A new alcohol dehydrogenase, reactive towards methanol, from Bacillus stearothermophilus (original) (raw)
Related papers
Journal of Biological Chemistry, 1991
Methanol dehydrogenase from the thermotolerant Bacillus sp. C 1 was studied by electron microscopy and image processing. Two main projections can be distinguished: one exhibits 5-fold symmetry and has a diameter of 15 nm, the other is rectangular with sides of 15 and 9 nm. Subsequent image processing showed that the 6-fold view possesses mirror symmetry. The rectangular views can be divided into two separate classes, one of which has 2-fold rotational symmetry. It is concluded that methanol dehydrogenase is a decameric molecule, and a tentative model is presented. The estimated molecular weight is 430,000, based on a subunit molecular weight of 43,000. The enzyme contains one zinc and one to two magnesium ions per subunit. N-terminal amino acid sequence analysis revealed substantial similarity with alcohol dehydrogenases from Saccharomyces cerevisiae, Zymomonas mobilis, Clostridium acetobutylicum, and Escherichia coli, which contain iron or zinc but no magnesium. In view of the aberrant structural and kinetic properties, it is proposed to distinguish the enzyme from common alcohol dehydrogenases (EC 1.1.1.1) by using the name NAD-dependent methanol dehydrogenase. All thermotolerant methanol-utilizing Bacillus spp. studied to date oxidize methanol by means of a novel NAD-dependent methanol dehydrogenase (MDH)' (1,2). The enzyme oxidizes primary C1-C4 alcohols and 1,3-propanediol and also catalyzes the NADH-dependent reduction of the corresponding aldehydes. Oxidation of alcohol substrates is strongly stimulated by a M , = 50,000 activator protein. Purification of the activator protein and its effects on the kinetics of alcohol oxidation are described in the accompanying paper (3). NAD-dependent alcohol dehydrogenases (ADH, EC 1.1.1.1) have been characterized from many sources (4). In general,
2011
The production of alcohol dehydrogenases with the capacity of oxidizing long chain aliphatic alcohols by mesophilic, thermophilic and hyperthermophilic strains induced by aliphatic alcohols and alkanes with 2-24 carbon atoms has been studied. Seven strains were tested: the mesophilic yeast Candida tropicalis ATCC20336, the thermophilic bacterium Thermus AB1and five hyperthermophilic bacterial strains: Thermus thermophilus HB27, HNI11, NR17, PRQ16 and PRQ25. Only C. tropicalis ATCC20336, Thermus AB1 and T. thermophilus PRQ25 produced alcohol dehydrogenases with the capacity of oxidizing aliphatic alcohols with 22 and 24 carbon atoms, after induction with 1-hexacosanol, 1-docosanol and 1-eicosanol, respectively. Higher initial reaction rates of oxidation of docosanol and eicosanol were obtained with the enzyme from C. tropicalis ATCC20336, with values of 196.9 and 218.8 mol NADH min −1 g of protein −1 , respectively.
