Vannelli T, Messmer M, Studer A, Vuilleumier S, Leisinger T
Mikrobiologisches Institut, Swiss Federal Institute of Technology, ETH-Zentrum, CH-8092 Zurich, Switzerland.
Proc Natl Acad Sci U S A. 1999 Apr 13;96(8):4615-20. doi: 10.1073/pnas.96.8.4615.
Methylobacterium sp. strain CM4, an aerobic methylotrophic alpha-proteobacterium, is able to grow with chloromethane as a carbon and energy source. Mutants of this strain that still grew with methanol, methylamine, or formate, but were unable to grow with chloromethane, were previously obtained by miniTn5 mutagenesis. The transposon insertion sites in six of these mutants mapped to two distinct DNA fragments. The sequences of these fragments, which extended over more than 17 kb, were determined. Sequence analysis, mutant properties, and measurements of enzyme activity in cell-free extracts allowed the definition of a multistep pathway for the conversion of chloromethane to formate. The methyl group of chloromethane is first transferred by the protein CmuA (cmu: chloromethane utilization) to a corrinoid protein, from where it is transferred to H4folate by CmuB. Both CmuA and CmuB display sequence similarity to methyltransferases of methanogenic archaea. In its C-terminal part, CmuA is also very similar to corrinoid-binding proteins, indicating that it is a bifunctional protein consisting of two domains that are expressed as separate polypeptides in methyl transfer systems of methanogens. The methyl group derived from chloromethane is then processed by means of pterine-linked intermediates to formate by a pathway that appears to be distinct from those already described in Methylobacterium. Remarkable features of this pathway for the catabolism of chloromethane thus include the involvement of a corrinoid-dependent methyltransferase system for dehalogenation in an aerobe and a set of enzymes specifically involved in funneling the C1 moiety derived from chloromethane into central metabolism.
甲基营养型芽孢杆菌CM4菌株是一种需氧甲基营养型α-变形杆菌，能够以氯甲烷作为碳源和能源进行生长。该菌株的突变体仍能利用甲醇、甲胺或甲酸生长，但不能利用氯甲烷生长，这些突变体先前是通过miniTn5诱变获得的。其中六个突变体中的转座子插入位点定位于两个不同的DNA片段。测定了这些片段的序列，其长度超过17 kb。通过序列分析、突变体特性以及无细胞提取物中酶活性的测定，确定了氯甲烷转化为甲酸的多步途径。氯甲烷的甲基首先由蛋白质CmuA（cmu：氯甲烷利用）转移至类咕啉蛋白，然后由CmuB将其从类咕啉蛋白转移至四氢叶酸。CmuA和CmuB均与产甲烷古菌的甲基转移酶具有序列相似性。在其C末端部分，CmuA也与类咕啉结合蛋白非常相似，这表明它是一种双功能蛋白，由两个结构域组成，而在产甲烷菌的甲基转移系统中这两个结构域是作为单独的多肽表达的。来自氯甲烷的甲基随后通过与蝶呤相关的中间体，经一条似乎与甲基营养型芽孢杆菌中已描述的途径不同的途径加工形成甲酸。因此，这条氯甲烷分解代谢途径的显著特征包括需氧生物中参与脱卤作用的类咕啉依赖性甲基转移酶系统，以及一组专门参与将来自氯甲烷的C1部分导入中心代谢的酶。
