Purification and characterization of osmoregulatory betaine aldehyde dehydrogenase of Escherichiacoli

The osmoregulatory NAD-dependent betaine aldehyde dehydrogenase (betaine aldehyde: NAD oxidoreductase, EC 1.2.1.8), of Escherichia coli, was purified to apparent homogeneity from an over-producing strain carrying the structural gene for the enzyme (betB) on the plasmid vector pBR322. Purification was achieved by ammonium sulfate fractionation of disrupted cells, followed by affinity chromatography on 5′-AMP Sepharose, gel-filtration and ion-exchange chromatography. The amino acid composition was determined. The dehydrogenase was found to be a tetramer with identical 55 kDa subunits. Both NAD and NADP could be used as cofactor for the dehydrogenase, but NAD was preferred. The dehydrogenase was highly specific for betaine aldehyde. None of the analogs tested functioned as a substrate, but several inhibited the enzyme competitively. The enzyme was not activated by salts at concentrations encountered during osmotic upshock, but it was salt tolerant, retaining 50% of maximal activity at 1.2 M K⁺. It is inferred that salt tolerance is an essential property for an enzyme participating in the cellular synthesis of an osmoprotectant.     

Carboxymethylhydroxymuconic semialdehyde dehydrogenase in the 4-hydroxyphenylacetate catabolic pathway of Escherichia coli

5-Carboxymethyl-2-hydroxymuconic semialdehyde dehydrogenase in the 4-hydroxyphenylacetate meta-cleavage pathway has been purified to 96% homogeneity. The native enzyme, which appears to be a tetramer, has an apparent molecular weight of 210000. The purified enzyme shows a narrow pH optimum at pH 7.8 and does not require ions for its catalytic activity. Under standard assay conditions the enzyme acts preferentially with NAD but reduces NADP at 11% of the rate observed for NAD, primarily because of a difference in K_m. Apparent K_m values are 6.4 μM for 5-carboxymethyl-2-hydroxymuconic semialdehyde and 52.2 μM for NAD.     

Substrate Specificity of 2-Ketogluc nate Reductase from Gluconobacti liquefaciens

The significant betaine aldehyde dehydrogenase activity was found in the cells of Pseudomonas aeruginosa A-16. The enzyme was inducibly formed and accumulated in the presence of choline, acetylcholine or betaine in the medium. The enzyme was purified approximately 620-fold with an overall recovery of 2.6% and proved to be homogeneous by ultracentrifugation. The molecular weight of the enzyme was determined as approximately 145,000 by gel filtration method. The enzyme had an isoelectric point around pH 5.1. The enzyme was quite specific for its substrate, betaine aldehyde. Both NADP and NAD functioned as coenzyme. The estimated values of Km at pH 7.4 and 25°C were 3.8 × 10^?4 m for betaine aldehyde, 8.9 × 10^?5 m for NADP and 2.2 × 10^?4 m for NAD.     

The effect of NADP on the subunit structure and activity of spinach chloroplast glyceraldehyde-3-phosphate dehydrogenase

Spinach chloroplast glyceraldehyde phosphate dehydrogenase (d-glyceraldehyde-3-phosphate: NADP oxidoreductase, phosphorylating; EC 1.2.1.13) is an equilibrium mixture of aggregates of a basic protomer (M_r about 145,000) and is active with both NADP and NAD. The enzyme is primarily “tetrameric” (M_r about 600,000), although minor amounts of smaller and larger oligomers are also found. Gel chromatography in buffer containing 30 μm NADP results in depolymerization of the enzyme, mainly to protomers. NAD does not dissociate and counteracts this effect of NADP.The apparent K_m values of the protomers are 7 μm (NADP) and 8 μm (NAD). The aggregates with a M_r > 10⁶ have properties similar to the protomers. The tetramer as first isolated has higher M_m values for NADP (380 μm) and NAD (48 μm), but its apparent affinity for NADP is further decreased by repeated gel filtrations in buffer or by a single one in buffer containing NAD. Such preparations display nonlinear kinetics when NADP is the varied substrate and have a K_m (NADP) of about 1.5–3.3 μm. All these effects are reversible.V values are apparently the same in all enzyme forms and the $\text{V (}\text{NADP}\text{)}\text{V (}\text{NAD}\text{)}$ ratio always approaches 2. Since, however, the enzyme is presumably dissociated by the NADP concentrations required for a “saturating” assay, the significance of V (NADP) seems questionable.     

