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

Abstract

UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) catalyzes the first committed step in the biosynthesis of the bacterial cell wall component peptidoglycan. The enzyme is the target of the antibiotic fosfomycin. A lysine residue (K22), strictly conserved in MurAs and the structurally and mechanistically 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 formation of the catalytically competent enzyme complex. Using site-directed mutagenesis, 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 inactive C115S mutant protein. It was found that all three K22 mutant proteins 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-sugar nucleotide substrate are not affected by the mutations, except for the K22E mutant protein. Similarly, binding of PEP was found to be unaffected in the K22 mutant proteins as demonstrated by tryptophan fluorescence quench titrations. On the other hand, the level of formation of a covalent adduct with either PEP or fosfomycin with the thiol group of cysteine 115 was diminished. The propensity to form an adduct with PEP decreased in the following order: wild type >> K22R > K22V > K22E. A comparable effect was found on the formation of the inhibitory covalent adduct of MurA and the antibiotic fosfomycin. These results are discussed in terms of an involvement of lysine 22 in a conformational change of MurA.

Structural and Functional Characterization of NikO, an Enolpyruvyl Transferase Essential in Nikkomycin Biosynthesis

“…4A (bottom panel, lanes 4 and 5), the test was clearly positive, indicating that NikO binds fosfomycin even in the absence of UMP. This finding is in contrast to the behavior of MurA whose reactivity toward fosfomycin is enhanced in the presence of UDPNAG (15,42) (Fig. 4A, bottom panel, lane 3).…”

“…The specific activity of NikO was 132 nmol P i min Ϫ1 mg Ϫ1 . For comparison, MurA from Enterobacter cloacae has a specific activity of 1300 nmol P i min Ϫ1 mg Ϫ1 and a k cat of 60 min Ϫ1 (42). Activity measurements with both NikO variants (C130A and D342A) showed that both amino acid exchanges lead to inactive enzyme (supplemental Fig.…”

Cloning, Expression and Characterization of UDP-N-Acetylglucosamine Enolpyruvyl Transferase (MurA) from Wolbachia Endosymbiont of Human Lymphatic Filarial Parasite Brugia malayi

“…Sequence and alignment analysis of w Bm-MurA with other bacterial MurA shows conservation of five important amino acid residues viz. Lys22, which participates in the formation of covalent adducts with PEP and fosfomycin [59], Cys115 and Asp305, which are involved in the product release and the final deprotonation step [60], Asp369 and Leu370 which facilitate interaction of fosfomycin with MurA [42] (all a.a. residues with ref. to E. coli ).…”

The structural biology of enzymes involved in natural product glycosylation

“…Conserved interactions include participation of K22 in the formation of covalent adducts with PEP and fosfomycin 207, 208 ; C115 (Figure 20D) in the participation of catalysis and product release 205 ; D305 in the final deprotonation from the C3 atom of the tetrahedral intermediate 209 ; and D369 and L370 for specific interactions with fosfomycin (both residues mutated in resistant strains) 210 .…”