Pentitol metabolism of Rhodobacter sphaeroides Si4: purification and characterization of a ribitol dehydrogenase.

The phototrophic bacterium Rhodobacter sphaeroides strain Si4 induced ribitol dehydrogenase (EC 1.1.1.56) when grown on ribitol- or xylitol-containing medium. This ribitol dehydrogenase was purified to apparent homogeneity by ammonium sulphate precipitation, affinity chromatography on Procion red, and chromatography on Q-Sepharose. For the native enzyme an isoelectric point of pH 6.1 and an apparent M(r) of 50,000 was determined. SDS-PAGE yielded a single peptide band of M(r) 25,000 suggesting a dimeric enzyme structure. The ribitol dehydrogenase was specific for NAD+ but unspecific as to its polyol substrate. In order of decreasing activity ribitol, xylitol, erythritol, D-glucitol and D-arabitol were oxidized. The pH optimum of substrate oxidation was 10, and that of substrate reduction was 6.5. The equilibrium constant of the interconversion of ribitol to D-ribulose was determined to be 0.33 nM at pH 7.0 and 25 degrees C. The Km-values determined for ribitol, ribulose, xylitol and NAD+ (in the presence of ribitol) were 6.3, 12.5, 77 and 0.077 mM, respectively. Because of the favourable Km for ribitol, a method for quantitative ribitol determination was elaborated.     

Purification and characterization of mannitol dehydrogenase from Aspergillus parasiticus.

Mannitol dehydrogenase, NADP specific (EC 1.1.1.138), was purified from mycelium of Aspergillus parasiticus (1-11-105 Whl). The enzyme had a molecular weight of 1.4 X 10(5) and was composed of four subunits of apparently equal size. The substrate specificity was limited to D-mannitol, D-glucitol, D-arabinitol, 1-deoxy-D-mannitol, and 1-deoxy-D-glucitol. Zinc ion was a powerful inhibitor of the enzyme, inhibition being competitive with respect to mannitol, with Ki and 1 microM. It is proposed that the stimulation of polyketide synthesis by zinc ion may be mediated in part by inhibition of mannitol dehydrogenase.     

The dihydrolipoamide dehydrogenase from the crenarchaeon Acidianus ambivalens

A dihydrolipoamide dehydrogenase (DLDH) was purified and characterized for the first time from a crenarchaeon, Acidianus ambivalens. The holoenzyme consists of two identical subunits with a molecular mass of 45.4 kDa per monomer. It contains FAD as a prosthetic group and uses NAD+ as the preferential substrate, but can also reduce NADP+. The Michaelis-Menten constants of the forward (NAD+ reduction) and reverse (NADH oxidation) reactions were KM (dihydrolipoamide)=0.70 mM, KM (NAD+)=0.71 mM, KM (lipoamide)=1.26 mM and KM (NADH)=3.15 microM. A comparative study of NADH:lipoamide oxidoreductase and NADH:K3[Fe(CN)6] oxidoreductase activities was performed, the optimal temperature and pH being different for each: 55 degrees C, pH 7 and 89 degrees C, pH 5.5, respectively. Although DLDH is generally part of the alpha-ketoacid dehydrogenase complexes in Bacteria and Eukarya, none of these complexes has yet been isolated from Sulfolobales. The metabolic role of DLDH in these organisms is discussed.     

Kinetic study of the catalytic mechanism of mannitol dehydrogenase from Pseudomonas fluorescens.

To characterize catalysis by NAD-dependent long-chain mannitol 2-dehydrogenases (MDHs), the recombinant wild-type MDH from Pseudomonas fluorescens was overexpressed in Escherichia coli and purified. The enzyme is a functional monomer of 54 kDa, which does not contain Zn(2+) and has B-type stereospecificity with respect to hydride transfer from NADH. Analysis of initial velocity patterns together with product and substrate inhibition patterns and comparison of primary deuterium isotope effects on the apparent kinetic parameters, (D)k(cat), (D)(k(cat)/K(NADH)), and (D)(k(cat)/K(fructose)), show that MDH has an ordered kinetic mechanism at pH 8.2 in which NADH adds before D-fructose, and D-mannitol and NAD are released in that order. Isomerization of E-NAD to a form which interacts with D-mannitol nonproductively or dissociation of NAD from the binary complex after isomerization is the slowest step (>/=110 s(-)(1)) in D-fructose reduction at pH 8.2. Release of NADH from E-NADH (32 s(-)(1)) is the major rate-limiting step in mannitol oxidation at this pH. At the pH optimum for D-fructose reduction (pH 7.0), the rate of hydride transfer contributes significantly to rate limitation of the catalytic cascade and the overall reaction. (D)(k(cat)/K(fructose)) decreases from 2.57 at pH 7.0 to a value of 相似文献   

