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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.