Sato Shusuke, Kudo Fumitaka, Kuzuyama Tomohisa, Hammerschmidt Friedrich, Eguchi Tadashi
Department of Chemistry , Tokyo Institute of Technology , 2-12-1 O-okayama , Meguro-ku, Tokyo 152-8551 , Japan.
Biotechnology Research Center, Collaborative Research Institute for Innovative Microbiology , The University of Tokyo , 1-1-1 Yayoi , Bunkyo-ku, Tokyo 113-8657 , Japan.
Biochemistry. 2018 Aug 21;57(33):4963-4966. doi: 10.1021/acs.biochem.8b00614. Epub 2018 Jul 12.
Fom3, a cobalamin-dependent radical S-adenosyl-l-methionine (SAM) methyltransferase, catalyzes C-methylation at the C2 position of cytidylylated 2-hydroxyethylphosphonate (HEP-CMP) to afford cytidylylated 2-hydroxypropylphosphonate (HPP-CMP) in fosfomycin biosynthesis. In this study, the Fom3 reaction product HPP-CMP was reanalyzed by chiral ligand exchange chromatography to confirm its stereochemistry. The Fom3 methylation product was found to be ( S)-HPP-CMP only, indicating that the stereochemistry of the C-methylation catalyzed by Fom3 is ( S)-selective. In addition, Fom3 reaction was performed with ( S)-[2-H]HEP-CMP and ( R)-[2-H]HEP-CMP to elucidate the stereoselectivity during the abstraction of the hydrogen atom from C2 of HEP-CMP. Liquid chromatography-electrospray ionization mass spectrometry analysis of the 5'-deoxyadenosine produced showed that the H atom of ( R)-[2-H]HEP-CMP was incorporated into 5'-deoxyadenosine but that from ( S)-[2-H]HEP-CMP was not. Retention of the H atom of ( S)-[2-H]HEP-CMP in HPP-CMP was also observed. These results indicate that the 5'-deoxyadenosyl radical stereoselectively abstracts the pro-R hydrogen atom at the C2 position of HEP-CMP and the substrate radical intermediate reacts with the methyl group on cobalamin that is located on the opposite side of the substrate from SAM. Consequently, it was clarified that the C-methylation catalyzed by Fom3 proceeds with inversion of configuration.
Fom3是一种依赖钴胺素的自由基S-腺苷-L-甲硫氨酸（SAM）甲基转移酶，在磷霉素生物合成中催化胞苷酰化2-羟乙基膦酸酯（HEP-CMP）的C2位发生C-甲基化反应，生成胞苷酰化2-羟丙基膦酸酯（HPP-CMP）。在本研究中，通过手性配体交换色谱法对Fom3反应产物HPP-CMP进行了重新分析，以确认其立体化学结构。结果发现Fom3甲基化产物仅为（S）-HPP-CMP，这表明Fom3催化的C-甲基化反应的立体化学是（S）-选择性的。此外，用（S）-[2-H]HEP-CMP和（R）-[2-H]HEP-CMP进行Fom3反应，以阐明从HEP-CMP的C2位提取氢原子过程中的立体选择性。对生成的5'-脱氧腺苷进行液相色谱-电喷雾电离质谱分析表明，（R）-[2-H]HEP-CMP的H原子被掺入到5'-脱氧腺苷中，而（S）-[2-H]HEP-CMP的H原子则未被掺入。还观察到（S）-[2-H]HEP-CMP的H原子保留在HPP-CMP中。这些结果表明，5'-脱氧腺苷自由基立体选择性地提取HEP-CMP C2位的前-R氢原子，底物自由基中间体与钴胺素上位于底物与SAM相对侧的甲基发生反应。因此，明确了Fom3催化的C-甲基化反应是构型翻转进行的。
