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Biochemical and Structural Analysis of FomD That Catalyzes the Hydrolysis of Cytidylyl (S)-2-Hydroxypropylphosphonate in Fosfomycin Biosynthesis
- Sato, Shusuke, Miyanaga, Akimasa, Kim, Seung-Young, Kuzuyama, Tomohisa, Kudo, Fumitaka, Eguchi, Tadashi
- Biochemistry 2018 v.57 no.32 pp. 4858-4866
- Lewis bases, Streptomyces, bacteria, biochemical pathways, biosynthesis, catalytic activity, cobalt, crystal structure, fosfomycin, hydrolysis, hydrophobic bonding, manganese, metal ions, moieties, multigene family, mutational analysis, phosphorus, proteins
- In fosfomycin biosynthesis, the hydrolysis of cytidylyl (S)-2-hydroxypropylphosphonate [(S)-HPP-CMP] to afford (S)-HPP is the only uncharacterized step. Because FomD is an uncharacterized protein with a DUF402 domain that is encoded in the fosfomycin biosynthetic gene cluster, FomD was hypothesized to be responsible for this reaction. In this 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 hydrolyzed 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 barrel and the two α-helices at the C-terminus and serve to presumably activate the phosphonate group of substrates for hydrolysis. Docking simulations with (S)-HPP-CMP suggested that the methyl group at the C2 position of the HPP moiety is recognized by a hydrophobic interaction with Trp68. Further mutational analysis suggested that a conserved Tyr107 among the DUF402 domain family of proteins activates a water molecule to promote nucleophilic attack on the phosphorus atom of the phosphonate moiety. These findings provide mechanistic insights into the FomD reaction and lead to a complete understanding of the fosfomycin biosynthetic pathway in Streptomyces.