Purification and characterization of an alcohol:N,N-dimethyl-4-nitrosoaniline oxidoreductase from the methanogen Methanosarcina barkeri DSM 804 strain Fusaro.
Cell-free extracts of Methanosarcina barkeri DSM 804 showed alcohol dehydrogenase activity under aerobic conditions when N,N-dimethyl-4-nitrosoaniline (NDMA) was used as an artificial electron acceptor. The NDMA-dependent alcohol dehydrogenase (NDMA-ADH) was purified to approximate homogeneity by column chromatography. It is most probably a homodimeric enzyme consisting of subunits of 45 kDa, the native molecular mass estimated by gel filtration being about 87 kDa. The purified protein had an isoelectric point of 4.3. It possesses a tightly but noncovalently bound NADP(H) cofactor. Each subunit contains 1 mol NADP(H)/mol, about 2 mol Zn2+/mol and significant amounts of magnesium. The purified enzyme preferably oxidized primary alcohols (including benzyl alcohol). NDMA-ADH from M. barkeri also catalyzed the stoichiometric dismutation of aldehydes, especially higher aliphatic aldehydes, to form equimolar amounts of the corresponding alcohol and acid without addition of an electron carrier. The enzyme did not catalyze the dehydrogenation of methanol or the disproportionation of formaldehyde and therefore is not directly involved in methanogenesis. An alignment of the N-terminal amino acid sequence of the enzyme with the sequences of other alcohol dehydrogenases from methanogenic and nonmethanogenic bacteria indicated no significant identity. Nevertheless there was a quite interesting sequence similarity in the first 30 N-terminal amino acids to plant cinnamyl alcohol dehydrogenase. NDMA-ADH from M. barkeri is a novel type of alcohol dehydrogenase in methanogenic bacteria.
Daussmann
T
T
Institut für Biotechnologie, Forschungszentrum Jülich, Germany.
Aivasidis
A
A
Wandrey
C
C
D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D000447:Aldehydes / Q000378:metabolism; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D007700:Kinetics; D008274:Magnesium / Q000032:analysis; D017021:Methanosarcina barkeri / Q000201:enzymology*; D008970:Molecular Weight; D009249:NADP / Q000032:analysis; D009603:Nitroso Compounds / Q000378:metabolism*; D016415:Sequence Alignment; D017421:Sequence Analysis; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D015032:Zinc / Q000032:analysis / Q000378:metabolism
D000447:Aldehydes; D004791:Enzyme Inhibitors; D009603:Nitroso Compounds; C008651:4-nitrosodimethylaniline; D009249:NADP; C046012:pentanal; D000429:Alcohol Oxidoreductases; C095705:alcohol - N, N'-dimethyl-4-nitrosoaniline oxidoreductase; D008274:Magnesium; D015032:Zinc