Effect of chain length on the activity of free and immobilized alcohol dehydrogenase towards aliphatic alcohols

As long-chain alcohol dehydrogenases are not easily available and seldom reported enzymes, it is worthwhile to appraise the potential of well known dehydrogenases, like horse liver alcohol dehydrogenases (HLAD), for the oxidation of long-chain aliphatic alcohols. Oxidation of docosanol (C₂₂) and tetracosanol (C₂₄) is of technological relevance within an industrial platform for the fractionation and upgrading of tall-oil from the Kraft pulping process. Results are presented on the characterization of free and immobilized HLAD with respect to their potential for oxidizing long-chain aliphatic alcohols. Enzyme activity with respect to chain length and pH is presented. Activity for both free and immobilized HLAD increased with pH up to 8.8, but behavior with respect to chain length varied from one biocatalyst to the other. Even though both biocatalysts were less active towards very long-chain aliphatic alcohols, immobilized HLAD had an activity on docosanol and tetracosanol higher than 50% of the value obtained with ethanol, butanol and octanol, which is encouraging and has not been previously reported. Investigation on thermophilic sources and further immobilization strategies are underway to obtain more active and stable catalysts amenable for working at high temperatures which is quite relevant in this case due to the poor solubility of substrates.     

Purification, characterization, and partial sequence of the glutathione-dependent formaldehyde dehydrogenase from Escherichia coli: a class III alcohol dehydrogenase.

The glutathione-dependent formaldehyde dehydrogenase from Escherichia coli has been purified to homogeneity and characterized. It is a 83,000-kDa homodimer containing 4 g-atom of zinc per dimer with a specific activity of 60 units/mg toward S-(hydroxymethyl)glutathione and NAD+ as substrates. Its isoelectric point, 4.4, is consistent with both its amino acid composition and chromatographic behavior on DEAE HPLC. The N-terminus is unblocked, and 47 residues from the N-terminus were sequenced. A computer search of the Swiss-Prot protein sequence data bank shows that the N-terminal sequence, [sequence; see text], is homologous with the mammalian class III alcohol dehydrogenases with 27 identities when compared to the human enzyme. Like the human, rat, and rabbit enzymes, it has high formaldehyde dehydrogenase activity in the presence of glutathione and catalyzes the oxidation of normal alcohols (ethanol, octanol, 12-hydroxydodecanoate) in a reaction that is not GSH-dependent. In addition, hemithiolacetals other than those formed from GSH, including omega-thiol fatty acids, also are substrates. The wide distribution and high degree of similarity of this enzyme to the plant and animal alcohol dehydrogenases suggest that the E. coli enzyme is closely related to the ancestor of the plant and animal dimeric zinc alcohol dehydrogenases.     

Expression of membrane-bound dehydrogenases from a mother of vinegar metagenome in Gluconobacter oxydans

Acetic acid bacteria are well-known for their membrane-bound dehydrogenases rapidly oxidizing a variety of substrates in the periplasm. Since many acetic acid bacteria have not been successfully cultured in the laboratory yet, studying membrane-bound dehydrogenases directly from a metagenome of vinegar microbiota seems to be a promising way to identify novel variants of these enzymes. To this end, DNA from a mother of vinegar was isolated, sequenced, and screened for membrane-bound dehydrogenases using an in silico approach. Six metagenomic dehydrogenases were successfully expressed using an expression vector with native promoters in the acetic acid bacterium strain Gluconobacter oxydans BP.9, which is devoid of its major native membrane-bound dehydrogenases. Determining the substrates converted by these enzymes, using a whole-cell DCPIP assay, revealed one glucose dehydrogenase with an enlarged substrate spectrum additionally oxidizing aldoheptoses, D-ribose and aldotetroses, one polyol dehydrogenase with an extreme diminished spectrum but distinguishing cis and trans-1,2-cyclohexandiol and a completely new secondary alcohol dehydrogenase, which oxidizes secondary alcohols with a hydroxyl group at position 2, as long as no primary hydroxyl group is present. Three further dehydrogenases were found with substrate spectra similar to known dehydrogenases of G. oxydans 621H.

