Stereochemical Course of Enzymatic Enolpyruvyl Transfer and Catalytic Conformation of the Active Site Revealed by the Crystal Structure of the Fluorinated Analogue of the Reaction Tetrahedral Intermediate Bound to the Active Site of the C115A Mutant of MurA

Abstract

MurA (UDP-GlcNAc enolpyruvyl transferase), the first enzyme in bacterial peptidoglycan biosynthesis, catalyzes the enolpyruvyl transfer from phosphoenolpyruvate (PEP) to the 3'-OH of UDP-GlcNAc by an addition-elimination mechanism that proceeds through a tetrahedral ketal intermediate. The crystal structure of the Cys115-to-Ala (C115A) mutant of Escherichia coli MurA complexed with a fluoro analogue of the tetrahedral intermediate revealed the absolute configuration of the adduct and the stereochemical course of the reaction. The fluorinated adduct was generated in a preincubation of wild-type MurA with (Z)-3-fluorophosphoenolpyruvate (FPEP) and UDP-GlcNAc and purified after enzyme denaturation. The fluorine substituent stabilizes the tetrahedral intermediate toward decomposition by a factor of 10(4)-10(6), facilitating manipulation of the adduct. The C115A mutant of MurA was utilized to avoid the microheterogeneity that arises in the wild-type MurA from the attack of Cys115 on C-2 of FPEP in competition with the formation of the fluorinated adduct. The crystal structure of the complex was determined to 2.8 A resolution, and the absolute configuration at C-2 of the adduct was found to be 2R. Thus, addition of the 3'-OH of UDP-GlcNAc is to the 2-si face of FPEP, corresponding to the 2-re face of PEP. Given the previous observation that, in D2O, the addition of D+ to C-3 of PEP proceeds from the 2-si face [Kim, D. H., Lees, W. J., and Walsh, C. T. (1995) J. Am. Chem. Soc. 117, 6380-6381], the addition across the double bond of PEP is anti. Also, because the overall stereochemical course has been shown to be either anti/syn or syn/anti [Lees, W. J., and Walsh, C. T. (1995) J. Am. Chem. Soc. 117, 7329-7337], it now follows that the stereochemistry of elimination of H+ from C-3 and Pi from C-2 of the tetrahedral intermediate of the reaction is syn.

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

“…The reaction mechanisms of both MurA and AroA have been studied intensively with pre-steady state experiments (11,14,15), site directed mutagenesis (11), substrate analogues (16)(17)(18), x-ray crystallography (7,19,20), fluorescence titration (21) and NMR experiments (6,22). Their mechanisms are similar in several aspects.…”

“…6). Both Duncan (19) and Bartlett (28) pointed out that phosphate could act as an intramolecular proton acceptor. Figure 6.…”

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

“…Inhibition of MurA by the antibiotic fosfomycin (3) or the viral maturation protein A 2 (4) results in bacterial cell lysis and death. The MurA reaction proceeds through a tetrahedral intermediate of substrates UDP-N-acetylglucosamine (UNAG) 3 and phosphoenolpyruvate (PEP) followed by elimination of inorganic phosphate to yield the enolpyruvyl product (EP-UNAG) (5,6). Enzyme mechanistic studies established that substrate PEP covalently reacts with the catalytically important residue Cys-115 to form a phospholactoyl adduct, suggesting that nucleophilic attack is a component of the reaction cycle (7)(8)(9).…”

Identification and Characterization of a MurA, UDP-N-Acetylglucosamine Enolpyruvyl Transferase from Cariogenic Streptococcus Mutans

“…Like other bacterial MurA, the inactivation was enhanced by the presence of UDPGlcNAc 9) since UDP-GlcNAc preincubation effects a conformation change in the enzyme 22) . From structural studies, it has been determined that E. coli MurA assumes a closed conformation in the presence of UDP-GlcNAc and fosfomycin 23) . Furthermore, the MurA is isolated from E. coli with PEP covalently bound to Cys115; addition of UDP-GlcNAc turns over the bound PEP to product, catalyzing the reaction 24) .…”