Crystal structure of fosfomycin resistance kinase FomA from Streptomyces wedmorensis

The fosfomycin resistance protein FomA inactivates fosfomycin by phosphorylation of the phosphonate group of the antibiotic in the presence of ATP and Mg(II). We report the crystal structure of FomA from the fosfomycin biosynthetic gene cluster of Streptomyces wedmorensis in complex with diphosphate and in ternary complex with the nonhydrolyzable ATP analog adenosine 5'-(beta,gamma-imido)-triphosphate (AMPPNP), Mg(II), and fosfomycin, at 1.53 and 2.2 angstrom resolution, respectively. The polypeptide exhibits an open alpha beta alpha sandwich fold characteristic for the amino acid kinase family of enzymes. The diphosphate complex shows significant disorder in loops surrounding the active site. As a result, the nucleotide-binding site is wide open. Binding of the substrates is followed by the partial closure of the active site and ordering of the alpha 2-helix. Structural comparison with N-acetyl-L-glutamate kinase shows several similarities in the site of phosphoryl transfer: 1) preservation of architecture of the catalytical amino acids of N-acetyl-L-glutamate kinase(Lys(9), Lys(216), and Asp(150) in FomA); 2) good super-position of the phosphate acceptor groups of the substrates, and 3) good super-position of the diphosphate molecule with the beta-and gamma-phosphates of AMPPNP, suggesting that the reaction could proceed by an associative in-line mechanism. However, differences in conformations of the triphosphate moiety of AMPPNP molecules, the long distance (5.1 angstrom) between the phosphate acceptor and donor groups in FomA, and involvement of Lys(18) instead of Lys(9) in binding with the gamma-phosphate may indicate a different reaction mechanism. The present work identifies the active site residues of FomA responsible for substrate binding and specificity and proposes their roles in catalysis.