Enzymatic Regeneration of NAD in Enantioselective Oxidation of Secondary Alcohols: Glutamate Dehydrogenase Versus NADH Dehydrogenase

To improve yield and productivity of ketose in NAD-dependent polyol oxidations, two enzymatic methods for regeneration of the oxidized coenzyme form have been compared and partly optimized for the batch conversion of xylitol into D-xylulose and D-sorbitol into D-fructose. Polyol oxidation was catalyzed by xylitol dehydrogenase from the yeast Galactocandida mastotermitis. Reduction of OM₂ (apparently to H₂O) by partially purified NADH dehydrogenase complex from Corynebacterium callunae could drive alcohol oxidations better than reductive amination of EaL-ketoglutarate by glutamate dehydrogenase. A fed-batch procedure was developed that overcame inhibition of glutamate dehydrogenase by α-ketoglutarate (K_is 25 mM), thus increasing the productivity of ketose almost 2-fold. For D-fructose production from D-sorbitol (0.1-0.3M) yields of < 90% and productivities up to 1.30g/(L.h) have been obtained. High conversion of up to 50g/L xylitol into D-xylulose for which xylitol dehydrogenase exhibits an about 80-fold higher specificity constant than for D-fructose required complexation of the ketose product with borate. In comparison with reductive amination by glutamate dehydrogenase, advantages of using NADH-dehydrogenase catalyzed regeneration of NAD for ketose production are (i) avoidance of byproduct formation, (ii) cheaper substrate (02 versus α-ketoglutarate), and (iii) easier process control (batch versus fed-batch).     

Enzymatic Production of Pure D-Mannitol at High Productivity

An enzymatic, NAD(H)-dependent process for the efficient production of D-mannitol from D-fructose as one single product is described and optimized with respect to productivity at high substrate conversion. Stereospecific reduction of D-fructose is catalyzed by recombinant mannitol dehydrogenase from Pseudomonas fluorescens DSM 50106, overexpressed in Escherichia coli. Regeneration of NADH is accomplished by formate dehydrogenase-mediated oxidation of formate into CO₂, thus avoiding byproduct formation and yielding total turnover numbers for the coenzyme of approximately 1000 for a single round of D-fructose conversion. In optimized batchwise reduction of D-fructose, a D-mannitol productivity of 2.25 g/(L h) was obtained for a final product concentration of 72g/L and a D-fructose conversion of 80%. D-Mannitol was crystallized from the ultrafiltered product solution in 97% purity and 85% recovery, thus also allowing reuse of enzymes for repeated batchwise production of D-mann!itol!.     

D-mannitol production by resting state whole cell biotrans-formation of D-fructose by heterologous mannitol and formate dehydrogenase gene expression in Bacillus megaterium

An in vivo system was developed for the biotransformation of D-fructose into D-mannitol by the expression of the gene mdh encoding mannitol dehydrogenase (MDH) from Leuconostoc pseudomesenteroides ATCC12291 in Bacillus megaterium. The NADH reduction equivalents necessary for MDH activity were regenerated via the oxidation of formate to carbon dioxide by coexpression of the gene fdh encoding Mycobacterium vaccae N10 formate dehydrogenase (FDH). High-level protein production of MDH in B. megaterium required the adaptation of the corresponding ribosome binding site. The fdh gene was adapted to B. megaterium codon usage via complete chemical gene synthesis. Recombinant B. megaterium produced up to 10.60 g/L D-mannitol at the shaking flask scale. Whole cell biotransformation in a fed-batch bioreactor increased D-mannitol concentration to 22.00 g/L at a specific productivity of 0.32 g D-mannitol (gram cell dry weight)(-1) h(-1) and a D-mannitol yield of 0.91 mol/mol. The nicotinamide adenine dinucleotide (NAD(H)) pool of the B. megaterium producing D-mannitol remained stable during biotransformation. Intra- and extracellular pH adjusted itself to a value of 6.5 and remained constant during the process. Data integration revealed that substrate uptake was the limiting factor of the overall biotransformation. The information obtained identified B. megaterium as a useful production host for D-mannitol using a resting cell biotransformation approach.     

