Purification and catalytic properties of L-valine dehydrogenase from Streptomyces cinnamonensis.

NAD+-dependent L-valine dehydrogenase was purified 180-fold from Streptomyces cinnamonensis, and to homogeneity, as judged by gel electrophoresis. The enzyme has an Mr of 88,000, and appears to be composed of subunits of Mr 41,200. The enzyme catalyses the oxidative deamination of L-valine, L-leucine, L-2-aminobutyric acid, L-norvaline and L-isoleucine, as well as the reductive amination of their 2-oxo analogues. The enzyme requires NAD+ as the only cofactor, which cannot be replaced by NADP+. The enzyme activity is significantly decreased by thiol-reactive reagents, although purine and pyrimidine bases, and nucleotides, do not affect activity. Initial-velocity and product-inhibition studies show that the reductive amination proceeds through a sequential ordered ternary-binary mechanism; NADH binds to the enzyme first, followed by 2-oxoisovalerate and NH3, and valine is released first, followed by NAD+. The Michaelis constants are as follows; L-valine, 1.3 mM; NAD+, 0.18 mM; NADH, 74 microM; 2-oxoisovalerate, 0.81 mM; and NH3, 55 mM. The pro-S hydrogen at C-4' of NADH is transferred to the substrate; the enzyme is B-stereospecific. It is proposed that the enzyme catalyses the first step of valine catabolism in this organism.     

Alanine dehydrogenase from Streptomyces fradiae. Purification and properties

Alanine dehydrogenase was purified to homogeneity from a cell-free extract of Streptomyces fradiae, which produces tylosin. The enzyme was purified 1180-fold to give a 21% yield, using a combination of hydrophobic chromatography and ion-exchange fast protein liquid chromatography. The relative molecular mass of the native enzyme was determined to be 210,000 or 205,000 by equilibrium ultracentrifugation or gel filtration, respectively. The enzyme is composed of four subunits, each of Mr 51,000. Using analytical isoelectric focusing the isoelectric point of alanine dehydrogenase was found to be 6.1. The Km were 10.0 mM for L-alanine and 0.18 mM for NAD+. Km values for reductive amination were 0.23 mM for pyruvate, 11.6 mM for NH4+ and 0.05 mM for NADH. Oxidative deamination of L-alanine proceeds through a sequential-ordered binary-ternary mechanism in which NAD+ binds first to the enzyme, followed by alanine, and products are released in the order ammonia, pyruvate and NADH.     

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.     

Purification and characterization of 3-hydroxyisobutyrate dehydrogenase from rabbit liver

3-Hydroxyisobutyrate dehydrogenase (3-hydroxy-2-methyl propanoate: NAD+ oxidoreductase, EC 1.1.1.31) was purified 1800-fold from rabbit liver by detergent extraction, differential solubility in polyethylene glycol and (NH4)2SO4, and column chromatography on DEAE-Sephacel, phenyl-Sepharose, CM(carboxymethyl)-Sepharose, Affi-Gel Blue, and Ultrogel AcA-34. The enzyme had a native Mr of 74,000 and appeared to be a homodimer with subunit Mr = 34,000. The enzyme was specific for NAD+. It oxidized both S-3-hydroxyisobutyrate and R-3-hydroxyisobutyrate, but the kcat/Km was approximately 350-fold higher for the S-isomer. Steady state kinetic analysis indicates an ordered Bi Bi reaction mechanism with NAD+ binding before 3-hydroxyisobutyrate. The enzyme catalyzed oxidation of S-3-hydroxyisobutyrate between pH 7.0 and 11.5 with optimal activity between pH 9.0 and 11.0. The enzyme apparently does not have a metal ion requirement. Essential sulfhydryl groups may be present at both the 3-hydroxyisobutyrate and NAD+ binding sites since inhibition by sulfhydryl-binding agents was differentially blocked by each substrate. The enzyme is highly sensitive to product inhibition by NADH which may play an important physiological role in regulating the complete oxidation of valine beyond the formation of 3-hydroxyisobutyrate.     

