title: {gamma}-Glutamylmethylamide is an essential intermediate in the metabolism of methylamine by Methylocella silvestris.
abstract: Methylocella silvestris BL2, a facultative methane utilizer, can grow on monomethylamine (MMA) as a sole carbon and nitrogen source. No activity of MMA dehydrogenase was detectable. Instead, this bacterium utilizes a methylated amino acid pathway (gamma-glutamylmethylamide [GMA] and N-methylglutamate [NMG]) for MMA metabolism. The activities of the two key enzymes in this pathway, GMA synthetase and NMG dehydrogenase, were found when the bacterium was grown on MMA. GMA was detected by high-performance liquid chromatography-mass spectrometry only when the bacterium was grown on MMA but not when it was grown on methanol. Proteomic analysis of soluble and membrane fractions of the proteome from MMA- and methanol-grown cultures revealed that an eight-gene cluster (Msil2632 to Msil2639) was induced by MMA and cotranscribed as an operon, as shown by reverse transcription-PCR. GMA-dissimilating enzyme activity was also detected when it was grown on MMA. Formaldehyde and ammonium production from GMA was dependent on glutamate but not on alpha-ketoglutarate. Marker exchange mutagenesis of a putative GMAS gene homologue (gmas, Msil2635) within this eight-gene cluster, with a kanamycin gene cassette, abolished growth of M. silvestris on MMA as either a sole carbon or a sole nitrogen source. Overall, our results suggest that gmas is essential in MMA metabolism by M. silvestris.
authors.affiliation: Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom.
authors.forename: Yin
authors.initials: Y
authors.lastname: Chen
authors.forename: Julie
authors.initials: J
authors.lastname: Scanlan
authors.forename: Lijiang
authors.initials: L
authors.lastname: Song
authors.forename: Andrew
authors.initials: A
authors.lastname: Crombie
authors.forename: M Tanvir
authors.initials: MT
authors.lastname: Rahman
authors.forename: Hendrik
authors.initials: H
authors.lastname: Schäfer
authors.forename: J Colin
authors.initials: JC
authors.lastname: Murrell
mesh_terms: D042062:Beijerinckiaceae / Q000201:enzymology / Q000254:growth & development / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003470:Culture Media; D015964:Gene Expression Regulation, Bacterial; D005971:Glutamates / Q000378:metabolism*; D008025:Ligases / Q000235:genetics / Q000378:metabolism; D013058:Mass Spectrometry; D008744:Methylamines / Q000378:metabolism*; D005810:Multigene Family; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism; D040901:Proteomics; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D003470:Culture Media; D005971:Glutamates; D008744:Methylamines; C476559:N-methylglutamate; C014574:gamma-glutamylmethylamide; C027451:methylamine; D010088:Oxidoreductases; D008025:Ligases
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