Characterization of the course of D-glucitol biotransformation by strains gluconobacter oxydans

Production strains Gluconobacter oxydans used in manufacture of L-sorbose from commercial D-glucitol were evaluated by HPLC determination of D-glucitol, L-sorbose, D-fructose, D-mannnitol, D-arabinitol, D-xylulose, and D-threo-2,5-hexodiulose in the course of fermentation. Serious differences were observed among the strains apparently identical by standard methods.     

D-Mannitol dehydrogenase from Absidia glauca. Purification, metabolic role, and subunit interactions.

When Absidia glauca was grown in minimal media with D-mannitol as the only source of carbon, an NAD+ specific D-mannitol dehydrogenase (EC 1.1.1.67) was induced. The crude extract also gave evidence of mannitol kinase, mannitol-1-phosphate dehydrogenase, phosphofructokinase, and L-iditol dehydrogenase activity. The heat labile purified preparation was judged enzymically homogeneous based on evidence derived from substrate specificity studies and activity staining, following disc gel electrophoresis. The enzymic monomer, with a weight of about 67000 daltons, slowly polymerizes when stored at -20 degrees C, giving a multiplicity of protein bands on electrophoresis distributed predominantly across a spectrum from dimer to pentamer, with enzymic activity resident predominantly in even multiples of the monomer. Depolymerization occurred rapidly (hours) when a frozen preparation was brought to and held between 4 and 20 degrees C. Aggregate fragmentation with sodium dodecyl sulfate showed a time-temperature dependence, terminating in a subunit component of 13000 daltons. pH optimum for polyol oxidation occurs at 9.6 (NaOH-glycine buffer) while ketose reduction proceeded most rapidly at pH 7.0-7.2 (phosphate buffer). A regulatory role is suggested for this enzyme based on dead-end inhibition by mannitol 1-phosphate, multiple enzyme forms, and its locus at the initiation site for mannitol utilization. The physiological relevance of low-temperature aggregation to regulatory control remains to be established.     

Enzymes of mannitol metabolism in the human pathogenic fungus Aspergillus fumigatus--kinetic properties of mannitol-1-phosphate 5-dehydrogenase and mannitol 2-dehydrogenase, and their physiological implications

The human pathogenic fungus Aspergillus fumigatus accumulates large amounts of intracellular mannitol to enhance its resistance against defense strategies of the infected host. To explore their currently unknown roles in mannitol metabolism, we studied A. fumigatus mannitol-1-phosphate 5-dehydrogenase (AfM1PDH) and mannitol 2-dehydrogenase (AfM2DH), each recombinantly produced in Escherichia coli, and performed a detailed steady-state kinetic characterization of the two enzymes at 25 °C and pH 7.1. Primary kinetic isotope effects resulting from deuteration of alcohol substrate or NADH showed that, for AfM1PDH, binding of D-mannitol 1-phosphate and NAD(+) is random, whereas D-fructose 6-phosphate binds only after NADH has bound to the enzyme. Binding of substrate and NAD(H) by AfM2DH is random for both D-mannitol oxidation and D-fructose reduction. Hydride transfer is rate-determining for D-mannitol 1-phosphate oxidation by AfM1PDH (k(cat) = 10.6 s(-1)) as well as D-fructose reduction by AfM2DH (k(cat) = 94 s(-1)). Product release steps control the maximum rates in the other direction of the two enzymatic reactions. Free energy profiles for the enzymatic reaction under physiological boundary conditions suggest that AfM1PDH primarily functions as a D-fructose-6-phosphate reductase, whereas AfM2DH acts in D-mannitol oxidation, thus establishing distinct routes for production and mobilization of mannitol in A. fumigatus. ATP, ADP and AMP do not affect the activity of AfM1PDH, suggesting the absence of flux control by cellular energy charge at the level of D-fructose 6-phosphate reduction. AfM1PDH is remarkably resistant to inactivation by heat (half-life at 40 °C of 20 h), consistent with the idea that formation of mannitol is an essential component of the temperature stress response of A. fumigatus. Inhibition of AfM1PDH might be a useful target for therapy of A. fumigatus infections.     

