Department of Biochemistry and Molecular Biology, Eberly College of Science, The Pennsylvania State University, University Park, Pennsylvania, USA.
J Bacteriol. 2012 Aug;194(16):4161-8. doi: 10.1128/JB.00593-12. Epub 2012 May 25.
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).
产甲烷八叠球菌的基因组编码了三个同源物，最初被注释为假设的融合钴胺素/甲基转移蛋白，它们在 CO 生长的细胞中高度升高，而在使用替代底物生长的细胞中则降低。仅基于缺失突变体的表型分析，先前的结论是，这些同源物是严格的二甲基硫醚：辅酶 M（CoM）甲基转移酶，不参与 CO 的代谢（E. Oelgeschlager 和 M. Rother，Mol. Microbiol. 72:1260-1272, 2009）。由 MA4383 编码的同源物（此处指定为 CmtA）通过在大肠杆菌中过表达该蛋白的生化特性进行了重新检查。用甲基钴胺（III）alamin 重建的纯化 CmtA 含有 1.0 ± 0.2 的钴与蛋白质摩尔比。紫外可见光谱是典型的甲基化钴胺素蛋白，在 370nm 和 420nm 处有吸收峰，在 450nm 至 600nm 之间有一个宽吸收带，在 525nm、490nm 和 550nm 处有吸收峰。用水合钴胺素重建的 CmtA 显示出甲基四氢甲酰基四氢叶酸：CoM（CH(3)-THMPT:HS-CoM）甲基转移酶活性（0.31 μmol/min/mg），CH(3)-THMPT 的表观 K(m)值为 135 μM，HS-CoM 的表观 K(m)值为 277 μM。在 CO 生长的细胞中，可溶性细胞部分与膜细胞部分之间的 CH(3)-THMPT:HS-CoM 甲基转移酶活性之比比甲醇生长的细胞高 15 倍。当用生长受限的 CO 分压培养时，缺失 CmtA 基因的突变株的生长速度和最终产量较低，表明 CmtA 在利用该底物生长时发挥作用。结果表明，CmtA 是一种可溶性 CH(3)-THSPT:HS-CoM 甲基转移酶，推测在产甲烷八叠球菌依赖 CO 的生长过程中补充膜结合的 CH(3)-THMPT:HS-CoM 甲基转移酶。因此，我们建议将 MA4384 编码的酶命名为 CmtA（细胞质甲基转移酶）。