A soluble protein factor from chinese cabbage converts indole-3-acetaldoxime to iaa

A soluble enzyme preparation from Chinese cabbage seedlings (Brassica campestris ssp. pekinensis) which catalyses the conversion of indole-3-acetaldoxime (IAOX) to IAA was partially purified by ion exchange chromatography. After purification enzyme activity was stable for more than 6 hr. Substrate kinetics showed a K_m value of 50 μM; the pH optimum was 7. The conversion of IAOX to IAA was increased by NAD, NADP or FAD, but none of them seemed to be a preferential co-substrate. Besides IAA some labelled indole-3-acetaldehyde (IAALD) could be extracted from the reaction mixture. Addition of unlabelled IAALD at 100 nmol/ml led to a significant inhibition of IAA formation while some label accumulated in the aldehyde, Indole-3-acetonitrile was never detected as a reaction product. The results are compared with those from earlier in vivo experiments and are discussed in view of their significance for IAA biosynthesis in the Brassicaceae.     

Purification and characterization of osmoregulatory betaine aldehyde dehydrogenase of Escherichia coli

The osmoregulatory NAD-dependent betaine aldehyde dehydrogenase (betaine aldehyde:NAD oxidoreductase, EC 1.2.1.8), of Escherichia coli, was purified to apparent homogeneity from an over-producing strain carrying the structural gene for the enzyme (betB) on the plasmid vector pBR322. Purification was achieved by ammonium sulfate fractionation of disrupted cells, followed by affinity chromatography on 5'-AMP Sepharose, gel-filtration and ion-exchange chromatography. The amino acid composition was determined. The dehydrogenase was found to be a tetramer with identical 55 kDa subunits. Both NAD and NADP could be used as cofactor for the dehydrogenase, but NAD was preferred. The dehydrogenase was highly specific for betaine aldehyde. None of the analogs tested functioned as a substrate, but several inhibited the enzyme competitively. The enzyme was not activated by salts at concentrations encountered during osmotic upshock, but it was salt tolerant, retaining 50% of maximal activity at 1.2 M K+. It is inferred that salt tolerance is an essential property for an enzyme participating in the cellular synthesis of an osmoprotectant.     

Partial purification and some properties of shikimate dehydrogenase from tomatoes

The partial purification of shikimate dehydrogenase (SDH) from tomato fruit was achieved by precipitation with ammonium sulphate, and chromatography on DEAE-cellulose and hydroxyapatite. The enzyme has a MW of 73000, shows an optimum at pH 9.1 and K_m values of 3.8 × 10^?5 M and 1.0 × 10^?5 M with shikimic acid and NADP as substrates. NADP could not be replaced by NAD. The tomato enzyme is competitively inhibited by protocatechuic acid with a K_i value of 7.7 × 10^?5 M. On the other hand, cinnamic acid derivatives and 2-hydroxybenzoic acid were ineffective. At 50° for 5 min the SDH is inactivated by 85%. The activity was inhibited by pCMB and N-ethylmaleimide, suggesting a requirement for SH groups. The inactivation plot of oxidation by pCMB was biphasic, and NADP decreased the reactivity of sulphydryl groups to the reagent. The activation energy was found to be 14.2kcal/mol. The properties of the SDH are discussed in relation to the enzymes from other sources.     

Hymenolepis microstoma: lactate and malate dehydrogenases of the adult worm.

