Aromatizing cyclohexa-1,5-diene-1-carbonyl-coenzyme A oxidase. Characterization and its role in anaerobic aromatic metabolism

Benzoyl-CoA reductases (BCRs) are key enzymes of anaerobic aromatic metabolism in facultatively anaerobic bacteria. The highly oxygen-sensitive enzymes catalyze the ATP-dependent reductive de-aromatization of the substrate, yielding cyclohexa-1,5-diene-1-carbonyl-CoA (1,5-dienoyl-CoA). In extracts from anaerobically grown denitrifying Thauera aromatica, we detected a benzoate-induced, benzoyl-CoA-forming, 1,5-dienoyl-CoA:acceptor oxidoreductase activity. This activity co-purified with BCR but could be partially separated from it by hydroxyapatite chromatography. After activity staining on native gels, a monomeric protein with a subunit molecular weight of M(r) 76,000 was identified. Mass spectrometric analysis of tryptic digests identified peptides from NADH oxidases/2,4-dienoyl-CoA reductases/"old yellow" enzymes. The UV-visible spectrum of the enriched enzyme suggested the presence of flavin and Fe/S-cofactors, and it was bleached upon the addition of 1,5-dienoyl-CoA. The enzyme had a high affinity for dioxygen as electron acceptor (K(m) = 10 microm) and therefore is referred to as 1,5-dienoyl-CoA oxidase (DCO). The likely product formed from dioxygen reduction was H(2)O. DCO was highly specific for 1,5-dienoyl-CoA (K(m) = 27 microm). The initial rate of DCO followed a Nernst curve with half-maximal activity at +10 mV. We propose that DCO provides protection for the extremely oxygen-sensitive BCR enzyme when the bacterium degrades aromatic compounds at the edge of steep oxygen gradients. The redox-dependent switch in DCO guarantees that DCO is only active during oxidative stress and circumvents futile de-aromatization/re-aromatization reactions catalyzed by BCR and DCO.     

Catechol 1,2-dioxygenase from Acinetobacter calcoaceticus: purification and properties.

Procedures for the purification of catechol 1,2-dioxygenase from extracts of Acinetobacter calcoaceticus strain ADP-96 are described. The purified enzyme was homogeneous as judged by ultracentrifugation and acrylamide gel electrophoresis. The enzyme contained 2 g-atoms of iron per mol of protein. The enzyme had a broad substrate specificity and catalyzed the oxidation of catechol, 4-methylcatechol, 3-methylcatechol, and 3-isopropyl catechol. The activity of the enzyme was inhibited by heavy metals, sulfhydryl inhibitors, and substrate analogues. The molecular weight of the enzyme was 85,000 as estimated by filtration on Bio-Gel agarose and 81,000 as estimated by sedimentation equilibrium analysis. The subunit size determined by sodium dodecyl sulfate-gel electrophoresis was 40,000. The amino terminal amino acid was methionine. The amino acid composition and spectral properties of 1,2-dioxygenase are also presented. Antisera prepared against the purified enzyme cross-reacted and inhibited enzyme activity in crude extracts from the other strain of A. calcoaceticus, but failed to cross-react and inhibit isofunctional enzyme from organisms of the genera Pseudomonas, Alcaligenes, and Nocardia.     

A novel NAD-dependent dehydrogenase,highly specific for 1,5-anhydro-D-glucitol,from Trichoderma longibrachiatum strain 11-3

A novel NAD-dependent dehydrogenase highly specific for 1,5-anhydro-D-glucitol (1,5-AG) was found in the cell extract of an imperfect fungus, Trichoderma longibrachiatum strain 11-3. This fungus used 1,5-AG as a sole carbon source for growth and transformed 1,5-AG into glucose. 1,5-AG dehydrogenase (AGH) was purified to homogeneity, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular mass of the purified enzyme was estimated to be 36 and 141 kDa by SDS-PAGE and by gel filtration, respectively, suggesting that the enzyme was homotetrameric. The enzyme was highly specific for 1,5-AG and did not exhibit activity with any sugar or sugar alcohol tested in this study other than 1,5-AG. A linear relationship between the initial rate of the enzyme reaction and the concentration of 1,5-AG at the physiological level was observed. The presence of glucose in abundance did not interfere with the relationship. The optimum temperature for the enzyme reaction was 50 degrees C, and the enzyme was stable at temperatures up to 70 degrees C. These results suggested that AGH is a novel enzyme and is useful for specifically diagnosing diabetes mellitus.     