Purification and properties of an extreme thermostable glutamate dehydrogenase from the archaebacterium Sulfolobus solfataricus

Glutamate dehydrogenase (L-glutamate:NAD(P)+ oxidoreductase, deaminating, EC 1.4.1.3.) of the extreme thermophilic archaebacterium Sulfolobus solfataricus was purified to homogeneity by (NH4)2SO4 fractionation, anion-exchange chromatography and affinity chromatography on 5'-AMP-Sepharose. The purified native enzyme had a Mr of about 270,000 and was shown to be a hexamer of subunit Mr of 44,000. It was active from 30 to 95 degrees C, with a maximum activity at 85 degrees C. No significant loss of enzyme activity could be detected, either after incubation of the purified enzyme at 90 degrees C for 60 min, or in the presence of 4 M urea or 0.1% SDS. The enzyme was catalytically active with both NADH and NADPH as coenzyme and was specific for 2-oxoglutarate and L-glutamate as substrates. With respect to coenzyme utilization the Sulfolobus solfataricus glutamate dehydrogenase resembled more closely the equivalent enzymes from eukaryotic organisms than those from eubacteria.     

Structure and properties of malic enzyme from Bacillus stearothermophilus

The malic enzyme (EC 1.1.1.38) gene of Bacillus stearothermophilus was cloned in Escherichia coli, and the enzyme was purified to homogeneity from the E. coli clone. In addition to the NAD(P)-dependent oxidative decarboxylation of L-malate, the enzyme catalyzes the decarboxylation of oxalacetate. The enzyme is a tetramer of Mr 200,000 consisting of four identical subunits of Mr 50,000. The pH optima for malate oxidation and pyruvate reduction are 8.0 and 6.0, respectively; and the optimum temperature is 55 degrees C. The enzyme strictly requires divalent metal cations for its activity, and the activity is enhanced 5-7 times by NH4+ and K+. Kinetic study shows that the values of the dissociation constant of the enzyme-coenzyme complex are 77 microM for NAD and 1.0 mM for NADP, indicating that the enzyme has a higher affinity for NAD than for NADP. The nucleotide sequence of the gene and its flanking regions was also found. A single open reading frame of 1434 base pairs encoding 478 amino acids was concluded to be that for the malic enzyme gene because the amino acid composition of the enzyme and the sequence of 16 amino acids from the amino terminus of the enzyme agreed well with those deduced from this open reading frame.     

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.     

Purification and properties of alanine dehydrogenase from Bacillus sphaericus.

1. The bacterial distribution of alanine dehydrogenase (L-alanine:NAD+ oxidoreductase, deaminating, EC 1.4.1.1) was investigated, and high activity was found in Bacillus species. The enzyme has been purified to homogeneity and crystallized from B. sphaericus (IFO 3525), in which the highest activity occurs. 2. The enzyme has a molecular weight of about 230 000, and is composed of six identical subunits (Mr 38 000). 3. The enzyme acts almost specifically on L-alanine, but shows low amino-acceptor specificity; pyruvate and 2-oxobutyrate are the most preferable substrates, and 2-oxovalerate is also animated. The enzyme requires NAD+ as a cofactor, which cannot be replaced by NADP+. 4. The enzyme is stable over a wide pH range (pH 6.0--10.0), and shows maximum reactivity at approximately pH 10.5 and 9.0 for the deamination and amination reactions, respectively. 5. Alanine dehydrogenase is inhibited significantly by HgCl2, p-chloromercuribenzoate and other metals, but none of purine and pyrimidine bases, nucleosides, nucleotides, flavine compounds and pyridoxal 5'-phosphate influence the activity. 6. The reductive amination proceeds through a sequential ordered ternary-binary mechanism. NADH binds first to the enzyme followed by ammonia and pyruvate, and the products are released in the order of L-ALANINE AND NAD+. The Michaelis constants are as follows: NADH (10 microM), ammonia (28.2 mM), pyruvate (1.7 mM), L-alanine (18.9 mM) and NAD+ (0.23 mM). 7. The pro-R hydrogen at C-4 of the reduced nicotinamide ring of NADH is exclusively transferred to pyruvate; the enzyme is A-stereospecific.     

Stable ammonia-specific NAD synthetase from Bacillus stearothermophilus: purification,characterization, gene cloning,and applications

