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

Leonid V. Bystrykh, Janet Vonck, Ernst F.J. van Bruggen, Jozef van Beeumen, Bart Samyn, Natalya I. Govorukhina, Nico Arfman, Johannis A. Duine, Lubbert Dijkhuizen

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    Abstract

    The quaternary protein structure of two methanol:N,N'-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 to 50 kDa. Both methanol:NDMA oxidoreductases possess a tightly but noncovalently bound NADP(H) cofactor at an NADPH-to-subunit molar ratio of 0.7. These cofactors are redox active toward alcohol and aldehyde substrates. Both enzymes contain significant amounts of Zn2+ and Mg2+ ions. The primary amino acid sequences of the A. methanolica and M. gastri MB19 methanol:NDMA oxidoreductases share a high degree of identity, as indicated by N-terminal sequence analysis (63% identity among the first 27 N-terminal amino acids), internal peptide sequence analysis, and overall amino acid composition. The amino acid sequence analysis also revealed significant similarity to a decameric methanol dehydrogenase of Bacillus methanolicus C1.

    Original languageEnglish
    Pages (from-to)1814-1822
    Number of pages9
    JournalJournal of Bacteriology
    Volume175
    Issue number6
    Publication statusPublished - Mar-1993

    Keywords

    • ALCOHOL-DEHYDROGENASE
    • ZYMOMONAS-MOBILIS
    • ESCHERICHIA-COLI
    • BIOLOGICAL MACROMOLECULES
    • SACCHAROMYCES-CEREVISIAE
    • THERMOTOLERANT BACILLUS
    • PROTEIN
    • IMAGES
    • GENE
    • PARTICLES

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