Purification and Characterization of an Alcohol: N,N‐dimethyl‐4‐nitrosoaniline Oxidoreductase from the Methanogen Methanosarcina Barkeri DSM 804 Strain Fusaro

Abstract

Cell-free extracts of Methanosarcinu bai-keri 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 Zn'+/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.

Stereoselective Carveol Dehydrogenase from Rhodococcus erythropolis DCL14

“…Examples are glyceraldehyde-3-phosphate dehydrogenase (58) and mammalian acetaldehyde dehydrogenase (56). The second group includes nicotinoprotein dehydrogenases that are, in vitro, only active when assayed in the presence of an artificial electron acceptor like N,N-dimethyl-4-nitrosoaniline or DCPIP (57,59,60). In vivo, these enzymes probably regenerate the cofactor by delivering the reduction equivalents directly to a component of an electron transport chain (61).…”

“…In vivo, these enzymes probably regenerate the cofactor by delivering the reduction equivalents directly to a component of an electron transport chain (61). Examples are methanol dehydrogenase from Amycolatopsis methanolica (57), short chain primary alcohol dehydrogenase from A. methanolica (60), and medium chain primary alcohol dehydrogenase from Methanosarcina barkeri (59). Being exclusively active with DCPIP, it is evident that CDH belongs to this second group of nicotinoprotein dehydrogenases.…”

Identification and functional characterization of a gene for the methanol : N,N'-dimethyl-4-nitrosoaniline oxidoreductase from Mycobacterium sp. strain JC1 (DSM 3803)

“…The Gram-positive bacterium Rhodococcus sp. NI86/21 (Nagy et al, 1995) and the methanogen Methanosarcina barkeri (Daussmann et al, 1997) also possess a DMNA-dependent nicotinoprotein alcohol : DMNA oxidoreductase, but the enzymes exhibit no activity with methanol as a substrate.…”

Effects of GroESL Coexpression on the Folding of Nicotinoprotein Formaldehyde Dismutase fromPseudomonas putidaF61

“…By contrast, the fact that nicotinoprotein FDM contains an NAD(H) tightly bound to each subunit suggests that incorporation of NAD(H) occurs during the folding of the subunit proteins. A similar process most likely also occurs with other nicotinoproteins, including UDP-galactose 4-epimerase from E. coli, 20) lactate-oxaloacetate transhydrogenase from Veillonella alcalescens, 21) glucose-fructose oxidoreductase from Zymomonas mobilis, 22) methanol:NDMA oxidoreductase from Amycolatopsis methanolica, 23) and alcohol dehydrogenase from Methanosarcina barkeri, 24) which raises the questions: how the tight binding of NAD(P) is done by these nicotinoproteins and what are the mechanistic implications of the strong binding interactions between the cofactor and the protein matrix. In vitro analysis of the refolding of denatured FDM in the presence of GroEL and GroES might help clarify the mechanism of NAD(H) binding and provide speciˆc details of the interaction between the coenzyme and the protein matrix.…”