Bacillus stearothermophilus H-804 isolated from a hot spring in Beppu, Japan, produced an ammonia-specific NAD synthetase (EC 6.3.1.5). The enzyme specifically used NH3 as an amide donor for the synthesis of NAD as it formed AMP and pyrophosphate from deamide-NAD and ATP. None of the l-amino acids tested, such as l-asparagine or l-glutamine, or other amino compounds such as urea, uric acid, or creatinine was used instead of NH3. Mg2+ was needed for the activity, and the maximum enzyme activity was obtained with 3 mM MgCl2. The molecular mass of the native enzyme was 50 kDa by gel filtration, and SDS-PAGE showed a single protein band at the molecular mass of 25 kDa. The optimum pH and temperature for the activity were from 9.0 to 10.0 and 60 degrees C, respectively. The enzyme was stable at a pH range of 7.5 to 9.0 and up to 60 degrees C. The Km for NH3, ATP, and deamide-NAD were 0.91, 0.052, and 0.028 mM, respectively. The gene encoding the enzyme consisted of an open reading frame of 738 bp and encoded a protein of 246 amino acid residues. The deduced amino acid sequence of the gene had about 32% homology to those of Escherichia coli and Bacillus subtilis NAD synthetases. We caused the NAD synthetase gene to be expressed in E. coli at a high level; the enzyme activity (per liter of medium) produced by the recombinant E. coli was 180-fold that of B. stearothermophilus H-804. The specific assay of ammonia and ATP (up to 25 microM) with this stable NAD synthetase was possible.     

Enzymes of vitamin B6 degradation. Purification and properties of isopyridoxal dehydrogenase and 5-formyl-3-hydroxy-2-methylpyridine-4-carboxylic-acid dehydrogenase

Two NAD+-dependent, highly specific pyridine-5-aldehyde dehydrogenases, 5-formyl-3-hydroxy-2-methylpyridine-4-carboxylic-acid (Compound 1) dehydrogenase and isopyridoxal dehydrogenase, were purified to homogeneity from Pseudomonas MA-1 and Arthrobacter Cr-7, respectively. Both enzymes are induced in response to growth of the organisms on pyridoxine and catalyze steps in the degradation of this compound by these organisms. Compound 1 dehydrogenase (Mr = 65,000) contains two subunits of equal size with methionine as the NH2-terminal amino acid and acts optimally at pH 7.8-8.5. It catalyzes with equal facility (turnover number = 400-670 s-1 molecule-1) both the oxidation of Compound 1 (Km = 65 microM) by NAD+ (Km = 25 microM) to 3-hydroxy-2-methylpyridine-4,5-dicarboxylic acid and the reduction of Compound 1 by NADH (Km = 20 microM) to 4-pyridoxic acid and appears to act as a true dismutase. The possible advantage to the organism of its ability to act as a dismutase is discussed briefly. No oxidation of 4-pyridoxic acid by this enzyme was observed. Isopyridoxal dehydrogenase (Mr = 242,000) contains four subunits of equal size, again with methionine at the NH2 terminus. At its optimal pH of 8.0-8.6, it catalyzes the oxidation of isopyridoxal (Km = 40 microM, turnover number = 10 s-1 molecule-1) by NAD+ (Km = 40 microM) to a mixture of 5-pyridoxic acid and 5-pyridoxolactone, which are produced in constant ratio throughout the course of the reaction. Formation of the two products, although unusual, is readily understandable in terms of the structure of isopyridoxal in solution or the structure of a possible acyl-enzyme intermediate in the oxidative reaction.     

Stereochemistry of the hydrogen transfer to NAD catalyzed by (S)alanine dehydrogenase from Bacillus subtilis.

The stereochemistry of the hydrogen transfer to NAD catalyzed by (S)alanine dehydrogenase [ (S)alanine: NAD oxidoreductase (EC 1.4.1.1) ] from B. subtilis was investigated. The label at C-2 of (S) [2,3--3H] alanine was enzymatically transferred to NAD, and the [4--3H]NADH produced isolated and the stereochemistry at C-4 investigated. It was found that the label was exclusively located at the (R) position which indicates that (S)alanine dehydrogenase is an A-type enzyme. This result was confirmed in an alternate way by reducing enzymatically [4--3H]NAD with non labeled (S)alanine and (S)alanine dehydrogenase and investigating the stereochemistry of the ]4--3H]NADH produced. As expected, the label was now exclusively located at the (S) position. This proves that (S)alanine dehydrogenase isolated from B. subtilis should be classified as an A-enzyme with regard to the stereochemistry of the hydrogen transfer to NAD.     

Formate dehydrogenase from the methane oxidizer Methylosinus trichosporium OB3b.

