Abstract. The enzymology of methanol utilization in thermotolerant methytotrophic Bacillus strains was investigated. In all strains an immunological]y related NAD-dependent methanol dehydrogenase was involved in the initial oxidation of methanol. In cells of Bacillus sp. C1 grown under methanol-limiting conditions this enzyme constituted a high percentage of total soluble protein. The methanol dehydrogenase from this organism was purified to homogeneity and characterized. In cell-free extracts the enzyme displayed biphasic kinetics towards methanol, with apparent Km values of 3.8 and 166 mM. Carbon assimilation was by way of the fructose-1,6-bisphosphate aldolase cleavage and transketolase/transaldolase rearrangement variant of the RuMP cycle of formaldehyde fixation. The key enzymes of the RuMP cycle, hexulose-6-phosphate synthase (HPS) and hexulose-6-phosphate isomerase (HPI), were present at very high levels of activity. Failure of whole cells to oxidize formate, and the absence of formaldehyde-and formate dehydrogenases indicated the operation of a non-linear oxidation sequence for formaldehyde via HPS. A comparison of the levels of methanol dehydrogenase and HPS in cells of Bacillus sp. C1 grown on methanol and glucose suggested that the synthesis of these enzymes is not under coordinate control.

The gene (mdh) Bacillus methanolicus Cl is a representative of the thermotolerant methanol-utilizing Bacillus spp. which oxidize methanol by use of an NAD-dependent methanol dehydrogenase (MDH) (3,5,9). The previously reported N-terminal amino acid sequence (27) has shown that the enzyme belongs to a novel family of NAD-dependent alcohol dehydrogenases (ADHs) which includes Zymomonas mobilis ADH2 (8), Saccharomyces cerevisiae ADH4 (29), Escherichia coli L-1,2-propanediol oxidoreductase (POR) (7), and the Clostridium acetobutylicum NADPH-dependent butanol dehydrogenase (ADH1) (31). This family of enzymes is different from the horse liver-type ADHs (long chain, type I) or Drosophila-type enzymes (short chain, type II) and will therefore be referred to as type III ADHs.The B. methanolicus MDH is a decameric enzyme with a subunit Mr of 43,000. Each subunit contains a noncovalently but tightly bound NAD(H) cofactor molecule, one zinc atom, and one or two magnesium atoms (27). The S. cerevisiae ADH4 enzyme also has been reported to contain zinc (29), while iron has been detected in the Z. mobilis ADH2 protein (8). The E. coli POR enzyme is stimulated by ferrous ions (25). Further data about the presence of metals in type III ADHs is lacking, and it remains to be decided whether the presence of magnesium in the Bacillus MDH is unique.The recently reported involvement of an activator protein, which stimulates NAD-dependent ADH activity of the purified MDH enzyme, certainly seems unique (4). In this respect, it is significant that in the other members of the type III ADH enzymes, the oxidative reaction does not play a metabolic role: all support a fermentative metabolic function in the respective wild-type organisms. Metabolic dependence on methanol requires a high flux of methanol oxidation: approximately twice as much carbon must be processed by this reaction compared with ethanol utilization. The activator protein has been suggested to play a dedicated role in the release of reducing equivalents from the bound NAD(H) cofactor (1). The occurrence of the activator protein in all methylotrophic Bacillus species investigated (4, 5) may thus form an interesting adaptation to increase the * Corresponding author. reaction rate of NAD-dependent ADHs in the oxidative direction.In this report, the cloning and nucleotide sequence analysis of the B. methanolicus Cl mdh gene are presented. The deduced amino acid sequence was found to share sipificant identity with the other known members of type III ADHs. The C-terminal part of the bifunctional E. coli fermentative ADH (10) could also be incorporated in this cluster. Sequence patterns, which may be related to the distinctive properties of these enzymes, such as NAD(P) and metal binding, have been analyzed and are discussed. Expression and properties of the B. methanolicus MDH in the E. coli host were investigated. MATERIALS AND METHODS

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