Role of the fused corrinoid/methyl transfer protein CmtA during CO-dependent growth of Methanosarcina acetivorans.
The genome of Methanosarcina acetivorans encodes three homologs, initially annotated as hypothetical fused corrinoid/methyl transfer proteins, which are highly elevated in CO-grown cells versus cells grown with alternate substrates. Based only on phenotypic analyses of deletion mutants, it was previously concluded that the homologs are strictly dimethylsulfide:coenzyme M (CoM) methyltransferases not involved in the metabolism of CO (E. Oelgeschlager and M. Rother, Mol. Microbiol. 72:1260 -1272, 2009). The homolog encoded by MA4383 (here designated CmtA) was reexamined via biochemical characterization of the protein overproduced in Escherichia coli. Purified CmtA reconstituted with methylcob(III)alamin contained a molar ratio of cobalt to protein of 1.0 ± 0.2. The UV-visible spectrum was typical of methylated corrinoid-containing proteins, with absorbance maxima at 370 and 420 nm and a band of broad absorbance between 450 and 600 nm with maxima at 525, 490, and 550 nm. CmtA reconstituted with aquocobalamin showed methyl-tetrahydromethanopterin:CoM (CH(3)-THMPT:HS-CoM) methyltransferase activity (0.31 μmol/min/mg) with apparent K(m) values of 135 μM for CH(3)-THMPT and 277 μM for HS-CoM. The ratio of CH(3)-THMPT:HS-CoM methyltransferase activity in the soluble versus membrane cellular fractions was 15-fold greater in CO-grown versus methanol-grown cells. A mutant strain deleted for the CmtA gene showed lower growth rates and final yields when cultured with growth-limiting partial pressures of CO, demonstrating a role for CmtA during growth with this substrate. The results establish that CmtA is a soluble CH(3)-THSPT:HS-CoM methyltransferase postulated to supplement the membrane-bound CH(3)-THMPT:HS-CoM methyltransferase during CO-dependent growth of M. acetivorans. Thus, we propose that the name of the enzyme encoded by MA4384 be CmtA (for cytoplasmic methyltransferase).
Department of Biochemistry and Molecular Biology, Eberly College of Science, The Pennsylvania State University, University Park, Pennsylvania, USA.
Venkata R
VR
Vepachedu
James G
JG
Ferry
D002248:Carbon Monoxide / Q000378:metabolism*; D003001:Cloning, Molecular; D003067:Coenzymes / Q000378:metabolism; D045728:Corrinoids / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D017353:Gene Deletion; D015870:Gene Expression; D007700:Kinetics; D015080:Mesna / Q000378:metabolism; D017020:Methanosarcina / Q000201:enzymology* / Q000254:growth & development* / Q000378:metabolism; D008780:Methyltransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D014805:Vitamin B 12 / Q000031:analogs & derivatives / Q000378:metabolism
D003067:Coenzymes; D045728:Corrinoids; D011994:Recombinant Proteins; D002248:Carbon Monoxide; C019476:mecobalamin; D008780:Methyltransferases; C048755:tetrahydromethanopterin S-methyltransferase; D015080:Mesna; D014805:Vitamin B 12