Formate dehydrogenase (NAD+ dependent) was isolated from the obligate methanotroph Methylosinus trichosporium OB3b. When the enzyme was isolated anaerobically, two forms of the enzyme were seen on native polyacrylamide gels, DE-52 cellulose and Sephacryl S-300 columns; they were approximately 315,000 and 155,000 daltons. The enzyme showed two subunits on sodium dodecyl sulfate-polyacrylamide gels. The Mr of the alpha-subunit was 53,800 +/- 2,800, and that of the beta-subunit was 102,600 +/- 3,900. The enzyme (Mr 315,000) was composed of these subunits in an apparent alpha 2 beta 2 arrangement. Nonheme iron was present at a concentration ranging from 11 to 18 g-atoms per mol of enzyme (Mr 315,000). Similar levels of acid-labile sulfide were detected. No other metals were found in stoichiometric amounts. When the enzyme was isolated aerobically, there was no cofactor requirement for NAD reduction; however, when isolated anaerobically, activity was 80 to 90% dependent on the addition of flavin mononucleotide (FMN) to the reaction mixture. Furthermore, the addition of formate to an active, anoxic solution of formate dehydrogenase rapidly inactivated it in the absence of an electron acceptor; this activity could be reconstituted approximately 85% by 50 nM FMN. Flavin adenine dinucleotide could not replace FMN in reconstituting enzyme activity. The Kms of formate dehydrogenase for formate, NAD, and FMN were 146, 200, and 0.02 microM, respectively. "Pseudomonas oxalaticus" formate dehydrogenase, which has physical characteristics nearly identical to those of the M. trichosporium enzyme, was also shown to be inactivated under anoxic conditions by formate and reactivated by FMN. The evolutionary significance of this similarity is discussed.     

Ammonia assimilation and synthesis of alanine, aspartate, and glutamate in Methanosarcina barkeri and Methanobacterium thermoautotrophicum

The mechanism of ammonia assimilation in Methanosarcina barkeri and Methanobacterium thermoautotrophicum was documented by analysis of enzyme activities, 13NH3 incorporation studies, and comparison of growth and enzyme activity levels in continuous culture. Glutamate accounted for 65 and 52% of the total amino acids in the soluble pools of M. barkeri and M. thermoautotrophicum. Both organisms contained significant activities of glutamine synthetase, glutamate synthase, glutamate oxaloacetate transaminase, and glutamate pyruvate transaminase. Hydrogen-reduced deazaflavin-factor 420 or flavin mononucleotide but not NAD, NADP, or ferredoxin was used as the electron donor for glutamate synthase in M. barkeri. Glutamate dehydrogenase activity was not detected in either organism, but alanine dehydrogenase activity was present in M. thermoautotrophicum. The in vivo activity of the glutamine synthetase was verified in M. thermoautotrophicum by analysis of 13NH3 incorporation into glutamine, glutamate, and alanine. Alanine dehydrogenase and glutamine synthetase activity varied in response to [NH4+] when M. thermoautotrophicum was cultured in a chemostat with cysteine as the sulfur source. Alanine dehydrogenase activity and growth yield (grams of cells/mole of methane) were highest when the organism was cultured with excess ammonia, whereas growth yield was lower and glutamine synthetase was maximal when ammonia was limiting.     

The novel disulfide reductase bis-gamma-glutamylcystine reductase and dihydrolipoamide dehydrogenase from Halobacterium halobium: purification by immobilized-metal-ion affinity chromatography and properties of the enzymes.

An NADPH-specific disulfide reductase that is active with bis-gamma-glutamylcystine has been purified 1,900-fold from Halobacterium halobium to yield a homogeneous preparation of the enzyme. Purification of this novel reductase, designated bis-gamma-glutamylcystine reductase (GCR), and purification of halobacterial dihydrolipoamide dehydrogenase (DLD) were accomplished with the aid of immobilized-metal-ion affinity chromatography in high-salt buffers. Chromatography of GCR on immobilized Cu2+ resin in buffer containing 1.23 M (NH4)2SO4 and on immobilized Ni2+ resin in buffer containing 4.0 M NaCl together effected a 120-fold increase in purity. Native GCR was found to be a dimeric flavoprotein of Mr 122,000 and to be more stable to heat when in buffer of very high ionic strength. DLD was chromatographed on columns of immobilized Cu2+ resin in buffer containing NaCl and in buffer containing (NH4)2SO4, the elution of DLD differing markedly in the two buffers. Purified DLD was found to be a heat-stable, dimeric flavoprotein of Mr 120,000 and to be very specific for NAD. The utility of immobilized-metal-ion affinity chromatography for the purification of halobacterial enzymes and the likely cellular function of GCR are discussed.     