Reconstitution and characterization of aminopyrrolnitrin oxygenase, a Rieske N-oxygenase that catalyzes unusual arylamine oxidation

Rieske oxygenases catalyze a wide variety of important oxidation reactions. Here we report the characterization of a novel Rieske N-oxygenase, aminopyrrolnitrin oxygenase (PrnD) that catalyzes the unusual oxidation of an arylamine to an arylnitro group. PrnD from Pseudomonas fluorescens Pf5 was functionally expressed in Escherichia coli, and the activity of the purified PrnD was reconstituted, which required in vitro assembly of the Rieske iron-sulfur cluster into the protein and the presence of NADPH, FMN, and an E. coli flavin reductase SsuE. Biochemical and bioinformatics studies indicated that the reconstituted PrnD contains a Rieske iron-sulfur cluster and a mononuclear iron center that are formed by residues Cys(69), Cys(88), His(71), His(91), Asp(323), His(186), and His(191), respectively. The enzyme showed a limited range of substrate specificity and catalyzed the conversion of aminopyrrolnitrin into pyrrolnitrin with K(m) = 191 microM and k(cat) = 6.8 min(-1). Isotope labeling experiments with (18)O(2) and H(2)(18)O suggested that the oxygen atoms in the pyrrolnitrin product are derived exclusively from molecular oxygen. In addition, it was found that the oxygenation of the arylamine substrates catalyzed by PrnD occurs at the enzyme active site and does not involve free radical chain reactions. By analogy to known examples of arylamine oxidation, a catalytic mechanism for the bioconversion of amino pyrrolnitrin into pyrrolnitrin was proposed. Our results should facilitate further mechanistic and crystallographic studies of this arylamine oxygenase and may provide a new enzymatic route for the synthesis of aromatic nitro compounds from their corresponding aromatic amines.     

Properties of the Membrane-Bound Quinoprotein Aldehyde Dehydrogenase from Acetobacter rancens

The n-alkane metabolizing strain Acetobacter rancens CCM 1774 possesses a dye-linked membrane-bound aldehyde dehydrogenase. The application of a sequential solubilization procedure at defined protein-detergent ratios allowed fast and effective purification without loss in enzyme activity. Both forms of aldehyde dehydrogenase—the membrane-bound and the solubilized enzyme— exhibited different properties, such as stability and electron transfer to cyto-chromes. By means of spectrophotometric investigations the presence of heme, FAD and other known groups in the purified enzyme could be excluded. Preliminary investigations with regard to the natural electron acceptor indicated the participation of PQQ in electron transfer. The fluorescence spectrum recorded for methanol extracts of the pure enzyme are comparable with those of adducts of PQQ. Inactivated aldehyde dehydrogenase could be reactivated by addition of these extracts, following saturation kinetics. Both enzyme forms catalyzed the oxidation of straight chain aldehydes, initial activities decreasing with increasing carbon chain length. Kinetic and inhibition experiments excluded a ping-pong mechanism as reported for other PQQ-enzymes. The membrane-bound enzyme should follow a compulsory-order mechanism in which the aldehyde substrate binds first whereas the purified enzyme follows a random-order mechanism.     

Purification and properties of a NADH-dependent 5,10-methylenetetrahydrofolate reductase from Peptostreptococcus productus

The methylenetetrahydrofolate reductase from the carbon-monoxide-utilizing homoacetogen Peptostreptococcus productus (strain Marburg) has been purified to apparent homogeneity. The purified enzyme catalyzed the oxidation of NADH with methylenetetrahydrofolate as the electron acceptor at a specific activity of 380 mumols.min-1 mg protein-1 (37 degrees C; pH 5.5). The apparent Km for NADH was near 10 microM. The apparent molecular mass of the enzyme was determined by gel filtration to be approximately 250.0 kDa. The enzyme consists of eight identical subunits with a molecular mass of 32 kDa. It contains 4 FAD/mol octamer which were reduced by the enzyme with NADH as the electron donor; iron could not be detected. Oxygen had no effect on the enzyme. Ultracentrifugation of cell extracts revealed that about 40% of the enzyme activity was recovered in the particulate fraction, suggesting that the enzyme is associated with the membrane. The enzyme also catalyzed the methylenetetrahydrofolate reduction with methylene blue as an artificial electron donor. The oxidation of methyltetrahydrofolate was mediated with methylene blue as the electron acceptor; neither NAD+ nor viologen dyes could replace methylene blue in this reaction. NADP(H) or FAD(H2) were not used to substrates for the reaction in either direction. The activity of the purified enzyme, which was proposed to be involved in sodium translocation across the cytoplasmic membrane, was not affected by the absence or presence of added sodium. The properties of the enzyme differ from those of the ferredoxin-dependent methylenetetrahydrofolate reductase of the homoacetogen Clostridium formicoaceticum and of the NADP(+)-dependent reductase of eucaryotes investigated so far.     