Lactate and malate dehydrogenases (EC 1.1.1.27 and EC 1.1.1.37, respectively) were precipitated with ammonium sulfate, redissolved in 100 mM phosphate buffer, and the kinetic parameters of each enzyme determined. Lactate dehydrogenase: The enzyme preparation had a specific activity of 0.35 μmole NADH oxidized/min/mg protein for pyruvate reduction, and 0.10 μmole NAD reduced/min/mg protein for lactate oxidation. K_m values for the substrates and cofactors were as follows: pyruvate = 0.51, mM; lactate = 3.8 mM; NADH = 0.011 mM; and NAD = 0.17 mM. NADPH, NADP, or d(?)-lactate would not replace NADH, NAD, or l(+)-lactate, respectively. The enzyme was relatively stable at 50 C for 45 min, but much less stable at 60 C; repeated freezing and thawing of the enzyme preparation had little effect on LDH activity. Both p-chloromercuribenzoate (p-CMB) and N-ethylmaleimide (NEM) significantly inhibited LDH activity. Polyacrylamide gel electrophoresis demonstrated the presence of at least two LDH isoenzymes in the unpurified enzyme preparation. The molecular weight was estimated at 160,000 by gel chromatography. Malate dehydrogenase: The enzyme preparation had a specific activity of 6.70 μmole NADH oxidized/min/mg protein for oxaloacetate reduction, and 0.52 μmole NAD reduced/ min/mg protein for malate oxidation. K_m values for substrates and cofactors were as follows: l-malate = 1.09 mM; oxaloacetate = 0.0059 mM; NADH = 0.017 mM; and NAD = 0.180 mM. NADP and NADPH would not replace NAD and NADH, respectively, d-malate was oxidized slowly when present in high concentrations (>100 mM). Significant substrate inhibition occurred with concentrations of l-malate and oxaloacetate above 40 mM and 0.5 mM, respectively. The enzyme was unstable at temperatures above 40 C, but repeated freezing and thawing of the enzyme preparation had little effect on MDH activity. Only p-CMB inhibited MDH activity. Polyacrylamide gel electrophoresis demonstrated the presence of at least three MDH isoenzymes in the unpurified enzyme preparation, and the molecular weight was estimated at 49,000 by gel chromatography.     

Making an Oriental equivalent of the yeast cytosolic aldehyde dehydrogenase as well as making one with positive cooperativity in coenzyme binding by mutations of glutamate 492 and arginine 480

Yeast has at least three partially characterized aldehyde dehydrogenases. Previous studies by gene disrupted in our laboratory revealed that the Saccharomyces cerevisiae cytosol ALDH1 played an important role in ethanol metabolism as did the class 2 mitochondrial enzyme. To date, few mutagenesis studies have been performed with the yeast enzymes. An important human variant of ALDH is one found in Asian People. In it, the glutamate at position 487 is replaced by a lysine. This glutamate interacts with an arginine (475) that is located in the subunit that makes up the dimer pair in the tetrameric enzyme. Sequence alignment shows that these two residues are located at positions 492 and 480, respectively, in the yeast class 1 enzyme which shares just 45% sequence identity with the human enzymes. Mutating glutamate 492 to lysine produced an enzyme with altered kinetic properties when compared to the wild-type glutamate-enzyme. The K_m for NADP of E492K increased to nearly 3600 μM compare to 40 μM for wild-type enzyme. The specific activity decreased more than 10-fold with respect to the recombinant wild-type yeast enzyme. Moreover, substituting a glutamine for a glutamate was not detrimental in that the E492Q had wild-type-like K_m for NADP and V_max. These properties were similar to the changes found with the human class 2 E487K mutant form. Further, mutating arginine 480 to glutamine produced an enzyme that exhibited positive cooperativity in NADP binding. The K_m for NADP increased 11-fold with a Hill coefficient of 1.6. The NADP-dependent activity of R480Q mutant was 60% of wild-type enzyme. Again, these results are very similar to what we recently showed to occur with the human enzyme [Biochemistry 39 (2000) 5295–5302]. These findings show that the even though the glutamate and arginine residues are not conserved, similar changes occur in both the human and the yeast enzyme when either is mutated.     

Nucleotide binding affinities of the intact proton-translocating transhydrogenase from Escherichia coli

Transhydrogenase (E.C. 1.6.1.1) couples the redox reaction between NAD(H) and NADP(H) to the transport of protons across a membrane. The enzyme is composed of three components. The dI and dIII components, which house the binding site for NAD(H) and NADP(H), respectively, are peripheral to the membrane, and dII spans the membrane. We have estimated dissociation constants (K_d values) for NADPH (0.87 μM), NADP⁺ (16 μM), NADH (50 μM), and NAD⁺ (100-500 μM) for intact, detergent-dispersed transhydrogenase from Escherichia coli using micro-calorimetry. This is the first complete set of dissociation constants of the physiological nucleotides for any intact transhydrogenase. The K_d values for NAD⁺ and NADH are similar to those previously reported with isolated dI, but the K_d values for NADP⁺ and NADPH are much larger than those previously reported with isolated dIII. There is negative co-operativity between the binding sites of the intact, detergent-dispersed transhydrogenase when both nucleotides are reduced or both are oxidised.     

