The generic position of 14 strains of gram-positive bacteria able to use methanol as a growth substrate was determined. All are obligately aerobic, thermotolerant organisms that are able to grow at temperatures of 35 to 60°C. Nine of the strains produce oval spores at a subterminal-to-central position in slightly swollen rod-shaped cells. DNA-DNA hybridization studies, 5s rRNA sequence analysis, and physiological characteristics revealed that all 14 strains cluster as a well-defined group and form a distinct new genospecies. Analysis of the 16s and 5s rRNA sequences indicated that this new species is distinct from BuciUus brevis but closely related to B.Jinnus and B. uzotoformuns. The name proposed for this new species is B. methunolicus. The type strain, PB1, has been deposited in the National Collection of Industrial and Marine Bacteria as NCIMB 13113.

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. Incubation of MDH with methanol and exogenous NAD (coenzyme) results in reduction of this NAD coenzyme. Both NAD species are not exchanged during catalysis. NAD thus plays two different and important roles in the MDH-catalyzed reaction, with the bound NAD cofactor acting as primary electron acceptor and the NAD coenzyme being responsible for reoxidation of the reduced cofactor. MDH obeys a ping-pong type reaction mechanism, which is consistent with such a temporary parking of reducing equivalents at the MDH-bound cofactor. Spectral studies show that, in the presence of exogenous NAD and Mgz+ ions, MDH interacts with a previously identified 50 00044, activator protein. The activator protein appears to facilitate the oxidation of the reduced NADH cofactor of MDH, which results in a strongly increased turnover rate of MDH.

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

HEKTOR, DelJl, TheExtracts of methanol-grown cells of Amycolutopsis rndkanolica and Mycubacterium gastri oxidized methanol and ethanol with concomitant reduction of NJV"'dimethyl-4-nitrosoaniline (NDMA), Anion-exchange chromatography revealed the presence of a single enzyme able to catalyse this activity in methanol-or ethanol-grown cells of M. gastri. A. methanolica, however, possessed two different enzymes, one of which was similar to the single enzyme found in M. gastvi. The methanol : NDM,4 oxidoreductases (MNO) were purified to homogeneity from methanolgrown cells of A. methanolicu and M . gastri. Both enzyme preparations showed similar relative molecular masses with subunits of Mr 50000 and 49000, and native enzymes of M, 268000 and 255000 (gel-filtration data for A. rnethnnulica and M. gastri, respectively). Both enzymes also displayed a similar substrate specificity. They were active with methanol and various other primary alcohols (yielding the corresponding aldehydes), poiyols and formaldehyde. In addition, the MNO enzymes produced methylformate from methanol plus formaldehyde, and catalyzed formaldehyde disrnutase and NADH-dependent formaldehyde reductase reactions. They did not possess NAD(P)'-or dye-linked alcohol dehydrogenase or oxidase activities,

Tetrazolium-dye-linked alcohol dehydrogenase (TD-ADH) of Arnycolatopsis methanolica could be resolved into three protein components, which have been purified. Each of the components has the ability to reconstitute TD-ADH activity when combined with the other two. Component 1 is identical to the previously characterized methanol:N,N'-dimethyl-4-nitrosoaniline oxidoreductase (MNO), a decameric protein with 50-kDa subunits, each carrying a tightly bound NADPH. Component 2 is a high molecular mass (> 640 kDa) protein with subunits of 44 kDa and 72 kDa, and which possesses a low tetrazoliumdye-linked NADH dehydrogenase activity. The protein contains a yellow chromophore of unknown identity. Component 3 is a low molecular mass (15 kDa) protein containing a 5'-deazaflavin and at least one other low-molecular-mass compound with properties similar, but not identical, to those of nicotinamide coenzymes. The results suggest that alcohol oxidation by the TD-ADH complex is camed out by component I (MNO), after which transfer of the reducing equivalents (mediated by component 3) occurs to component 2, which (in vitro) is linked to the tetrazolium dye. Fractionation of A. methanolica extracts showed that most of the 5'-deazaflavin was present in component 3. Other gram-positive bacteria having a TD-ADH complex also produced 5'-deazaflavin. It is concluded that oxidation of primary aliphatic alcohols by A. rnethanolica, and probably also by other gram-positive bacteria containing MNO or TD-ADH, proceeds via TD-ADH. The likeliness of 5'-deazaflavin participation in this process is discussed.Keywords: dye-linked alcohol dehydrogenase; methanol :N,Ni-dimethyl-4-nitrosoaniline oxidoreductase ; 5'-deazaflavin ; niethanol ; formaldehyde.The enzymology of methanol oxidation in gram-positive bacteria is much more complicated than that in their gram-negative counterparts, where this conversion is catalysed by the pyrroloquinoline-quinone-containing methanol dehydrogenase. This complexity is illustrated by the diverse methanol-oxidizing activities described for the actinomycete Amycolatopsis methanolica. Kato et al. (1975) reported that extracts of methanolgrown cells exhibit phenazine methosulphate/2,6-dichloroindophenol (C1,Ind) methanol dehydrogenase activity. Subsequently, Duine et al. (1984) reported the presence of a methanol dehydrogenase (MeOH-DH) in a multienzyme complex, displaying a C1,Ind-dependent activity that was stimulated by NAD addition. However, both these assays were difficult to reproduce. It was only after the development of a reproducible assay (van Ophem et al., 1991) sible. Typical features of the assay are that only a few tetrazolium dyes are active [e.g. 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)], it is stimulated by high phosphate or sulphate concentrations, and the relationship between the amount of extract and activity is non-linear. The latter two points suggested also this tetrazolium-dye-linked alcohol dehydrogenase (TD-ADH) activity originates from a multienzyme complex.TD-ADH a...