     

Cytochemical research on cysts of the sarcosporidian Sarcocystis bovicanis. II. Oxidation-reduction enzymes

By the means of light-microscopic cytological enzymatic methods, the presence of several enzymes (NAD.H and NADP.H-tetrazolium reductases, in addition to alcohol, succinate, isocitrate, glucose-6-phosphate, beta-hydroxybutyrate and glutamate dehydrogenases) has been studied in the tissue cysts of S. bovicanis. A mixed character of oxidative metabolism in the cyst stages is suggested, the involvement of gluconeogenesis being proposed. Neither beta-hydroxybutyrate nor alcohol dehydrogenase activity was demonstrated indicating the absence or a very low rate of lipid metabolism, and suggesting that the process of glycolysis may end with malate formation. From the low activity level of succinate dehydrogenases it is concluded that the citric acid cycle plays presumably a secondary role, if at all, in the energy supply of the cyst stages. Also, a low activity of glucose-6-phosphate dehydrogenases is pointed out. Thus, it is proposed that glycolysis may be primary, if not the only, oxidative pathway in the cyst stages.     

Characterization of Proteinases Excreted by Bacillus subtilis Marburg Strain during Sporulation

S ummary . This study has characterized 3 proteolytic enzymes during sporulation by Bacillus subtilis Marburg strain when grown in nutrient broth. A method of purification is described which permits the separation of 2 different proteinases: one belonging to the metal enzyme group and the other to the serine enzyme group. The third enzyme, probably an esterase, showed a high esterolytic activity, but only low proteolytic activity. Determination of the 3 enzymes in a mixture was accomplished by using specific substrates and inhibitors. They were excreted simultaneously between the end of the growth phase until the appearance of the prespores. During this entire period, 20% of the total proteolytic activity was due to the metal proteinase; 80% of the proteolytic activity and 15% of the esterolytic activity was due to the serine proteinase; 85% of the esterolytic activity was the result of the esterase. These findings will contribute to a more complete phenotypic characterization of those mutants of sporulation that appear to be involved in the production of extracellular hydrolytic enzymes.     

Long-chain alcohol and aldehyde dehydrogenase activities in Acinetobacter calcoaceticus strain HO1-N.

Three alcohol dehydrogenases have been identified in Acinetobacter calcoaceticus sp. strain HO1-N: an NAD(+)-dependent enzyme and two NADP(+)-dependent enzymes. One of the NADP(+)-dependent alcohol dehydrogenases was partially purified and was specific for long-chain substrates. With tetradecanol as substrate an apparent Km value of 5.2 microM was calculated. This enzyme has a pI of 4.5 and a molecular mass of 144 kDa. All three alcohol dehydrogenases were constitutively expressed. Three aldehyde dehydrogenases were also identified: an NAD(+)-dependent enzyme, an NADP(+)-dependent enzyme and one which was nucleotide independent. The NAD(+)-dependent enzyme represented only 2% of the total activity and was not studied further. The NADP(+)-dependent enzyme was strongly induced by growth of cells on alkanes and was associated with hydrocarbon vesicles. With tetradecanal as substrate an apparent Km value of 0.2 microM was calculated. The nucleotide-independent aldehyde dehydrogenase could use either Würster's Blue or phenazine methosulphate (PMS) as an artificial electron acceptor. This enzyme represents approximately 80% of the total long-chain aldehyde oxidizing activity within the cell when the enzymes were induced by growing the cells on hexadecane. It is particulate but can be solubilized using Triton X-100. The enzyme has an apparent Km of 0.36 mM for decanal.     

