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Genetics of the glutamate‐mediated methylamine utilization pathway in the facultative methylotrophic beta‐proteobacterium Methyloversatilis universalis FAM5
Author(s) -
Latypova Ekaterina,
Yang Song,
Wang YiShun,
Wang Tiansong,
Chavkin Theodore A.,
Hackett Murray,
Schäfer Hendrik,
Kalyuzhnaya Marina G.
Publication year - 2010
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.2009.06989.x
Subject(s) - biology , glutamate dehydrogenase , biochemistry , gene , glutamate synthase , glutamine synthetase , methylamine , metabolic pathway , genetics , enzyme , protein subunit , glutamine amidotransferase , gene cluster , atp synthase , glutamate receptor , glutamine , amino acid , receptor
Summary The ability of some microbial species to oxidize monomethylamine via glutamate‐mediated pathways was proposed in the 1960s; however, genetic determinants of the pathways have never been described. In the present study we describe a gene cluster essential for operation of the N ‐methylglutamate pathway in the methylotrophic beta‐proteobacterium Methyloversatilis universalis FAM5. Four major polypeptides from protein fractions displaying high activities of N ‐methylglutamate synthetase, N ‐methylglutamate dehydrogenase and γ‐glutamylmethylamide synthetase were selected for mass spectrometry‐based identification. The activities of enzymes were associated with the presence of peptides identified as ferredoxin‐dependent glutamate synthase (GltB2), large subunit of putative heterotetrameric sarcosine oxidase (SoxA) and glutamine synthetase type III (GSIII) respectively. A gene cluster (8.3 kb) harbouring gltB2, soxA and gsIII ‐like genes was amplified from M. universalis FAM5, sequenced and assembled. Two partial and six complete open reading frames arranged in the order soxBDAG‐gsIII‐gltB132 were identified and subjected to mutational analysis, functional and metabolic profiling. We demonstrated that gltB ‐like and sox ‐like genes play a key role in methylamine utilization and encode N ‐methylglutamate synthetase and N ‐methylglutamate dehydrogenase respectively. Metabolic, enzymatic and mutational analyses showed that the gsIII ‐like gene encodes γ‐glutamylmethylamide synthetase; however, this enzyme is not essential for oxidation of methylamine.