Purification, Characterization, and Gene Cloning of Purine Nucleosidase from Ochrobactrum anthropi

A bacterium, Ochrobactrum anthropi, produced a large amount of a nucleosidase when cultivated with purine nucleosides. The nucleosidase was purified to homogeneity. The enzyme has a molecular weight of about 170,000 and consists of four identical subunits. It specifically catalyzes the irreversible N-riboside hydrolysis of purine nucleosides, the K_m values being 11.8 to 56.3 μM. The optimal activity temperature and pH were 50°C and pH 4.5 to 6.5, respectively. Pyrimidine nucleosides, purine and pyrimidine nucleotides, NAD, NADP, and nicotinamide mononucleotide are not hydrolyzed by the enzyme. The purine nucleoside hydrolyzing activity of the enzyme was inhibited (mixed inhibition) by pyrimidine nucleosides, with K_i and K_i′ values of 0.455 to 11.2 μM. Metal ion chelators inhibited activity, and the addition of Zn²⁺ or Co²⁺ restored activity. A 1.5-kb DNA fragment, which contains the open reading frame encoding the nucleosidase, was cloned, sequenced, and expressed in Escherichia coli. The deduced 363-amino-acid sequence including a 22-residue leader peptide is in agreement with the enzyme molecular mass and the amino acid sequences of NH₂-terminal and internal peptides, and the enzyme is homologous to known nucleosidases from protozoan parasites. The amino acid residues forming the catalytic site and involved in binding with metal ions are well conserved in these nucleosidases.     

Binding of pyridine coenzymes to the β-subunit of the voltage sensitive potassium channels

The β-subunit of the voltage-sensitive K⁺ channels shares 15–30% amino acid identity with the sequences of aldo–keto reductases (AKR) genes. However, the AKR properties of the protein remain unknown. To begin to understand its oxidoreductase properties, we examine the pyridine coenzyme binding activity of the protein in vitro. The cDNA of K_vβ2.1 from rat brain was subcloned into a prokaryotic expression vector and overexpressed in Escherichia coli. The purified protein was tetrameric in solution as determined by size exclusion chromatography. The protein displayed high affinity binding to NADPH as determined by fluorometric titration. The K_D values for NADPH of the full-length wild-type protein and the N-terminus deleted protein were 0.1±0.007 and 0.05±0.006 M, respectively — indicating that the cofactor binding domain is restricted to the C-terminus, and is not drastically affected by the absence of the N-terminus amino acids, which form the ball and chain regulating voltage-dependent inactivation of the α-subunit. The protein displayed poor affinity for other coenzymes and the corresponding values of the K_D for NADH and NAD were between 1–3 μM whereas the K_D for FAD was >10 μM. However, relatively high affinity binding was observed with 3-acetyl pyridine NADP, indicating selective recognition of the 2′ phosphate at the binding site. The selectivity of K_vβ2.1 for NADPH over NADP may be significant in regulating the K⁺ channels as a function of the cellular redox state.     

Substrate-dependent activation of uridine diphosphoglucose dehydrogenase from the dimorphic fungusMucor rouxii

As part of an investigation on polyuronide synthesis in the dimorphic fungusMucor rouxii, we have studied the synthesis from UDP-glucose (UDPG) of the activated sugar precursor UDPGlucuronic acid. UDPG dehydrogenases from yeastlike and mycelial forms ofM. rouxii were partially purified by ammonium sulfate fractionation and ultrafiltration. The enzyme preparations were largely free of enzymes which could degrade the substrates or products of the reaction. In the assay, enzymes from both sources exhibited slow activation over a period of several minutes. The rate of activation was dependent on both the enzyme concentration and the concentration of UDPG and NAD. The enzyme from the mycelial form had akm for UDPG of 490 μm, aK_m for NAD of 297 μm, and a molecular weight at pH 7.0 of 440,000 as estimated by gel chromatography. In contrast the enzyme from the yeastlike form had akm for UDPG of 168 μm, aK_m for NAD of 740 μm, and a molecular weight at pH 7.0 of 330,000. At pH 8.0, both enzymes dissociated to give forms of lower molecular weight. At this pH, UDPG alone could activate the enzyme. This activation was accompanied by the association of the lower-molecular-weight forms to give the higher-molecular-weight forms seen at pH 7.0. Association was prevented by heat treatment. The heat-treated enzymes could not produce UDPGlucuronic acid.     

