Nicotinoprotein [NAD(P)‐containing] alcohol/aldehyde oxidoreductases

Abstract

Extracts of Gram-positive bacteria like Rhodococcus rhodochrous, Rhodococcus erythropolisand Amycolatopsis methanolica, but not those of several Gram-negative ones, showed dehydrogenase activity for ethanol as well as for methanol when 4-nitroso-N, N-dimethylaniline (NDMA) was used as electron acceptor. Chromatography of extracts of the first two organisms revealed one activity for both substrates, that of A. methanolica two activities, one of which is able to oxidize methanol and has been purified (Bystrykh, L. V., Govorukhina, N. I., van Ophem, P. W., Hektor, H. J., Dijkhuizen, L. and Duine, J. A., unpublished results). The other, indicated as NDMA-dependent alcohol dehydrogenase (NDMA-ADH), was purified to homogeneity. It is a trimeric enzyme consisting of subunits of 39 kDa and one firmly bound NAD as cofactor. Although NDMA-ADH shows structural similarity with the long-chain, zinc-containing, NAD(P)-dependent alcohol dehydrogenases with respect to the N-terminal sequence up to residue 41 (56% identity with horse liver alcohol dehydrogenase), the enzymes are catalytically different since NDMA-ADH is unable to use NAD(P)(H) as a coenzyme and NAD(P)-dependent alcohol dehydrogenases are inactive with NDMA (in the absence of NAD). Comparison of the NDMA-ADH properties with those of the methanol-oxidizing enzyme of A. methanolica, Mycobacterium gastri and Bacillus methanolica C1, and formaldehyde dismutase of Pseudomonas putida F61 revealed large differences in structural as well as catalytic properties, in spite of the fact that all are nicotinoproteins [enzymes which have bound NAD(P) as a cofactor]. It is concluded, therefore, that NDMA-ADH is a novel type of nicotinoprotein alcohol dehydrogenase.In the search for a methanol-oxidizing activity in Amycolatopsis methanolica, an assay able to detect alcohol dehydrogenases having NAD(P) as cofactor was applied, using 4-nitroso-N, N-dimethylaniline (NDMA) as electron acceptor [ 1). However, upon applying anion-exchange chromatography on extracts exhibiting such a property, two separate activities were found in the eluted fractions, one showing activity with alcohols including methanol, the other excluding methanol. The enzyme responsible for the first activity [the Correspondence to J. A. Duine,

Metaproteogenomic analysis of microbial communities in the phyllosphere and rhizosphere of rice

“…This finding is in agreement with observations on other plant species and underlines the importance of one-carbon metabolism for this genus upon phyllosphere colonization (Sy et al, 2005;Delmotte et al, 2009;Schmidt et al, 2010). Furthermore, a methanol:NDMA oxidoreductase was identified and assigned to Amycolatopsis (von Ophem et al, 1993), suggesting that other bacteria may also benefit from plant-released methanol in the rice phyllosphere, even though they are apparently less numerous.…”

Formaldehyde dismutase activities in Gram-positive bacteria oxidizing methanol

“…methanolicu on a QSepharose column revealed the presence of two proteins active with NDMA and alcohols. One of these was active with both methanol and ethanol, whereas the other one was specific for ethanol (Van Ophem et al, 1993). In cells of M .…”

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

“…MDO activity was assayed by measuring the methanol-dependent reduction of DMNA (e 440 535 400 M 21 cm 21 ) as described by van Ophem et al (1993). Methanol or other substrates were added to the reaction mixture to a final concentration of 50 mM.…”

“…Among Gram-positive methylotrophic bacteria, Bacillus methanolicus C1 uses NAD-dependent nicotinoprotein MDH (Arfman et al, 1989(Arfman et al, , 1997Hektor et al, 2002), and Amycolatopsis methanolica, Mycobacterium gastri MB19, Rhodococcus rhodochrous LMD 89.129 and Rhodococcus erythropolis DSM 1069 use N,N9-dimethyl-4-nitrosoaniline (DMNA)-dependent nicotinoprotein methanol : DMNA oxidoreductase (MDO) (Bystrykh et al, 1993a, b;van Ophem et al, 1993) for the oxidation of methanol. This MDO is similar to the NAD-dependent MDH of B. methanolicus C1 in quaternary structure, cofactor composition, tightly but non-covalently bound NAD(P)(H) and enzymic properties (Bystrykh et al, 1993b;Hektor et al, 2002;van Ophem et al, 1993;Vonck et al, 1991). The MDO also has enzymic properties different from those of the B. methanolicus C1 MDH: MDO exhibits DMNA-dependent MDH, NADHdependent formaldehyde reductase and formaldehyde dismutase activities (Hektor et al, 2000), while B. methanolicus C1 MDH only displays NAD-dependent MDH and NADH-dependent formaldehyde reductase activities (Arfman et al, 1989).…”

“…Gram-negative methylotrophic bacteria have NAD(P)-independent MDHs that contain pyrroloquinoline quinone (PQQ) as a prosthetic group (Anthony, 2000). Among Gram-positive methylotrophic bacteria, Bacillus methanolicus C1 uses NAD-dependent nicotinoprotein MDH (Arfman et al, 1989(Arfman et al, , 1997Hektor et al, 2002), and Amycolatopsis methanolica, Mycobacterium gastri MB19, Rhodococcus rhodochrous LMD 89.129 and Rhodococcus erythropolis DSM 1069 use N,N9-dimethyl-4-nitrosoaniline (DMNA)-dependent nicotinoprotein methanol : DMNA oxidoreductase (MDO) (Bystrykh et al, 1993a, b;van Ophem et al, 1993) for the oxidation of methanol. This MDO is similar to the NAD-dependent MDH of B. methanolicus C1 in quaternary structure, cofactor composition, tightly but non-covalently bound NAD(P)(H) and enzymic properties (Bystrykh et al, 1993b;Hektor et al, 2002;van Ophem et al, 1993;Vonck et al, 1991).…”