L-erythrulose production by oxidative fermentation is catalyzed by PQQ-containing membrane-bound dehydrogenase

Thermotolerant Gluconobacter frateurii CHM 43 was selected for L-erythrulose production from mesoerythritol at higher temperatures. Growing cells and the membrane fraction of the strain rapidly oxidized mesoerythritol to L-erythrulose irreversibly with almost 100% of recovery at 37 degrees C. L-Erythrulose was also produced efficiently by the resting cells at 37 degrees C with 85% recovery. The enzyme responsible for mesoerythritol oxidation was found to be located in the cytoplasmic membrane of the organism. The EDTA-resolved enzyme required PQQ and Ca2+ for L-erythrulose formation, suggesting that the enzyme catalyzing meso-erythritol oxidation was a quinoprotein. Quinoprotein membrane-bound mesoerythritol dehydrogenase (QMEDH) was solubilized and purified to homogeneity. The purified enzyme showed a single band in SDS-PAGE of which the molecular mass corresponded to 80 kDa. The optimum pH of QMEDH was found at pH 5.0. The Michaelis constant of the enzyme was found to be 25 mM for meso-erythritol as the substrate. QMEDH showed a broad substrate specificity toward C3-C6 sugar alcohols in which the erythro form of two hydroxy groups existed adjacent to a primary alcohol group. On the other hand, the cytosolic NAD-denpendent meso-erythritol dehydrogenase (CMEDH) of the same organism was purified to a crystalline state. CMEDH showed a molecular mass of 60 kDa composed of two identical subunits, and an apparent sedimentation constant was 3.6 s. CMEDH catalyzed oxidoreduction between mesoerythritol and L-erythrulose. The oxidation reaction was observed to be reversible in the presence of NAD at alkaline pHs such as 9.0-10.5. L-Erythrulose reduction was found at pH 6.0 with NADH as coenzyme. Judging from the catalytic properties, the NAD-dependent enzyme in the cytosolic fraction was regarded as a typical pentitol dehydrogenase of NAD-dependent and the enzyme was independent of the oxidative fermentation of L-erythrulose production.     

Oxygen-dependent inactivation of ribulose 1,5-bisphosphate carboxylase/oxygenase in crude extracts of Rhodospirillum rubrum and establishment of a model inactivation system with purified enzyme.

Ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase (RuBPC/O) was inactivated in crude extracts of Rhodospirillum rubrum under atmospheric levels of oxygen; no inactivation occurred under an atmosphere of argon. RuBP carboxylase activity did not decrease in dialyzed extracts, indicating that a dialyzable factor was required for inactivation. The inactivation was inhibited by catalase. Purified RuBPC/O is relatively oxygen stable, as no loss of activity was observed after 4 h under an oxygen atmosphere. The aerobic inactivation catalyzed by endogenous factors in crude extracts was mimicked by using a model system containing purified enzyme, ascorbate, and FeSO4 or FeCl3. Dithiothreitol was found to substitute for ascorbate in the model system. Preincubation of the purified enzyme with RuBP led to enhanced inactivation, whereas Mg2+ and HCO3- significantly protected against inactivation. Unlike the inactivation catalyzed by endogenous factors from extracts of R. rubrum, inactivation in the model system was not inhibited by catalase. It is proposed that ascorbate and iron, in the presence of oxygen, generate a reactive oxygen species which reacts with a residue at the activation site, rendering the enzyme inactive.     

The bacterial oxidation of vitamin B6. 4-Pyridoxic acid dehydrogenase: a membrane-bound enzyme from Pseudomonas MA-1