Enzymic synthesis of lignin precursors. Purification of properties of the S-adenosyl-l-methionine: caffeic acid 3-O- methyltransferase from soybean cell suspension cultures.

A 3-O-methyltransferase which catalyzes the methylation of caffeic acid to ferulic acid using S-adenosyl-l-methionine as methyl donor has been isolated and purified about 60-fold from cell suspension cultures of soybean (Glycine max L., var. Mandarin). The enzyme utilized, in addition to caffeic acid (K_m = 133 μM), 5-hydroxyferulic acid (K_m = 55 μM), 3,4,5-trihydroxy-cinnamic acid (K_m = 100 μM), and protocatechualdehyde (K_m = 50 μM) as substrates. Methylation proceeded only in the meta position. The enzyme was unable to catalyze the methylation of ferulic acid, of ortho-, meta-, and para-coumaric acids, and of the flavonoid compounds quercetin and luteolin. The methylation of caffeic acid and 5-hydroxyferulic acid showed a pH optimum at 6.5–7.0. No stimulation of the reaction velocity was observed when Mg²⁺ ions were added. EDTA did not inhibit the reaction. The K_m for S-adencsyl-l-methionine was 15 μm. S-Adenosyl-l-homocysteine was a potent competitive inhibitor of S-adenosyl-l-methionine (K_i = 6.9 μM).     

Purification and characterization of a new glucose dehydrogenase from vegetative cells of Bacillus megaterium

A new type of glucose dehydrogenase was purified from vegetative cells of Bacillus megaterium IAM1030. The characteristics of the vegetative-cell enzyme were investigated and compared with the enzyme from sporulating cells of B. megaterium IWG3. They are very similar in the following points: molecular size (M_r 120,000), subunit composition (homo tetramer), pH-activity profile with an optimum pH at around 8, pH-stability profile with a stable pH range of 6.0–7.5 (at 25°C, for 30 min), substrate specificity (specific for d-glucose and 2-deoxy-d-glucose), and the affinity for glucose (a K_m value of 11–12 mM at pH 8.0, 2.5 mM NAD). They are a little different in the following points: slower mobility for the vegetative-cell enzyme in polyacrylamide-gel electrophoresis at pH 8, immunological determinants (some of them are common), and higher heat resistance for the vegetative-cell enzyme at pH 6.5. They are quite different in their affinity for NAD and NADP. The K_m values for NAD are 0.1 mM for the vegetative-cell enzyme and 1.0 mM for the spore enzyme, while the values for NADP are 7.1 mM for the vegetative-cell enzyme and 0.09 mM for the spore enzyme, at pH 8.0, 0.1 M d-glucose. These results suggest that B. megaterium has at least two types of glucose dehydrogenase.     

Interaction of human aldehyde dehydrogenase with aromatic substrates and ligands

The substrate benzaldehyde (but not propionaldehyde) could elute aldehyde dehydrogenase from a p-hydroxyacetophenone-affinity column, and inhibit the esterase activity (K_i=47 μM), indicating that this simple aromatic aldehyde binds to the free enzyme and possibly in the substrate-binding site. Thus, the kinetic mechanism for aldehyde dehydrogenase might be dependent upon which aldehyde is used in the reaction. Chloramphenicol which also elutes the enzyme from the affinity column, shows a discriminatory effect by inhibiting the ALDH1 oxidation of benzaldehyde and activating that of propionaldehyde while showing no effect when assayed with hexanal or cyclohexane–carboxaldehyde. Chloramphenicol is an uncompetitive inhibitor against NAD when benzaldehyde is the substrate. We propose that this drug might interact with both the benzaldehyde and NAD binding sites.     

Glycine betaine aldehyde dehydrogenase from Bacillus subtilis: characterization of an enzyme required for the synthesis of the osmoprotectant glycine betaine