Group III alcohol dehydrogenase from Pectobacterium atrosepticum: insights into enzymatic activity and organization of the metal ion-containing region

NAD(P)⁺-dependent alcohol dehydrogenases (ADH) are widely distributed in all phyla. These proteins can be assigned to three nonhomologous groups of isozymes, with group III being highly diverse with regards to catalytic activity and primary structure. Members of group III ADHs share a conserved stretch of amino acid residues important for cofactor binding and metal ion coordination, while sequence identities for complete proteins are highly diverse (<20 to >90 %). A putative group III ADH PaYqhD has been identified in BLAST analysis from the plant pathogenic enterobacterium Pectobacterium atrosepticum. The PaYqhD gene was expressed in the heterologous host Escherichia coli, and the recombinant protein was purified in a two-step purification procedure to homogeneity indicating an obligate dimerization of monomers. Four conserved amino acid residues involved in metal ion coordination were substituted with alanine, and their importance for catalytic activity was confirmed by circular dichroism spectrum determination, in vitro, and growth experiments. PaYqhD exhibits optimal activity at 40 °C with short carbon chain aldehyde compounds and NADPH as cofactor indicating the enzyme to be an aldehyde reductase. No oxidative activities towards alcoholic compounds were detectable. EDTA completely inhibited catalytic activity and was fully restored by the addition of Co²⁺. Activity measurements together with sequence alignments and structure analysis confirmed that PaYqhD belongs to the butanol dehydrogenase-like enzymes within group III of ADHs.     

Enzymic synthesis of lignin precursors. Further studies on cinnamyl-alcohol dehydrogenase from soybean-cell-suspension cultures

Isoenzyme 2 of cinnamyl-alcohol dehydrogenase from soybean suspension cultures was purified about 3800-fold to apparent homogeneity by an improved purification procedure involving biospecific elution of the enzyme from a NADP+-agarose column. On sodium dodecylsulfate gels the dehydrogenase showed only one protein band with Mr 40 000 +/- 500. The enzyme is strongly inhibited by thiol reagents. Various metal chelators as well as the nonchelating 7,8-benzoquinoline also inhibited enzyme activity. Inhibition by 10 mM 1,10-phenanthroline could be partially reversed by addition of Zn2+. 1,10-Phenanthroline and 7,8-benzoquinoline are non-competitive inhibitors with respect to NADP+. The presence of zinc in the dehydrogenase was proved by atomic absorption spectroscopy and by specific incorporation of 65Zn into the enzyme. In steady-state kinetics inhibition patterns were obtained which are consistent with an ordered bi-bi mechanism in which NADP(H) is the first substrate to bind and the last product released. The cinnamyl-alcohol dehydrogenase belongs to the A-specific dehydrogenases and removes the pro-R hydrogen from coniferyl alcohol. The enzyme shows many similarities with alcohol dehydrogenases from horse and rat liver and from yeast.     

Purification of subtilisin by single-step affinity chromatography

The dye 4-(4-aminophenylazo)phenylarsonic acid was coupled to activated CH-Sepharose 4B and the resulting affinity matrix was shown to be highly efficient for the purification of subtilisin. A crystalline subtilisin was purified to homogeneity using this affinity chromatography procedure with a purification fold of 1.4 and with an enzyme activity yield of 98%. Similarly subtilisin from a crude enzyme preparation was purified to 211 fold by this single step procedure with 94% recovery of the enzyme activity. The purified enzymes were shown to be homogeneous by polyacrylamide gel electrophoresis.     

Purification and kinetic analysis of the two recombinant arogenate dehydrogenase isoforms of Arabidopsis thaliana.

