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Methanol metabolism in thermotolerant methylotrophic Bacillus strains involving a novel catabolic NAD-dependent methanol dehydrogenase as a key enzyme

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Abstract

The enzymology of methanol utilization in thermotolerant methylotrophic Bacillus strains was investigated. In all strains an immunologically 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 K m 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.

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Abbreviations

RuMP:

ribulose monophosphate

HPS:

hexulose-6-phosphate synthase

HPI:

hexulose-6-phosphate isomerase

MDH:

methanol dehydrogenase

ADH:

acohol dehydrogenase

PQQ:

pyrroloquinoline, quinone

DTT:

dithiothreitol

NBT:

nitrobluetetrazolium

PMS:

phenazine methosulphate

DCPIP:

dichlorophenol indophenol

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Author notes

  1. W. Clement

    Present address: DMV Campina bv, P.O. Box 13, NL-5460 BA, Veghel, The Netherlands

Authors and Affiliations

  1. Department of Microbiology, University of Groningen, NL-9751 NN, Haren, The Netherlands

    N. Arfman, W. Clement, R. J. van Oosterwijk, G. E. de Vries, W. Harder, M. M. Attwood & L. Dijkhuizen

  2. Department of Microbiology, University of Sheffield, S10 2TN, Sheffield, UK

    E. M. Watling

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  1. N. Arfman
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  4. R. J. van Oosterwijk
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  5. G. E. de Vries
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  6. W. Harder
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  7. M. M. Attwood
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  8. L. Dijkhuizen
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Arfman, N., Watling, E.M., Clement, W. et al. Methanol metabolism in thermotolerant methylotrophic Bacillus strains involving a novel catabolic NAD-dependent methanol dehydrogenase as a key enzyme. Arch. Microbiol. 152, 280–288 (1989). https://doi.org/10.1007/BF00409664

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  • DOI: https://doi.org/10.1007/BF00409664

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