A highly specific inducible membrane-bound 4-pyridoxic acid dehydrogenase has been solubilized and purified to apparent homogeneity from Pseudomonas MA-1 grown with pyridoxine as a sole source of carbon and nitrogen. The undenatured enzyme migrates as a single band on gel electrophoresis; denatured preparations show two barely resolved bands (Mr = 63,000 and 61,000). Undenatured preparations aggregate readily, as evidenced by Mr values of 148,000, 470,000, and greater than 670,000 obtained by density gradient centrifugation or by gel filtration under various conditions. The enzyme contains FAD but no Fe or acid-labile S; an average minimum molecular weight of 131,000 was calculated from the FAD content. In the presence of 2,6-dichloroindophenol, the enzyme dehydrogenates 4-pyridoxic acid to the corresponding aldehyde; this reaction is not inhibited by CN-. At the pH optimum of 8.0, a Vm of approximately 7.0 mumol min-1 mg-1 and a Km of 9 microM were obtained. 2,6-Dichloroindophenol, phenazine methosulfate, and menadione are effective electron acceptors; ubiquinones are less active, while NAD, FAD, and O2 are inactive. However, in membrane fractions, oxygen supports 4-pyridoxic acid oxidation via a CN--sensitive electron transport chain, indicating that the dehydrogenase probably is coupled to ATP generation in such preparations.     

Production of 5-keto-d-gluconate by acetic acid bacteria is catalyzed by pyrroloquinoline quinone (PQQ)-dependent membrane-bound d-gluconate dehydrogenase

Gluconobacter suboxydans IFO 12528 was selected as the best strain for 5-keto-d-gluconate (5KGA) production by oxidative fermentation. 5KGA was markedly accumulated by the strain during cultivation in a medium containing d-glucose and/or d-gluconate. The resting cells and the membrane fraction also catalyzed 5KGA formation with a minimal formation of 2-keto-d-gluconate (2KGA), an alternative keto-d-gluconate from d-gluconate. The membrane fraction of the organism was confirmed to contain a membrane-bound d-gluconate dehydrogenase (GADH) catalyzing d-gluconate oxidation to 5KGA of which optimum pH and temperature were found at pH 4 and 15°C, respectively. After treating the membrane fraction with EDTA allowing conversion from holo-GADH to the apoenzyme, 5KGA-forming GADH was confirmed to be a pyrroloquinoline quinone (PQQ)-dependent enzyme by the fact that the enzyme activity was restored by the addition of CaCl₂ and PQQ. The 5KGA-forming GADH was totally distinct from 2KGA-forming GADH in which a covalently bound FAD functions as coenzyme. 5KGA-forming GADH was well solubilized from the membrane fraction with n-octyl-β-d-thioglucoside and 5KGA formation was favourably catalyzed at relatively lower temperature, while 2KGA-forming enzyme was solubilized with Triton X-100 and relatively higher temperatures was optimum for 2KGA formation. These results are completely discrepant from the conclusion proposed by Klasen et al. [R. Klasen, S. Bringer-Mayer, H. Sahm, J. Bacteriol., 177, 1995, 2637] claiming that 5KGA was produced by d-gluconate oxidation catalyzed by NADP-dependent cytoplasmic 5KGA reductase from Gluconobacter species at fairly alkaline pH such as 10.     

Isolation and Sequence Analysis of Bovine αsl-Casein cDNA Clone

Nucleoside oxidase, a novel nucleoside oxidizing enzyme has been purified from a crude extract of Pseudomonas maltophilia LB-86 by a ammonium sulfate fractionation, heat treatment, column chromatography on DEAE-Toyopearl, and gel filtration twice on Sephacryl S-200. The overall purification was approximately 60-fold with a yield of 35 %. The purified enzyme gave a single protein band on acrylamide gel electrophoresis.

Reaction products from inosine were identified as inosine-5′-aldehyde and inosine-5′-carboxylic acid. The enzyme catalyzed the oxidation of inosine to inosine-5′-carboxylic acid via inosine-5′-aldehyde using molecular oxygen as a primary electron acceptor with no formation of hydrogen peroxide.     

Ribulose 1,5-bisphosphate carboxylase from the thermophilic, acidophilic alga, Cyanidium caldarium (Geitler). Purification, characterisation and thermostability of the enzyme.

An an initial stage in the study of proteins from thermophilic algae, the enzyme ribulose 1,5-bisphosphate carboxylase 2-phospho-D-glycerate carboxylyase (dimerizing, EC 4.1.1.39) was purified 11-fold from the thermophilic alga Cyandium caldarium, with a 24% recovery. This purified enzyme appeared homogeneous on polyacrylamide gels and could be dissociated into two subunit types of molecular weights 55,000 and 14,900. The optimal assay temperature was 42.5 degrees C, whilst enzyme purified from Chlorella spp. showed maximum activity at 35 degrees C. The thermostability of Cyanidium ribulose 1,5-bisphosphate carboxylase was considerably greater than that of the Chlorella enzyme, and the presence of Mg2+ and HCO-3 further enhanced this heat stability. A break in the Arrhenius plot occured at 20 degrees C for Chlorella ribulose 1,5-bisphosphate carboxylase and 36 degrees C for the enzyme from Cyanidium. It is suggested that the thermostability of Cyanidium ribulose 1,5-bisphosphate carboxylase is a result of an inherent stability of the enzyme molecule which permits efficient CO2 fixation at high temperatures but results in low activity in the mesophilic temperature range.     