Production of the compatible solute glycine betaine from its precursors choline or glycine betaine aldehyde confers a considerable level of tolerance against high osmolarity stress to the soil bacterium Bacillus subtilis. The glycine betaine aldehyde dehydrogenase GbsA is an integral part of the osmoregulatory glycine betaine synthesis pathway. We strongly overproduced this enzyme in an Escherichia coli strain that expressed a plasmid-encoded gbsA gene under T7φ10 control. The recombinant GbsA protein was purified 23-fold to apparent homogeneity by fractionated ammonium sulfate precipitation, ion-exchange chromatography on Q-Sepharose, and subsequent hydrophobic interaction chromatography on phenyl-Sepharose. Molecular sieving through Superose 12 and sedimentation centrifugation through a glycerol gradient suggested that the native enzyme is a homodimer with 53.7-kDa subunits. The enzyme was specific for glycine betaine aldehyde and could use both NAD⁺ and NADP⁺ as cofactors, but NAD⁺ was strongly preferred. A kinetic analysis of the GbsA-mediated oxidation of glycine betaine aldehyde to glycine betaine revealed K _m values of 125 μM and 143 μM for its substrates glycine betaine aldehyde and NAD⁺, respectively. Low concentrations of salts stimulated the GbsA activity, and the enzyme was highly tolerant of high ionic conditions. Even in the presence of 2.4 M KCl, 88% of the initial enzymatic activity was maintained. B. subtilis synthesizes high levels of proline when grown at high osmolarity, and the presence of this amino acid strongly stimulated the GbsA activity in vitro. The enzyme was stimulated by moderate concentrations of glycine betaine, and its activity was highly tolerant against molar concentrations of this osmolyte. The high salt tolerance and its resistance to its own reaction product are essential features of the GbsA enzyme and ensure that B. subtilis can produce high levels of the compatible solute glycine betaine under conditions of high osmolarity stress. Received: 2 May 1997 / Accepted: 2 July 1997     

Localization and characteristics of rat liver mitochondrial aldehyde dehydrogenases.

Two NAD-dependent aldehyde dehydrogenase enzymes from rat liver mitochondria have been partially purified and characterized. One enzyme (enzyme I) has molecular weight of 320,000 and has a broad substrate specificity which includes formaldehyde; NADP is not a cofactor for this enzyme. This enzyme has K_m values for most aldehydes in the micromolar range. The isoelectric point was found to be 6.06. A second enzyme (enzyme II) has a molecular weight of 67,000, a K_m value for most aldehydes in the millimolar range but no activity toward formaldehyde. NADP does serve as a coenzyme, however. The isoelectric point is 6.64 for this enzyme. By utilization of the different substrate properties of these two enzymes it was possible to demonstrate a time-dependent release from digitonin-treated liver mitochondria. The high K_m, low molecular weight enzyme (enzyme II) is apparently in the intermembrane space while the low K_m, high molecular weight enzyme (enzyme I) is in the mitochondrial matrix and is most likely responsible for oxidation of acetaldehyde formed from ethanol.     

The pyridine nucleotide cycle: presence of a nicotinamide mononucleotide-specific amidohydrolase in Propionibacterium shermanii

A specific nicotinamide mononucleotide amidohydrolase which catalyzes the stoichiometric conversion of NMN to nicotinate mononucleotide and ammonia has been partially purified from an extract of $\text{Propionibacterium}\text{shermanii}$. The reaction has optimum activity at pH 5.6, a K_m of 70 μM, and an experimental activation energy of 14.5 Kcal/mole. The enzyme appears to be highly specific for NMN. Neither free nicotinamide nor NAD, NADH, NADP, NADPH compete with NMN. Numerous substances such as isonicotinic acid hydrazide and quinolinic acid are also without effect. It can be stored at ?15° in 12% glycerol, but is somewhat unstable in the absence of this solvent. The enzyme is composed of a heatstable and a heat-sensitive subunit. This enzyme considerably simplifies the pyridine nucleotide cycle, and may, besides this salvage function for NAD, play a role in B₁₂ biosynthesis and in the bacterial DNA ligase reaction.     

Alteration of Coenzyme Specificity of Lactate Dehydrogenase from Thermus thermophilus by Introducing the Loop Region of NADP(H)-Dependent Malate Dehydrogenase

Previously we found that replacement of seven amino acid residues in a loop region markedly shifted the coenzyme specificity of malate dehydrogenase from NAD(H) toward NADP(H). In the present study, we replaced the seven amino acid residues in the corresponding region of an NAD(H)-dependent lactate dehydrogenase with those of NADP(H)-dependent malate dehydrogenase, and examined the coenzyme specificity of the resulting mutant enzyme. Coenzyme specificity was significantly shifted by 399-fold toward NADPH when k _cat?K _m ^coenzyme was used as the measure of coenzyme specificity. The effect of the replacements on coenzyme specificity is discussed based on in silico simulation of the three-dimensional structure of the lactate dehydrogenase mutant.