Role of the loop containing residue 115 in the induced-fit mechanism of the bacterial cell wall biosynthetic enzyme MurA

The induced-fit mechanism in Enterobacter cloacae MurA has been investigate d by kinetic studies and X-ray crystallography. The antibiotic fosfomycin, an irreversible inhibitor of MurA, induced a structural change in UDP-N-ace tylglucosamine (UDPGlcNAc)liganded enzyme with a time dependence similar to that observed for the inactivation progress. The mechanism of action of fo sfomycin on MurA appeared to be of the bimolecular type, the overall rate c onstants of inactivation and structural change. being k(inact)(*) = 104 M-1 s(-1) and k(struc)(*) = 85 M-1 s(-1), respectively. Fosfomycin as well as the second MurA substrate, phosphoenolpyruvate (PEP), are known to interact with the side chain of Cys115. Like wildtype MurA, the catalytically inact ive single-site mutant protein Cys115Ser structurally interacted with UDPGl cNAc in a rapidly reversible reaction. However, in contrast to wild-type en zyme, binding of PEP to mutant protein induced a rate-limited, biphasic str uctural change. Fosfomycin did not affect the structure of the mutant prote in. The crystal structure of unliganded Cys115Ser MurA at 1.9 Angstrom reso lution revealed that the overall conformation of the loop comprising residu es 112-121 is not influenced by the mutation. However, other than Cys115 in wild-type MurA, Ser115 exhibits two distinct side-chain conformations. A d etailed view on the loop revealed the existence of an elaborate hydrogen-bo nding network mainly supplied by water molecules, presumably stabilizing it s conformation in the unliganded state. The comparison between the known cr ystal structures of MurA, together with the kinetic data obtained, suggest intermediate conformational states in the MurA reaction, in which the loop undergoes multiple structural changes upon ligand binding.