title: 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: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.
authors.affiliation: Department of Microbiology, University of Groningen, Haren, The Netherlands.
authors.forename: L V
authors.initials: LV
authors.lastname: Bystrykh
authors.forename: J
authors.initials: J
authors.lastname: Vonck
authors.forename: E F
authors.initials: EF
authors.lastname: van Bruggen
authors.forename: J
authors.initials: J
authors.lastname: van Beeumen
authors.forename: B
authors.initials: B
authors.lastname: Samyn
authors.forename: N I
authors.initials: NI
authors.lastname: Govorukhina
authors.forename: N
authors.initials: N
authors.lastname: Arfman
authors.forename: J A
authors.initials: JA
authors.lastname: Duine
authors.forename: L
authors.initials: L
authors.lastname: Dijkhuizen
mesh_terms: D039903:Actinobacteria / Q000201:enzymology* / Q000648:ultrastructure; D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000648:ultrastructure; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003067:Coenzymes / Q000378:metabolism; D007091:Image Processing, Computer-Assisted; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009161:Mycobacterium / Q000201:enzymology* / Q000648:ultrastructure; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
chemical_list: D000596:Amino Acids; D003067:Coenzymes; D000429:Alcohol Oxidoreductases; C095705:alcohol - N, N'-dimethyl-4-nitrosoaniline oxidoreductase