The present study reports the first purification and kinetic characterization of two plant arogenate dehydrogenases (EC 1.3.1.43), an enzyme that catalyses the oxidative decarboxylation of arogenate into tyrosine in presence of NADP. The two Arabidopsis thaliana arogenate dehydrogenases TyrAAT1 and TyrAAT2 were overproduced in Escherichia coli and purified to homogeneity. Biochemical comparison of the two forms revealed that at low substrate concentration TyrAAT1 is four times more efficient in catalyzing the arogenate dehydrogenase reaction than TyrAAT2. Moreover, TyrAAT2 presents a weak prephenate dehydrogenase activity whereas TyrAAT1 does not. The mechanism of the dehydrogenase reaction catalyzed by these two forms has been investigated using steady-state kinetics. For both enzymes, steady-state velocity patterns are consistent with a rapid equilibrium, random mechanism in which two dead-end complexes, E-NADPH-arogenate and E-NADP-tyrosine, are formed.     

Covalent fixation of NAD+ to dehydrogenases and properties of the modified enzymes

Starting from 6-chloropurine riboside and NAD+, different reactive analogues of NAD+ have been obtained by introducing diazoniumaryl or aromatic imidoester groups via flexible spacers into the nonfunctional adenine moiety of the coenzyme. The analogues react with different amino-acid residues of dehydrogenases and form stable amidine or azobridges, respectively. After the formation of a ternary complex by the coenzyme, the enzyme and a pseudosubstrate, the reactive spacer is anchored in the vicinity of the active site. Thus, the coenzyme remains covalently attached to the protein even after decomposition of the complex. On addition of substrates the covalently bound coenzyme is converted to the dihydro-form. In enzymatic tests the modified dehydrogenases show 80-90% of the specific activity of the native enzymes, but they need remarkably higher concentrations of free NAD+ to achieve these values. The dihydro-coenzymes can be reoxidized by oxidizing agents like phenazine methosulfate or by a second enzyme system. Various systems for coenzyme regeneration were investigated; the modified enzymes were lactate dehydrogenase from pig heart and alcohol dehydrogenase from horse liver; the auxiliary enzymes were alcohol dehydrogenase from yeast and liver, lactate dehydrogenase from pig heart, glutamate dehydrogenase and alanine dehydrogenase. Lactate dehydrogenase from heart muscle is inhibited by pyruvate. With alanine dehydrogenase as the auxiliary enzyme, the coenzyme is regenerated and the reaction product, pyruvate, is removed. This system succeeds to convert lactate quantitatively to L-alanine. The thermostability of the binary enzyme systems indicates an interaction of covalently bound coenzymes with both dehydrogenases; both binding sites seem to compete for the coenzyme. The comparison of dehydrogenases with different degrees of modifications shows that product formation mainly depends on the amount of incorporated coenzyme.     

A comparative study of the effect of pyrazole on the activity of enzymes in the alcohol/polyol dehydrogenase family]

A comparative study of the effect of pyrazol, an inhibitor of the coenzyme-binding site of alcohol dehydrogenases, on the activity of enzymes of the alcohol/polyol dehydrogenase group has been carried out. Commercial preparations of alcohol dehydrogenases from the cytoplasm of horse liver cells and yeast cells, as well as the enzyme from the cytoplasm of Trichosporon pullulans cells was completely inhibited by 1 mM pyrazol, while alcohol dehydrogenases from Candida utilis and Saccharomyces carlsbergensis were inhibited only by 25% and the enzymes from Saccharomyces cerevisiae and Torulopsis candida by 30 and 38%, respectively. The inhibition degree of alcohol dehydrogenases from the cytoplasm of liver cells of various mammals (bull, calf, rat, gopher) and birds (hen, pheasant, duck) varied from 12 to 42% in the presence of 1 mM pyrazol. The activity of sorbitol dehydrogenase from the liver cytoplasm of these mammals and birds changed neither in the presence of 1 mM pyrazol, nor in the case of a 15-fold increase of the inhibitor concentration. Possible structural differences in the coenzyme-binding site of the active center of the enzymes under study are discussed.     