Solubilization, partial purification and properties of N-methylglutamate dehydrogenase from Pseudomonas aminovorans.

1. Extracts of amine-grown Pseudomonas aminovorans contained a particle-bound N-methylglutamate dehydrogenase (EC 1.5.99.5). The enzyme was not present in succinate-grown cells, and activity appeared before growth began in succinate-grown cells which had been transferred to methylamine growth medium. 2. Membrane-containing preparations from methylamine-grown cells catalysed an N-methylglutamate-dependent uptake of O2 or reduction of cytochrome c, which was sensitive to inhibitors of the electron-transport chain. 3. N-Methylglutamate dehydrogenase activity with phenazine methosulphate or 2,6-dichlorophenol-indophenol as electron acceptor could be solubilized with 1% (w/v) Triton X-100. The solubilized enzyme was much less active with cytochrome c as electron acceptor and did not sediment in 1 h at 150000g. Solubilization was accompanied by a change in the pH optimum for activity. 4. The solubilized enzyme was partially purified by Sepharose 4B and hydroxyapatite chromatograpy to yield a preparation 22-fold increased in specific activity over the crude extract. 5. The partially-purified enzyme was active with sarcosine, N-methylalanine and N-methylaspartate as well as with N-methylglutamate. Evidence suggesting activity with N-methyl D-amino acids as well as with the L-forms was obtained. 6. The enzyme was inhibited by p-chloromercuribenzoate, iodoacetamide and by both ionic and non-ionic detergents. 2-Oxoglutarate and formaldehyde were also inhibitors. 7. Kinetic analysis confirmed previous workers' observations of a group transfer (Ping Pong) mechanism. 8. Spectral observations suggested that the partially purified preparation contained flavoprotein and a b-type cytochrome. 9. The role of the enzyme in the oxidation of methylamine is discussed.     

Purification and properties of protoporphyrinogen oxidase from an anaerobic bacterium, Desulfovibrio gigas.

Protoporphyrinogen oxidase has been solubilized from plasma membranes of Desulfovibrio gigas. The enzyme was purified to apparent homogeneity with single silver-stained protein bands on isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gels. This protoporphyrinogen oxidase has a molecular weight (Mr) of 148,000 and is composed of three dissimilar subunits of Mrs 12,000, 18,500, and 57,000, which are held together by sulfhydryl bonds. Unlike other protoporphyrinogen oxidases, which use molecular oxygen as an electron acceptor, this enzyme does not couple to oxygen. The protoporphyrinogen oxidase donates electrons to 2,6-dichlorophenol-indophenol but not to NAD+, NADP+, flavin adenine dinucleotide, or flavin mononucleotide. The natural physiological electron acceptor of the protoporphyrinogen oxidase from D. gigas is unknown. By using 2,6-dichlorophenol-indophenol as the electron acceptor, the Km and Vmax values for oxidation of protoporphyrinogen were determined to be 21 microM and 8.38 nmol/min per 70 micrograms of protein, respectively. The catalytic rate constant, Kcat, was calculated to be 17.7 mol of protoporphyrin formed per mole of enzyme per min of incubation, and the Kcat/Km was 0.84. Energies of activation were calculated from Arrhenius plots with 7,429 cal (ca. 31,080 J)/mol per degree below 10 degrees C and 1,455 cal (ca. 6,088, J)/mol per degree above 10 degrees C. Optimum enzyme activity was at 23 degrees C, and inhibition was observed with both N-ethylmaleimide and iodoacetamide.     

Activation of Pseudomonas cytochrome oxidase by limited proteolysis with subtilisin

Oxidized Pseudomonas cytochrome oxidase (ferrocytochrome c2: oxygen oxidoreductase; E.C.1.9.3.2) can be digested with subtilisin under controlled conditions that convert the original parent polypeptide chain (Mr on SDS gels approximately equal to 60,000) to a slightly smaller species (Mr on SDS gels approximately equal to 58,000). Under the conditions used (0.33% subtilisin, w/w, pH 7.4), the product formed from the oxidase was relatively stable to further digestion. Cytochrome oxidase activity was assayed at intervals during proteolysis by following the rate of oxidation of Pseudomonas ferrocytochrome c-551 by the enzyme in the presence of oxygen. The activity increased to a plateau that was more than two times the value for an untreated control. These observations suggest that clipping a small peptide from Pseudomonas cytochrome oxidase either facilitates the rate-limiting electron transfer between the intraprotein heme c and heme d1, enhances the interaction of the enzyme with ferrocytochrome c-551, or both.     

