Lysine 22 in UDP-N-acetylglucosamine enolpyruvyl transferase from Enterobacter cloacae is crucial for enzymatic activity and the formation of covalent adducts with the substrate phosphoenolpyruvate and the antibiotic fosfomycin

UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) catalyzes the first committed step in the biosynthesis of the bacterial cell wall component pep eidoglycan. The enzyme is the target of the antibiotic fosfomycin. A lysine residue (K22), strictly conserved in MurAs and the structurally and mechan istically related 5-enolpyruvylshikimate 3-phosphate synthases (EPSPS), is located near the active center of the enzyme. This residue is thought to be involved directly in the binding of the substrate phosphoenolpyruvate (PEP ) and also to participate in the conformational change leading to the forma tion of the catalytically competent enzyme complex. Using site-directed mut agenesis, we have replaced this lysine with arginine (K22R), valine (K22V), and glutamate (K22E). These mutant proteins were expressed, purified, and characterized in comparison to wild-type MurA and a previously described in active C115S mutant protein. It was found that all three K22 mutant protein s had less than 0.5% of the wild-type activity. Using isothermal titration calorimetry, it could be shown that the binding parameters for the UDP-suga r nucleotide substrate are not affected by the mutations, except for the K2 2E mutant protein. Similarly, binding of PEP was found to be unaffected in the K22 mutant proteins as demonstrated by tryptophan fluorescence quench t itrations. On the other hand, the level of formation of a covalent adduct w ith either PEP or fosfomycin with the thiol group of cysteine 115 was dimin ished. The propensity to form an adduct with PEP decreased in the following order: wild type much greater than K22R > K22V > K22E. A comparable effect was found on the formation of the inhibitory covalent adduct of MurA and t he antibiotic fosfomycin. These results are discussed in terms of an involv ement of lysine 22 in a conformational change of MurA.