Identification of lactaldehyde dehydrogenase and glycolaldehyde dehydrogenase as functions of the same protein in Escherichia coli

Lactaldehyde dehydrogenase is an enzyme involved in the aerobic metabolism of fucose in wild type Escherichia coli, and glycolaldehyde dehydrogenase is an enzyme involved in the metabolism of ethylene glycol in mutant cells able to utilize this glycol. Both enzyme sources display oxidative activity on either substrate with a constant ratio between these activities. We have found that both enzymatic activities present the same electrophoretic mobility when crude extracts were electrophoresed in polyacrylamide gels and the gels stained for enzyme activities. Furthermore, both enzymatic activities co-chromatograph in a DEAE-Sephadex column. If lactaldehyde dehydrogenase of wild type cells is purified near homogeneity and the purification procedure is screened for both aldehydes as substrates, only one enzyme is apparent, giving again a constant ratio between lactaldehyde and glycolaldehyde dehydrogenase activities. Genetic evidence of the fact that both activities are functions of the same protein is provided by the observation that mutation to thermosensitivity for the production of lactaldehyde dehydrogenase affected in the same way the production of glycolaldehyde dehydrogenase. Glycolaldehyde dehydrogenase from mutant cells is purified in a procedure coincident with the lactaldehyde dehydrogenase purification, yielding a single enzyme electrophoretically indistinguishable from the purified lactaldehyde dehydrogenase. Peptide mapping of the purified preparation after digestion with chymotrypsin or Staphylococcus aureus protease V8 gives an indistinguishable band pattern between both enzymes.     

Sex differences in the control of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. Interaction of estrogen, testosterone and insulin in the regulation of enzyme levels in vivo and in cultured hepatocytes

Control of the activities of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and malate dehydrogenase was investigated in intact rats and in hepatocyte cultures. 1) Adult females had 2-fold greater activities of hepatic glucose-6-phosphate- and 6-phosphogluconate dehydrogenases than adult males, but similar activities of malate dehydrogenase. Castrated males showed decreased activities of all three enzymes in comparison to age- and weight-matched intact controls. In starved animals the activities of all three enzymes decreased significantly. After refeeding with nonpurified diet the activities returned to the prestarved levels in females, but increased to clearly higher values in intact and castrated males. 2) Estrogen levels were in the same range in immature and adult male and female rats. Testosterone levels were highest in adult males, clearly lower in adult females (1/8) and immature males (1/8), still lower in immature females (1/15) and lowest in castrated males (1/40). A simple correlation of the sex differences in these hormone levels to sex differences in glucose-6-phosphate- and 6-phosphogluconate dehydrogenase activities was not apparent. 3) In serum-free, dexamethasone-supplemented 48-h cultures of hepatocytes from both male and female rats the basal activities of glucose-6-phosphate dehydrogenase were the same; they were increased 2-3 fold by insulin alone, 1.5 fold by estrogen alone and 4-5 fold by insulin plus estrogen. Apparently sex differences did not persist in 48-h cell cultures. 4) In 48-h cultures of male hepatocytes, then used as the experimental model, insulin alone increased the activity not only of glucose-6-phosphate dehydrogenase but also of 6-phosphogluconate and malate dehydrogenases.(ABSTRACT TRUNCATED AT 250 WORDS)     

Coenzymic activity of NADP derivatives alkylated at 2'-phosphate and 6-amino groups

