17 ホスホマイシン生合成におけるC-メチル化機構 (口頭発表の部)

抄録

Fosfomycin is a clinically approved broad-spectrum antibiotic produced by Streptomyces and Pseudomonas. The structural feature of fosfomycin is the characteristic phosphonate group that is attached to the C1 of methyl oxirane. Although five genes were identified to be involved in the fosfomycin biosynthesis in Streptomyces by genetic analysis and in vitro enzymatic analysis with recombinant enzymes, the biosynthetic mechanism that links between the 2-hydroxyethylphosphonate (HEP) and 2-hydroxypropylphosphonate (HPP) intermediates was elusive. In this study, functional analysis of a cobalamin-dependent radical S-adenosyl-L-methionine C-methyltransferase Fom3 that is coded in the fosfomycin biosynthetic gene cluster was carried out. When cytidylylated 2-hydroxyethylphosphonate (HEP-CMP) was incubated in the presence of SAM, methylcobalamin (MeCbl), methylviologen, NADH and dithiothreitol (DTT) with reconstituted Fom3, the production of cytidylylated 2-hydroxypropylphosphonate (HPP-CMP) was clearly observed. The production of 5'-deoxyadenosine (5'-dA) was also observed to indicate that the 5'-deoxyadenosyl radical was generated to abstract the hydrogen atom of the substrate HEP-CMP. In the presence of “HEP” instead of HEP-CMP, the production of 5'-dA was not detected, confirming that HEP is not an appropriate substrate of Fom3. This result suggests that the CMP moiety of HEP-CMP is critical for substrate recognition by Fom3. Therefore, HEP-CMP is likely an intermediate in the fosfomycin biosynthetic pathway. The Fom3 reaction product was also isolated to confirm the chemical structure by NMR. In the ¹H-NMR spectrum of the product, two sets of doublet methyl signals were observed around 1.15 ppm to indicate that Fom3 produced a mixture of two diastereomers. The ratio of the (S)- and (R)-HPP-CMP in the Fom3 reaction product was almost equal, thereby revealing that there is no stereoselectivity during the C-methylation. The production of S-adenosyl-L-homocysteine (SAH) was also observed, indicating that MeCbl is regenerated by SAM for the catalytic cycle. The production ratio of HPP-CMP, 5'-dA and SAH was almost equal. Based on these results, we propose the Fom3 reaction mechanism as follows. At first, 5'-deoxyadenosyl radical generated from the reductive cleavage of SAM abstracts one of the hydrogen atoms at C2 of HEP-CMP to generate the substrate radical intermediate, which reacts with the methyl group on MeCbl at the opposite side from SAM and the [4Fe-4S] cluster to give (S)- and (R)-HPP-CMP. The generated cob(II)alamin is reduced by DTT to cob(I)alamin, which is methylated in the presence of SAM to regenerate MeCbl.