Role of the Loop Containing Residue 115 in the Induced-Fit Mechanism of the Bacterial Cell Wall Biosynthetic Enzyme MurA

Abstract

The induced-fit mechanism in Enterobacter cloacae MurA has been investigated by kinetic studies and X-ray crystallography. The antibiotic fosfomycin, an irreversible inhibitor of MurA, induced a structural change in UDP-N-acetylglucosamine (UDPGlcNAc)-liganded enzyme with a time dependence similar to that observed for the inactivation progress. The mechanism of action of fosfomycin on MurA appeared to be of the bimolecular type, the overall rate constants of inactivation and structural change being = 104 M(-1) s(-1) and = 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 wild-type MurA, the catalytically inactive single-site mutant protein Cys115Ser structurally interacted with UDPGlcNAc in a rapidly reversible reaction. However, in contrast to wild-type enzyme, binding of PEP to mutant protein induced a rate-limited, biphasic structural change. Fosfomycin did not affect the structure of the mutant protein. The crystal structure of unliganded Cys115Ser MurA at 1.9 A resolution revealed that the overall conformation of the loop comprising residues 112-121 is not influenced by the mutation. However, other than Cys115 in wild-type MurA, Ser115 exhibits two distinct side-chain conformations. A detailed view on the loop revealed the existence of an elaborate hydrogen-bonding network mainly supplied by water molecules, presumably stabilizing its conformation in the unliganded state. The comparison between the known crystal 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.

Recruitment of genes and enzymes conferring resistance to the nonnatural toxin bromoacetate

“…The inactivation rate of MurA with fosfomycin is also dependent upon UDP-GlcNAc (8). Surprisingly, the second-order inactivation rate of MurA by bromoacetate, under saturating UDPGlcNAc at pH 8.0, is only 9-fold slower than by fosfomycin under saturating UDP-GlcNAc at pH 6.9 (104 M −1 s −1 ) (26).…”

Phosphate Analogues as Probes of the Catalytic Mechanisms of MurA and AroA, Two Carboxyvinyl Transferases

“…They concluded that PEP inside the catalytic center was protonated and the enzyme stabilized an oxacarbenium ion upon binding with UDP-GicNAc. The substrate-induced conformational change was later demonstrated in both crystal structure (7,25,26) and fluorescence experiments (21).…”

“…PEP also interacts with it to form a covalently linked phospholactoyl-enzyme adduct. Cys115 is located in a loop which undergoes a large conformational change as the enzyme binds to the substrates (26). Site-directed mutagenesis experiments suggest it acts as a general acid in protonating the C3 position in the addition step, and a general base for decomposition of THI in both forward and backward directions (11).…”

Functional Consequence of Covalent Reaction of Phosphoenolpyruvate with UDP-N-acetylglucosamine 1-Carboxyvinyltransferase (MurA)

“…However, the discovery that Cys-115 could be replaced by an aspartate without loss of activity (a mutation found in some bacteria including Mycobacteria) led to the conclusion that Cys-115 functions as a general acid-base catalyst and not as a nucleophile in the reaction with PEP (10). Cys-115 is a member of a 10-residue loop in the N-terminal globular domain that undergoes drastic conformational changes upon binding of UNAG by approaching the interdomain cleft and positioning the Cys-115 side chain into the PEP binding site (11)(12)(13)(14)(15). Cys-115 has received considerable attention as the target of covalent modification by fosfomycin (12) and other electrophilic natural products such as terreic acid (16) and cnicin (17).…”