Electron microscopic analysis and structural characterization of novel NADP(H)-containing methanol: N,N'-dimethyl-4-nitrosoaniline oxidoreductases from the gram-positive methylotrophic bacteria Amycolatopsis methanolica and Mycobacterium gastri MB19

Abstract

The quaternary protein structure of two methanol:NN'-dimethyl-4-nitrosoaniline (NDMA) oxidoreductases purified from Amycolatopsis methanolica and Mycobacterium gastri MB19 was analyzed by electron microscopy and image processing. The enzymes are decameric proteins (displaying fivefold symmetry) with estimated molecular masses of 490 to 500 kDa based on their subunit molecular masses of 49 268,000 (6). Here, we present the quaternary structure of the A. methanolica and M. gastri MNOs as analyzed by electron microscopy and image processing. In addition, their cofactor and metal compositions, as well as the amino acid sequences of the N termini and several internal peptide fragments, are compared with those of various alcohol dehydrogenases. MATERIALS AND METHODSGrowth conditions and enzyme purification. The growth of A. methanolica and M. gastri MB19 and the purification of the MNO enzymes from these organisms were performed as described by Bystrykh et al. (6).Electron microscopy. Specimens for electron microscopy were prepared by applying a drop of MNO, at a concentration of 0.5 mg/ml, to grids covered with a carbon-coated Formvar film which had been treated by a glow discharge in pentylamine immediately before being used. The specimens were blotted with filter paper and negatively stained with a solution of 2% (wt/vol) sodium silicotungstate or 1% (wt/vol) uranyl acetate.Electron microscopy was performed with a Philips CM12 or a JEOL JEM 1200 EX, both operating at 80 kV. With both microscopes, a low-dose system was used for focussing on an area adjacent to the area to be imaged, in order to avoid 1814 on May 7, 2018 by guest

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

“…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).…”

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

“…Electrophoresis in the presence of SDS revealed one band with 39 kDa. Electron microscopy of the enzyme did not show the presence of decamers, as has been observed with MFF [2] and methanol dehydrogenase from Bacillus methanolica C1 [20].…”

“…2), thereby resembling the absorption spectra of MFF [2] and of methanol dehydrogenase (N. Arfman et al, unpublished results), but not that of (rabbit muscle) glyceraldehyde-3-phosphate dehydrogenase (which also contains tightly bound NAD) which has an absorption maximum at 360nm [24]. Samples of enzyme denaturated by heating were inactive in the pyrroloquinoline quinone assay and also flavins were absent, as judged from the absorption spectrum.…”

“…Alignment with MFF [2] showed 18 % identity with methanol dehydrogenase from Bacillus methanolica C1 7 % identity [20], with factor-dependent formaldehyde dehydrogenase 23 % identity [16], and with horse liver alcohol dehydrogenase 56 % identity [25 ] (Scheme 1).…”

Gene Expression Analysis of Methylotrophic Oxidoreductases Involved in the Oligotrophic Growth ofRhodococcus erythropolisN9T-4

“…These enzymes are known to be involved in methanol oxidation in methylotrophic bacteria, 6) and NDMA-dependent MDHs have only been found in Gram-positive methylotrophic bacteria. 5,7) These results led us to think that oligotrophic growth would involve methanol metabolism in N9T-4, although this bacterium cannot utilize methanol as the sole carbon source. We attempted to search for other genes involved in methanol metabolism in the draft genome sequence of N9T-4 which has recently been determined by our group.…”