Genetics of the glutamate-mediated methylamine utilization pathway in the facultative methylotrophic beta-proteobacterium Methyloversatilis universalis FAM5.
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 gamma-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 gamma-glutamylmethylamide synthetase; however, this enzyme is not essential for oxidation of methylamine.
Latypova
Ekaterina
E
Department of Chemical Engineering, University of Washington, Seattle, WA 98195, USA.
Yang
Song
S
Wang
Yi-Shun
YS
Wang
Tiansong
T
Chavkin
Theodore A
TA
Hackett
Murray
M
Schäfer
Hendrik
H
Kalyuzhnaya
Marina G
MG
D000409:Alanine / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics; D020562:Betaproteobacteria / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D004252:DNA Mutational Analysis / Q000379:methods; D020869:Gene Expression Profiling / Q000379:methods; D005970:Glutamate Synthase / Q000235:genetics / Q000378:metabolism; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D005973:Glutamine / Q000378:metabolism; D007700:Kinetics; D008744:Methylamines / Q000378:metabolism*; D005810:Multigene Family; D016366:Open Reading Frames; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism
D001426:Bacterial Proteins; D008744:Methylamines; D010446:Peptide Fragments; D005973:Glutamine; D018698:Glutamic Acid; C027451:methylamine; D005970:Glutamate Synthase; D000409:Alanine