Purification of an inducible L-valine dehydrogenase of Streptomyces coelicolor A3(2)

Valine dehydrogenase (VDH) from Streptomyces coelicolor A3(2) was purified from cell-free extracts to apparent homogeneity. The enzyme had an Mr 41,000 in denaturing conditions and an Mr 70,000 by gel filtration chromatography, indicating that it is composed of two identical subunits. It oxidized L-valine and L-alpha-aminobutyric acid efficiently, L-isoleucine and L-leucine less efficiently, and did not act on D-valine. It required NAD+ as cofactor and could not use NADP+. Maximum dehydrogenase activity with valine was at pH 10.5 and the maximum reductive amination activity with 2-oxoisovaleric acid and NH4Cl was at pH 9. The enzyme exhibited substrate inhibition in the forward direction and a kinetic pattern with NAD+ that was consistent with a sequential ordered mechanism with non-competitive inhibition by valine. The following Michaelis constants were calculated from these data: L-valine, 10.0 mM; NAD+, 0.17 mM; 2-oxoisovalerate, 0.6 mM; and NADH, 0.093 mM. In minimal medium, VDH activity was repressed in the presence of glucose and NH4+, or glycerol and NH4+ or asparagine, and was induced by D- and L-valine. The time required for full induction was about 24 h and the level of induction was 2- to 23-fold.     

Valine dehydrogenase from Streptomyces fradiae: purification and properties

Valine dehydrogenase (VDH) was purified to homogeneity from cell-free extract of Streptomyces fradiae, which produces tylosin. The enzyme was purified 1508-fold in a 17.7% yield using a combination of hydrophobic chromatography and ion-exchange fast protein liquid chromatography. The Mr of the native enzyme was determined to be 218,000 and 215,000, by equilibrium ultracentrifugation and size-exclusion high-performance liquid chromatography, respectively. The enzyme is composed of 12 subunits of Mr 18,000. Using analytical isoelectric focusing the isoelectric point of VDH was found to be 4.7. Oxidative deamination of L-valine was optimal at pH 10.6. Reductive amination of 2-oxoisovalerate was optimal at pH 8.8. The Michaelis constants (Km) were 1 mM for L-valine and 0.029 mM for NAD+. Km values for reductive amination were 0.80 mM for 2-oxoisovalerate, 0.050 mM for NADH and 22 mM for NH4+.     

Potato tuber succinate semialdehyde dehydrogenase: purification and characterization

Succinate semialdehyde dehydrogenase (SSADH) has been purified from potato tubers with 39% yield, 832-fold purification, and a specific activity of 6.5 units/mg protein. The final preparation was homogeneous as judged from native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel filtration on Sepharose 6B gave a relative molecular mass (Mr) of 145,000 for the native enzyme. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave a single polypeptide band of Mr 35,000. Thus the enzyme appears to be a tetramer of identical subunits. Chromatofocusing of the enzyme gave a pI of 8.7. The enzyme was maximally active at pH 9.0 in 100 mM sodium pyrophosphate buffer. In 100 mM Tris-HCl buffer, pH 9.0, the enzyme gave only 20% of the activity found in pyrophosphate buffer and had a shorter linear rate. The enzyme was specific for succinate semialdehyde (SSA) as substrate and could not utilize acetaldehyde, glyceraldehyde 3-phosphate, malonaldehyde, lactate, or ethanol as substrates. The enzyme was also specific for NAD+ as cofactor and NADP+ and 3-acetylpyridine adenine dinucleotide could not serve as cofactors. Potato SSADH had a Km of 4.6 microM for SSA when assayed in pyrophosphate buffer and was inhibited by that substrate at concentrations greater than 120 microM. The Km for NAD+ was found to be 31 microM. The enzyme required exogenous addition of a thiol compound for maximal activity and was inhibited by the thiol-directed reagents p-hydroxymercuribenzoate, dithionitrobenzoate, and N-ethyl-maleimide, by heavy metal ions Hg2+, Cu2+, Cd2+, and Zn2+, and by arsenite. These results indicate a requirement of a SH group for catalytic activity.     

