Lexan Lhu
Analysis of methyltransferase activity in carbon monoxide-grown Methanosarcina acetivorans
Isolated from marine sediments, the anaerobe Methanosarcina acetivorans is a methanogenic member of the archaea domain (Sowers, et al., 1984). M. acetivorans is capable of producing methane from acetate, methanol, and methylamines and was more recently found to produce methane from carbon monoxide (Rother and Metcalf, 2004 ). Reductions of these substrates to CH4 occur through a succession of methylreductase and methyltransferase reactions using the methyl carrier coenzyme tetrahydrosarcinapterin (THSPT). Many of the transformations involved in CO metabolism remain poorly characterized. A better understanding of CO metabolism is critical to balancing the global carbon cycle and to revealing steps behind a metabolic pathway that evolved in primitive life when Earth's atmosphere contained elevated CO levels.
My research is in collaboration with Dr. J. G. Ferry in the BMMB department, whose previous work using proteomic and Quantitative Real Time-PCR (RT-PCR) methods led to a proposed 8-step CO-to-CH4 pathway in M. acetivorans (Lessner, et al., 2006). The proposed pathway for CO metabolism begins with the initial oxidation of CO to CO2 via CO dehydrogenase, with concomitant reductions of coenzymes ferredoxin (Fd) and F420. These reduced coenzymes provide the electrons needed by methylreductases to reduce CO2 to methane. CO2 is first bound to the methyl carrier methanofuran (MF) creating CHO-MF. The formyl group is transferred to THSPT (forming CHO-THSPT) and reduced to CH3-THMPT. The methyl is next transferred to coenzyme M (CoM) by CH3-THMPT:CoM methyltransferase and finally CH3-CoM reductase completes the reduction to CH4.
Pathway proposed for the conversion of CO to acetate and methane by M. acetivorans. From Lessner, et al., 2006. Click on the image to view a larger version.
Analyses of the M. acetivorans proteome and genome (published at www.tigr.org) suggest the presence of novel methyltransferase activity which might play an important role during CO metabolism. Proteins putatively involved in CH3-THMPT:CoM methyl transfer contain a soluble corrinoid protein domain that is conserved in the well-studied, membrane bound, Na+-translocating CH3-THMPT:CoM methyltransferases widely found across all species of methanogenic archaea (Gottschalk, and Thauer, 2001). To reinforce and further study the proposed methanogenic pathway associated with CO reduction, RT-PCR is being used to determine whether the putative CH3-THMPT:CoM methyltransferase proteins are up-regulated during growth on CO. Methyltransferase activity assays are also being performed on various fractions from CO-grown M. acetivorans cell lysates (total lysate, membrane fraction, soluble fraction), and these are being compared to acetate-grown cells. Select proteins believed to be associated with soluble CO methyltransferase activity will be purified from M. acetivorans and/or a heterologous expression system, as well as deleted from the M. acetivorans genome, to better characterize their activities and relevance to CH3-THMPT:CoM methyl transfer during CO metabolism.
- Gottschalk, G., and Thauer RK., (2001). Biochimica et Biophysica Acta, 1505: 28-36
- Lessner, DJ., Li, L., Li, Q., Rejtar, T., Andreev, VP., Reichlen, M., Hill, K., Moran, JJ., Karger, BL., and Ferry JG., (2006). PNAS, 103:17921-17926
- Rother, M., and Metcalf, WW., (2004). PNAS, 101:16929-16934
- Sowers, KR., Baron, SF., and Ferry, JG., (1984). Applied and Environmental Microbiology, 47:971-978