Purification and some properties of cyclohexylamine oxidase from a Pseudomonas sp.

Cyclohexylamine oxidase was purified 90-fold from cell-free extracts of Pseudomonas sp. capable of assimilating sodium cyclamate. The purified enzyme was homogeneous in disc electrophoresis, and the molecular weight was found to be approximately 80,000 by gel filtration. The enzyme catalyzed the following reaction: cyclohexylamine+O2+H2O leads to cyclohexanone+NH3+H2O2. The enzyme thus can be classified as an amine oxidase; it utilized oxygen as the ultimate electron acceptor. The pH optimum of the reaction was 6.8 and the apparent Km value for cyclohexylamine was 2.5 X 10(-4) M. The enzyme was highly specific for the deamination of alicyclic primary amines such as cyclohexylamine, but was found to be inactive toward ordinary amines used as substrates for amine oxidases. The enzyme solution was yellow in color and showed a typical flavoprotein spectrum; the addition of cyclohexylamine under anaerobic conditions caused reduction of the flavin in the native enzyme. The flavin of the prosthetic group was identified as FAD by thin layer chromatography. The participation of sulfhydryl groups in the enzymic action was also suggested by the observation that the enzyme activity was inhibited in the presence of PCMB and could be recovered by the addition of glutathione.     

Increased susceptibility of ribulose-1,5-bisphosphate carboxylase/oxygenase to proteolytic degradation caused by oxidative treatments

The susceptibility of the chloroplastic enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase to proteolysis by trypsin, chymotrypsin, proteinase K, and papain is enhanced by oxidative treatments including spontaneous oxidation of cysteines. Proteinases exhibit a high specificity for the oxidized inactive form of the carboxylase, cleaving its large subunit. Treatment of the inactive enzyme with dithiothreitol results in partial recovery of both carboxylase activity and resistance to proteolysis. This behavior may explain the specific degradation of ribulose-1,5-bisphosphate carboxylase/oxygenase that occurs in vivo during leaf senescence.     

Substrate Specificity of the Purified Primary Alcohol Dehydrogenases from Methanol-Oxidizing Bacteria

Hyphomicrobium strain WC, Pseudomonas strain TP-1, and Pseudomonas strain W1 are capable of growth on methanol as the sole source of carbon and energy. Methanol-grown cells of each organism contain a primary alcohol dehydrogenase that has been purified to homogeneity. Each enzyme has a molecular weight of 120,000 and shows an in vitro requirement for phenazine methosulfate and ammonium ions for enzymatic activity. Normal aliphatic alcohols are oxidized rapidly by each enzyme. The presence of a methyl group on the carbon atom adjacent to the primary alcohol group lowers the enzymatic activity. This effect is reduced as the methyl substituent is moved further away from the hydroxyl group. The effect of other substituents on enzymatic activity is reported. Methanol, formaldehyde, and to a limited extent acetaldehyde are oxidized by the primary alcohol dehydrogenases. Higher aldehydes are not oxidized. A possible explanation for this specificity, with regard to aldehydes, is presented in terms of degree of hydration of the aldehyde.     

Purification and properties of the membrane-bound NADH oxidase of a facultatively anaerobic alkaliphile

A membrane-bound NADH oxidase of an anaerobic alkaliphile, M-12 (a strain of Amphibacillus sp.), was solubilized with decanoyl N-methylglucamide and purified by chromatography on DEAE-Sepharose and hydroxyapatite. The purified enzyme appears to consist of a single polypeptide component with an apparent molecular mass of 56 kDa. The enzyme catalyzed the oxidation of NADH with the formation of H₂O₂ and exhibited a specific activity of 46 μmol NADH min^–1 (mg protein)^–1. NADPH did not serve as a substrate for the enzyme. The K _m for NADH was estimated to be 0.05 mM. The enzyme exhibited a pH dependence for activity, with a pH optimum at approximately 9.5. The enzyme required a high concentration of salt and exhibited maximum activity in the presence of 600 mM NaCl. Received: 3 August 1998 / Accepted: 23 December 1998