The missing link in the fungal L-arabinose catabolic pathway,identification of the L-xylulose reductase gene

The fungal L-arabinose pathway consists of five enzymes, aldose reductase, L-arabinitol 4-dehydrogenase, L-xylulose reductase, xylitol dehydrogenase, and xylulokinase. All the genes encoding the enzymes of this pathway are known except for that of L-xylulose reductase (EC 1.1.1.10). We identified a gene encoding this enzyme from the filamentous fungus Trichoderma reesei (Hypocrea jecorina). The gene was named lxr1. It was overexpressed in the yeast Saccharomyces cerevisiae, and the enzyme activity was confirmed in a yeast cell extract. Overexpression of all enzymes of the L-arabinose pathway in S. cerevisiae led to growth of S. cerevisiae on L-arabinose; i.e., we could show that the pathway is active in a heterologous host. The lxr1 gene encoded a protein with 266 amino acids and a calculated molecular mass of 28 428 Da. The LXRI protein is an NADPH-specific reductase. It has activity with L-xylulose, D-xylulose, D-fructose, and L-sorbose. The highest affinity is toward L-xylulose (K(m) = 16 mM). In the reverse direction, we found activity with xylitol, D-arabinitol, D-mannitol, and D-sorbitol. It requires a bivalent cation for activity. It belongs to the protein family of short chain dehydrogenases. The enzyme is catalytically similar and homologous in sequence to a D-mannitol:NADP 2-dehydrogenase (EC 1.1.1.138).     

Purification and properties of polyol dehydrogenase from Cephalosporium chrysogenus.

A polyol dehydrogenase of broad specificity was purified 178-fold from extracts of the filamentous fungus Cephalosporium chrysogenum. The enzyme was found to act as an oxido-reductase in two substrate-coenzyme systems: D-sorbitol (or xylitol)-nicotinamide-adenine dinucleotide (NAD) and D-mannitol-nicotinamide adenine dinucleotide phosphate (NADP). The dehydrogenase was composed of five isozymes, which, as a mixture, exhibited these properties: Km to D-sorbitol and D-mannitol, 7.15 to 10(-2) M; PH optimum, 9 to 10; molecular weight, 300,000 subunit weight, 29,000; PI, 5.8 to 7.5. The NADP-linked activity was labile to treatment with heat or ethylenediaminetetraacetic acid. Mixed substrate assays support the hypothesis that both NAD-, and NADP-linked activities are associated with isozymes of a single dehydrogenase.     

Purification and characterization of a novel form of 20 alpha-hydroxysteroid dehydrogenase from Clostridium scindens.

We have purified a steroid-inducible 20 alpha-hydroxysteroid dehydrogenase from Clostridium scindens to apparent homogeneity. The final enzyme preparation was purified 252-fold, with a recovery of 14%. Denaturing and nondenaturing polyacrylamide gradient gel electrophoresis showed that the native enzyme (Mr, 162,000) was a tetramer composed of subunits with a molecular weight of 40,000. The isoelectric point was approximately pH 6.1. The purified enzyme was highly specific for adrenocorticosteroid substrates possessing 17 alpha, 21-dihydroxy groups. The purified enzyme had high specific activity for the reduction of cortisone (Vmax, 280 nmol/min per mg of protein; Km, 22 microM) but was less reactive with cortisol (Vmax, 120 nmol/min per mg of protein; Km, 32 microM) at pH 6.3. The apparent Km for NADH was 8.1 microM with cortisone (50 microM) as the cosubstrate. Substrate inhibition was observed with concentrations of NADH greater than 0.1 mM. The purified enzyme also catalyzed the oxidation of 20 alpha-dihydrocortisol (Vmax, 200 nmol/min per mg of protein; Km, 41 microM) at pH 7.9. The apparent Km for NAD+ was 526 microM. The initial reaction velocities with NADPH were less than 50% of those with NADH. The amino-terminal sequence was determined to be Ala-Val-Lys-Val-Ala-Ile-Asn-Gly-Phe-Gly-Arg. These results indicate that this enzyme is a novel form of 20 alpha-hydroxysteroid dehydrogenase.     

