrogenases are entirely consistent with the direct transfer of NADH between E1 and E2 whenever the two enzymes transfer hydrogen via opposite faces (A and B) of the nicotinamide ring. Whenever the two enzymes are both A or both B, NADH transfer occurs only via the aqueous solvent. Some mechanistic inferences and their possible physiological significance are discussed.     

Evidence against tight channelling of NADH in hepatocytes

Tritiated substrates at tracer levels were incubated with rat hepatocytes plus 10 mM L-lactate, and the yields of tritium in glucose and water, as well as the tritium distribution on C-6 and C-4 of glucose, determined. Substrates of cytosolic type A NAD-linked dehydrogenases showed some preferential labeling of C-6 of glucose (the pathway involving type A malate dehydrogenase), whereas substrates of cytosolic type B NAD-linked dehydrogenases showed some preferential labeling of C-4 of glucose (the pathway involving type B glyceraldehyde-3P dehydrogenase). The results found are consistent with a classical diffusion model of NADH metabolism, and are at odds with the Srivastava hypothesis (based on isolated enzyme studies) which indicated that direct transfer of NADH can occur between many NAD-linked enzymes but only when they are of opposite (A or B) specificity.     

Fructose 1,6-bisphosphate-dependent L-lactate dehydrogenase from Thermus aquaticus YT-1, an extreme thermophile: activation by citrate and modification reagents and comparison with Thermus caldophilus GK24 L-lactate dehydrogenase

Heat-stable fructose 1,6-bisphosphate-dependent L-lactate dehydrogenase [EC 1.1.1.27] was purified from an extremely thermophilic bacterium, Thermus aquaticus YT-1. The amino acid composition and NH2-terminal 34 amino acid sequence of the enzyme were determined. Its NH2-terminal sequence shows high homology with those of Thermus caldophilus GK24 (82% identity) and some other bacterial L-lactate dehydrogenases (44-53% identity), indicating the close phylogenic relationship of the two Thermus species. At the same time, the two Thermus L-lactate dehydrogenases were found not to be identical not only chemically but also kinetically and immunologically. Citrate activated the T. aquaticus enzyme in the weak acidic pH region, while fructose 1,6-bisphosphate did in both acidic and neutral pH regions. The maximum activity obtained with citrate at pH 5.0 was about 2.5 times higher than that in the presence of fructose 1,6-bisphosphate at pH 6.7. The enzymes modified with 2,3-butanedione, acetic anhydride and diethyl pyrocarbonate in the presence of both NADH and oxamate were desensitized to fructose 1,6-bisphosphate, and the modified enzymes were active even in the absence of fructose 1,6-bisphosphate. All of the modified enzymes examined were still activated by citrate similarly to the native enzyme. These results suggest that the mechanism of activation by citrate is different from that by fructose 1,6-bisphosphate, and that the citrate-binding site is different from the fructose 1,6-bisphosphate-binding site.