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The complex structure of FomD with CDP
Structural highlights
FunctionPublication Abstract from PubMedIn 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 Mn(2+) or Co(2+). 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 beta-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 alpha-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. Biochemical and Structural Analysis of FomD That Catalyzes the Hydrolysis of Cytidylyl ( S)-2-Hydroxypropylphosphonate in Fosfomycin Biosynthesis.,Sato S, Miyanaga A, Kim SY, Kuzuyama T, Kudo F, Eguchi T Biochemistry. 2018 Aug 14;57(32):4858-4866. doi: 10.1021/acs.biochem.8b00690., Epub 2018 Jul 25. PMID:30010320[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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