Purification and properties of NADP-dependent glutamate dehydrogenase from Streptomyces fradiae

Streptomyces fradiae has two chromatographically distinct forms of glutamate dehydrogenase (GDH): one GDH utilizes NAD as coenzyme, the other uses NADP. The intracellular level of both GDHs is strongly regulated by the nitrogen source in the growth medium. NADP-dependent GDH was purified to homogeneity from crude extracts of S. fradiae. The Mr of the native enzyme was determined to be 200,000 by size-exclusion high-performance liquid chromatography whereas after sodium dodecyl sulphate-polyacrylamide gel electrophoresis one major band of Mr 49,000 was found, suggesting that the enzyme is a tetramer. The enzyme was highly specific for the substrates 2-oxoglutarate and L-glutamate, and required NADP, which could not be replaced by NAD, as a cofactor. The pH optimum was 9.2 for oxidative deamination of glutamate and 8.4 for reductive amination of 2-oxoglutarate. The Michaelis constants (Km) were 28.6 mM for L-glutamate and 0.12 mM for NADP. Km values for reductive amination were 1.54 mM for 2-oxoglutarate, 0.07 mM for NADPH and 30.8 mM for NH+4. The enzyme activity was significantly reduced by adenine nucleotides, particularly ATP.     

Partial purification, substrate specificity and regulation of alpha-L-glycerolphosphate dehydrogenase from Saccharomyces carlsbergensis.

alpha-L-Glycerolphosphate dehydrogenase (sn-glycerol-3-phosphate:NAD+ 2-oxidoreductase, EC 1.1.1.8) from Saccharomyces carlsbergensis was purified 400-fold. The enzyme preparation is free of interfering activities, such as glyceraldehyde phosphate dehydrogenase, alcohol dehydrogenase, triose phosphate isomerase and glycerolphosphatase. At pH 7.0 it is specific for NADH (Km = 0.027 mM with 0.8 mM dihydroxyacetone phosphate) and dihydroxyacetone phosphate (Km = 0.2 mM with 0.2 mM NADH). Between pH 5.0 and 6.0 the enzyme functions with NADPH, but only at 7% of the rate with NADH. Various anions (I- greater than SO42- greater than Br- greater than Cl-) act as inhibitors competing with the substrate dihydroxyacetone phosphate. Inorganic phosphate (Ki = 0.1 mM), pyrophosphate and arsenate are strong inhibitors. The nucleotides ATP and ADP are also inhibitory, but their action seems to be of the same type as the general anion competition (Ki = 0.73 mM for ATP). The results are consistent with the notion that the enzyme may regulate the redox potential of the NAD+/NADH couple during fermentation.     

Purification and characterization of benzaldehyde dehydrogenase I from Acinetobacter calcoaceticus.

Benzaldehyde dehydrogenase I was purified from Acinetobacter calcoaceticus by DEAE-Sephacel, phenyl-Sepharose and f.p.l.c. gel-filtration chromatography. The enzyme was homogeneous and completely free from the isofunctional enzyme benzaldehyde dehydrogenase II, as judged by denaturing and non-denaturing polyacrylamide-gel electrophoresis. The subunit Mr value was 56,000 (determined by SDS/polyacrylamide-gel electrophoresis). Estimations of the native Mr value by gel-filtration chromatography gave values of 141,000 with a f.p.l.c. Superose 6 column, but 219,000 with Sephacryl S300. Chemical cross-linking of the enzyme subunits indicated that the enzyme is tetrameric. Benzaldehyde dehydrogenase I was activated more than 100-fold by K+, Rb+ and NH4+, and the apparent Km for K+ was 11.2 mM. The pH optimum in the presence of K+ was 9.5 and the pI of the enzyme was 5.55. The apparent Km values for benzaldehyde and NAD+ were 0.69 microM and 96 microM respectively, and the maximum velocity was approx. 110 mumol/min per mg of protein. Various substituted benzaldehydes were oxidized at significant rates, and NADP+ was also used as cofactor, although much less effectively than NAD+. Benzaldehyde dehydrogenase I had an NAD+-activated esterase activity with 4-nitrophenol acetate as substrate, and the dehydrogenase activity was inhibited by a range of thiol-blocking reagents. The absorption spectrum indicated that there was no bound cofactor or prosthetic group. Some of the properties of the enzyme are compared with those of other aldehyde dehydrogenases, specifically the very similar isofunctional enzyme benzaldehyde dehydrogenase II from the same organism.