Identity of alanine:glyoxylate aminotransferase with alanine:2-oxoglutarate aminotransferase in rat liver cytosol

Rat liver soluble fraction contained 3 forms of alanine: glyoxylate aminotransferase. One with a pI of 5.2 and an Mr of approx. 110,000 was found to be identical with cytosolic alanine:2-oxoglutarate aminotransferase. The pI 6.0 enzyme with an Mr of approx. 220,000 was suggested to be from broken mitochondrial alanine:glyoxylate aminotransferase 2 and the pI 8.0 enzyme with an Mr of approx. 80,000 enzyme from broken peroxisomal and mitochondrial alanine:glyoxylate aminotransferase 1. These results suggest that the cytosolic alanine: glyoxylate aminotransferase activity is due to cytosolic alanine: 2-oxoglutarate aminotransferase.     

Purification and characterization of a paired basic residue-specific pro-opiomelanocortin converting enzyme from bovine pituitary intermediate lobe secretory vesicles

Pro-opiomelanocortin (adrenocorticotropin/endorphin prohormone) is processed to yield active hormones by cleavages at paired basic amino acid residues. In this study, an enzyme that specifically cleaves at the paired basic residues of this prohormone has been purified from bovine pituitary intermediate lobe secretory vesicles, the intracellular processing site of proopiomelanocortin. This enzyme, named pro-opiomelanocortin converting enzyme, has been characterized as a glycoprotein of Mr approximately 70,000. It has an apparent isoelectric point between 3.5 and 4.0. The pH optimum of the pro-opiomelanocortin converting enzyme is between 4 and 5, but the enzyme is highly active at the intravesicular pH of 5.1-5.6. The enzyme specifically cleaved the Lys-Arg pairs of pro-opiomelanocortin to yield Mr = to 21,000-23,000 ACTH, beta-lipotropin, Mr 13,000 and 4,500 ACTH, beta-endorphin, and a Mr = 16,000 NH2-terminal glycopeptide, the products synthesized by the pituitary intermediate lobe in situ. NH2- and COOH-terminal analysis of the products indicated that the pro-opiomelanocortin converting enzyme cleaves the peptide bond either between the Lys and Arg or on the carboxyl side of the Arg at Lys-Arg pairs of pro-opiomelanocortin. The intracellular localization, pH optimum, and cleavage specificity of the enzyme suggest that it may function as a pro-opiomelanocortin processing enzyme in the pituitary intermediate lobe in vivo.     

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+.     

An extracellular monoADP-ribosyl transferase activity in Entamoeba histolytica trophozoites

Due to the important role of monoADP-ribosyl transferases in physiological and pathological events, we investigated whether the protozoan parasite Entamoeba histolytica had monoADP-ribosyl transferase activity. Reactions were initiated using ameba-free medium as the source of both enzyme and ADP-ribosylation substrate(s) and [32P]NAD+ as source of ADP-ribose. Proteins were analyzed by electrophoresis, and [32P]-labeled proteins were detected by autoradiography. Using the crude extracellular medium, a major labeled product of Mr 37.000 was observed. The yield of this product was reduced markedly using medium from Brefeldin A-treated trophozoites, indicating that the extracellular monoADP-ribosyl transferase and/or its substrate depended on vesicular transport. The labeling of the 37-kDa substrate was dependent on reaction time, temperature, pH, and the ratio of unlabeled NAD+ to [32P]NAD+. After two purification steps, several new substrates were observed, perhaps due to their enrichment. The reaction measured ADP-ribosylation since [14C-carbonyl]NAD+ was not incorporated into ameba substrates and a 75-fold molar excess of ADP-ribose caused no detectable inhibition of the monoADP-ribosyl transferase reaction. On the basis of sensitivity to NH2OH, the extracellular monoADP-ribosyl transferase of E. histolytica may be an arginine-specific enzyme. These results demonstrate the existence in E. histolytica of at least one extracellular monoADP-ribosyl transferase, whose localization depends upon a secretion process.     

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.     

CO oxidoreductase from Streptomyces strain G26 is a molybdenum hydroxylase.

CO oxidoreductase was purified to 95% homogeneity from crude mycelial extracts of Streptomyces G26. The purified preparation has a specific activity of 25.7 units/mg, a 13-fold improvement on crude soluble mycelial extracts. The native enzyme (Mr 282,000) is composed of non-identical subunits of Mr 110,000 and 33,000. It is a molybdenum hydroxylase containing 1.6 mol of FAD, 7.3 mol of Fe, 8.3 mol of acid-labile sulphide and 1.3 mol of Mo per mol of enzyme. Purified CO oxidoreductase catalyses the reduction of benzyl viologen, confirming the previously reported ability of this enzyme to interact with low-potential acceptors. Cytochrome c reduction cannot be accounted for entirely by non-enzymic reduction by superoxide radicals. NAD+ and NADP+ are not reduced, nor is clostridial ferredoxin.