Coenzymic activities of the following NADP derivatives were investigated: 2'-O-(2-carboxyethyl)phosphono-NAD (I), N6-(2-carboxyethyl)-NADP (II), 2'-O-(2-carboxyethyl)phosphono-N6-(2-carboxyethyl)-NAD (III), 2'-O-[N-(2-aminoethyl)carbamoylethyl]phosphono-NAD (IV), N6-[N-(2-aminoethyl)carbamoylethyl]-NADP (Va), 2',3'-cyclic NADP, and 3'-NADP. Derivatives I and IV show the effects of modification at the 2'-phosphate group, and derivatives II and Va show those at the 6-amino group of NADP. As for enzymes, alcohol, isocitrate, 6-phosphogluconate, glucose, glucose-6-phosphate, and glutamate dehydrogenases were used. These enzymes were grouped on the basis of the ratio of the activities for NAD and NADP into NADP-specific enzymes (ratio less than 0.01), NAD(P)-specific enzymes (0.01 less than ratio less than 100), and NAD-specific enzymes (ratio greater than 100). For NADP-specific enzymes, modifications at the 2'-phosphate group of NADP caused great loss of cofactor activity. The relative cofactor activities (NADP = 100%) of derivatives I and IV for these enzymes were 0.5-20 and 0.01-0.5%, respectively. On the other hand, NAD(P)-specific enzymes showed several types of responses to the NADP derivatives. The relative cofactor activities of I and IV for Leuconostoc mesenteroides and Bacillus stearothermophilus glucose-6-phosphate dehydrogenases and beef liver glutamate dehydrogenase were 60-200%; whereas, for B. megaterium glucose dehydrogenase and L. mesenteroides alcohol dehydrogenase, the values were 0.8-8%. For NAD-specific enzymes, these values were 20-50%. The relative cofactor activities of 2',3'-cyclic NADP and 3'-NADP were very low (less than 0.2%) except for beef liver glutamate dehydrogenase, B. stearothermophilus glucose-6-phosphate dehydrogenase, and horse liver alcohol dehydrogenase. Kinetic studies showed that the losses of the cofactor activity of NADP by these modifications were mainly due to the increase of the Km value. The mechanisms of coenzyme specificity of dehydrogenases are discussed. Unlike the 2'-phosphate group, the 6-amino group is common to NAD and NADP, and the effects of modification at the 6-amino group were independent of the coenzyme specificity of enzymes used for the assay. Derivatives II and Va had high relative cofactor activities (65-130%) for most of the enzymes except for isocitrate and glucose dehydrogenases (less than 1%) and L. mesenteroides alcohol dehydrogenase (20-60%). The cofactor activity of derivative III was generally lower than those of I and II.     

Purification of Alcohol Dehydrogenase fromEntamoeba histolyticaandSaccharomyces cerevisiaeUsing Zinc-Affinity Chromatography

We have developed a single-step method for the purification of NADP⁺-dependent alcohol dehydrogenase fromEntamoeba histolyticaand NAD⁺-dependent alcohol dehydrogenase fromSaccharomyces cerevisiae.It is based on the affinity for zinc of both enzymes. The amebic enzyme was purified almost 800 times with a recovery of 54% and the yeast enzyme was purified 30 times with a recovery of 100%. The kinetic constants of the purified enzymes were similar to those reported for other purification methods. With mammalian alcohol dehydrogenase, we obtained a 40-kDa band suggestive of purified alcohol dehydrogenase, but we failed to retain enzymatic activity in this preparation. Our results suggest that the described method is more applicable to the purification of tetrameric alcohol dehydrogenases.     

Improved enzyme screening by automated fast protein liquid chromatography

The assay for NADH-dependent dehydrogenases in crude extracts is often interfered with non-specific reactions. Therefore a screening for such enzymes is hampered by high blank values. To overcome such problems we chromatographed crude extracts on a fast protein liquid chromatography system during part of an enzyme screening for 2-hydroxyisocaproate dehydrogenases and lactate dehydrogenases. The automated chromatography procedure presented consists of a combination of gel filtration and ion-exchange chromatography. The total time needed to perform one cycle of the two-column purification, including the equilibration and regeneration steps, is about 35 min. The procedure described separates the desired enzyme, 2-hydroxyisocaproate dehydrogenase, totally from any interfering activity such as NADH-oxidase and also from the second enzyme of interest, the lactate dehydrogenase. Besides the elimination of the side reactions the desired enzymes are purified up to 20-fold.     