Stereopecificity and Other Properties of a Novel Secondary-Alcohol-Specific Alcohol Dehydrogenase
European Journal of Biochemistry, 1981
NAD-dependent alcohol dehydrogenase from the methanol-grown Methylcoccus sp. CRL MI (type I membrane), Methylosinus zrichosporium OB3b (type I1 membrane), Mc.thylobucierium organophillum CRL 26 (type I1 membrane, facultative methylotroph), Psrudomonas sp. ATCC 21 439, and Pichia pusioris Y-55 are secondaryalcohol-specific and that from P. pu.stori.v Y-7556 is not. This novel secondary-alcohol-specific alcohol dehydrogenase (secondary-alcohol dehydrogenase) has been purified from methanol-grown Pseudomonus sp. ATCC 21 439. Secondary-alcohol dehydrogenase shows a single protein band on acrylamide gel electrophoresis and has a molecular weight of 95000. It consists of two subunits of M,48000 daltons and two atoms of zinc per molecule of enzyme protein. It oxidizes secondary alcohols, notably 2-propanol and 2-butanol. Primary alcohols are not oxidized. The pH and temperature optima for secondary-alcohol dehydrogenase are 8-9, and 30-35 'C, respectively. The activation energy calculated is 82.8 kJ. Secondary-alcohol dehydrogenase also catalyzes the reduction of methyl ketones to their corresponding 2-alcohols in the presence of NADH (a reverse reaction). The K,,,
2004
Oxidation of C,-C, primary alcohols in thermotolerant Bacillus methanolicus strains is catalyzed by an NAD-dependent methanol dehydrogenase (MDH), composed of ten identical 43 000-M, subunits. Each MDH subunit contains a tightly, but non-covalently, bound NAD (H) molecule, in addition to 1 Znz+ and 1-2 Mg2+ ions. The NAD (H) cofactor is oxidized and reduced by formaldehyde and methanol, respectively, while it remains bound to the enzyme.
Purification and comparative studies of alcohol dehydrogenases
Comparative Biochemistry and Physiology Part B: Comparative Biochemistry, 1987
Al~tract--1. Alcohol dehydrogenases from various animal and plant sources were purified by a common procedure which employed DEAE, Sephadex-G100 and affinity chromatographies.
Applied and environmental …, 1988
Thermoanaerobacter ethanolicus (ATCC 31550) has primary and secondary alcohol dehydrogenases. The two enzymes were purified to homogeneity as judged from sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration. The apparent MrS of the primary and secondary alcohol dehydrogenases are 184,000 and 172,000, respectively. Both enzymes have high thermostability. They are tetrameric with apparently identical subunits and contain from 3.2 to 5.5 atoms of Zn per subunit. The two dehydrogenases are NADP dependent and reversibly convert ethanol and 1-propanol to the respective aldehydes. The V1m values with ethanol as a substrate are 45.6 ,umol/min per mg for the primary alcohol dehydrogenase and 13 ,umol/min per mg for the secondary alcohol dehydrogenase at pH 8.9 and 60°C. The primary enzyme oxidizes primary alcohols, including up to heptanol, at rates similar to that of ethanol. It is inactive with secondary alcohols. The secondary enzyme is inactive with 1-pentanol or longer chain alcohols. Its best substrate is 2-propanol, which is oxidized 15 times faster than ethanol. The secondary alcohol dehydrogenase is formed early during the growth cycle. It is stimulated by pyruvate and has a low Km for acetaldehyde (44.8 mM) in comparison to that of the primary alcohol dehydrogenase (210 mM). The latter enzyme is formed late in the growth cycle. It is postulated that the secondary alcohol dehydrogenase is largely responsible for the formation of ethanol in fermentations of carbohydrates by T. ethanolicus.
Bioscience, Biotechnology, and Biochemistry, 2003
High NAD-dependent alcohol dehydrogenase (ADH) activity was found in the cytoplasm when a membranebound, quinoprotein, ADH-deˆcient mutant strain of Acetobacter pasteurianus SKU1108 was grown on ethanol. Two NAD-dependent ADHs were separated and puriˆed from the supernatant fraction of the cells. One (ADH I) is a trimer, consisting of an identical subunit of 42 kDa, while the other (ADH II) is a homodimer, having a subunit of 31 kDa. One of the two ADHs, ADH II, easily lost the activity during the column chromatographies, which could be stabilized by the addition of DTT and MgCl 2 in the column buŠer. ADH I but not ADH II contained approximately one zinc atom per subunit. The N-terminal amino acid analysis indicated that ADH I and ADH II have homology to the longchain and short-chain ADH families, respectively. ADH I showed a preference for primary alcohols, while ADH II had a preference for secondary alcohols. The two ADHs showed clear diŠerence in their kinetics on ethanol, acetaldehyde, NAD, and NADH. The physiological function of both ADH I and ADH II are also discussed.