Biochemical and Structural Analysis of FomD That Catalyzes the Hydrolysis of Cytidylyl ( S)-2-Hydroxypropylphosphonate in Fosfomycin Biosynthesis.

In fosfomycin biosynthesis, the hydrolysis of cy-tidylyl (S)-2-hydroxypropylphosphonate ((S)-HPP-CMP) to afford (S)-HPP is the only uncharac-terized step. Since FomD is an uncharacterized pro-tein with a DUF402 domain that is encoded in the fosfomycin biosynthetic gene cluster, FomD was hypothesized to be responsible for this reaction. In the present study, FomD was found to hydrolyze (S)-HPP-CMP to give (S)-HPP and CMP efficiently in the presence of Mn2+ or Co2+. FomD also hydro-lyzed cytidylyl 2-hydroxyethylphosphonate (HEP-CMP), which is a biosynthetic intermediate before C-methylation. The kcat/KM value of FomD with (S)-HPP-CMP was 10-fold greater than that with HEP-CMP, suggesting that FomD hydrolyzes (S)-HPP-CMP rather than HEP-CMP in bacteria. The crystal structure of FomD showed that this protein adopts a barrel-like fold, which consists of a large twisted antiparallel β-sheet. This is a key structural feature of the DUF402 domain-containing proteins. Two metal cations are located between the FomD...