Merging protein, gene and genomic data: the evolution of the MDR-ADH family

Multiple members of the MDR-ADH (MDR: Medium-chain dehydrogenases/reductases; ADH: alcohol dehydrogenase) family are found in vertebrates, although the enzymes that belong to this family have also been isolated from bacteria, yeast, plant and animal sources. Initial understanding of the physiological roles and evolution of the family relied on biochemical studies, protein alignments and protein structure comparisons. Subsequently, studies at the genetic level yielded new information: the expression pattern, exon-intron distribution, in silico-derived protein sequences and murine knockout phenotypes. More recently, genomic and EST databases have revealed new family members and the chromosomal location and position in the cluster of both the first and new forms. The data now available provide a comprehensive scenario, from which a reliable picture of the evolutionary history of this family can be made.     

Overexpression, purification and characterization of Mycobacterium bovis BCG alcohol dehydrogenase.

A previous study of the effect of zinc deprivation on Mycobacterium bovis BCG pointed out the potential importance of an alcohol dehydrogenase for maintaining the hydrophobic character of the cell envelope. In this report, the effect of the overexpression of the M. bovis BCG alcohol dehydrogenase (ADH) in Mycobacterium smegmatis and M. bovis BCG is described. The purification of the enzyme was performed to apparent homogeneity from overexpressing M. bovis BCG cells and its kinetic parameters were determined. The enzyme showed a strong preference for both aromatic and aliphatic aldehydes while the corresponding alcohols were processed 100-1000-fold less efficiently. The best kcat/Km values were found with benzaldehyde > 3-methoxybenzaldehyde > octanal > coniferaldehyde. A phylogenetic analysis clearly revealed that the M. bovis BCG ADH together with the ADHs from Bacillus subtilis and Helicobacter pylori formed a sister group of the class C medium-chain alcohol dehydrogenases, the plant cinnamyl alcohol dehydrogenases (CADs). Comparison of the kinetic properties of our ADH with some related class C enzymes indicated that the mycobacterial enzyme substrate profile resembled that of the CADs involved in plant defence rather than those implicated in lignification. A possible role for the M. bovis BCG ADH in the biosynthesis of the lipids composing the mycobacterial cell envelope is proposed.     

Purification and properties of malate dehydrogenase from Pseudomonas testosteroni.

Nicotinamide adenine dinucleotide-linked malate dehydrogenase has been purified from Pseudomonas testosteroni (ATCC 11996). The purification represents over 450-fold increase in specific activity. The amino acid composition of the enzyme was determined and found to be quite different from the composition of the malate dehydrogenases from animal sources as well as from Escherichia coli. Despite this difference, however, the data show that the enzymatic properties of the purified enzyme are remarkably similar to those of other malate dehydrogenases that have been previously studied. The Pseudomonas enzyme has a molecular weight of 74,000 and consists of two subunits of identical size. In addition to L-malate, the enzyme slowly oxidizes other four-carbon dicarboylates having an alpha-hydroxyl group of S configuration such as meso- and (-) tartrate. Rate-determining steps, which differ from that of the reaction involving L-malate, are discussed for the reaction involving these alternative substrates. Oxidation of hydroxymalonate, a process previously undetected with other malate dehydrogenases, is demonstrated fluorometrically. Hydroxymalonate and D-malate strongly enhance the fluorescence of the reduced nicotinamide adenine dinucleotide bound to the enzyme. The enzyme is A-stereospecific with respect to the coenzyme. Malate dehydrogenase is present in a single form in the Pseudomonas. The susceptibility of the enzyme to activation or inhibition by its substrates-particularly the favoring of the oxidation of malate at elevated concentrations-strongly resembles the properties of the mitochondrial enzymes. The present study reveals that whereas profound variations in chemical composition have occurred between the prokaryotic and eukaryotic enzymes, the physical and catalytic properties of malate dehydrogenase, unlike lactate dehydrogenase, are well conserved during the evolutionary process.