UDP-N-acetylmuramic Acid (UDP-MurNAc) Is a Potent Inhibitor of MurA (Enolpyruvyl-UDP-GlcNAc Synthase) † Shehadeh Mizyed, ‡,§ Anna Oddone, ‡ Bartosz Byczynski, ‡ Donald W. Hughes, ‡ and Paul J. Berti* ,‡,| Departments of Chemistry and Biochemistry and the Antimicrobial Research Centre, McMaster UniVersity, 1280 Main Street West, Hamilton, Ontario L8S 4M1, Canada ReceiVed October 27, 2004; ReVised Manuscript ReceiVed December 27, 2004 ABSTRACT: Purified recombinant MurA (enolpyruvyl-UDP-GlcNAc synthase) overexpressed in Escherichia coli had significant amounts of UDP-MurNAc (UDP-N-acetylmuramic acid) bound after purification. UDP-MurNAc is the product of MurB, the next enzyme in peptidoglycan biosynthesis. About 25% of MurA was complexed with UDP-MurNAc after five steps during purification that should have removed it. UDP-MurNAc isolated from MurA was identified by mass spectrometry, NMR analysis, and comparison with authentic UDP-MurNAc. Subsequent investigation showed that UDP-MurNAc bound to MurA tightly, with K d,UDP-MurNAc ) 0.94 ( 0.04 μM, as determined by fluorescence titrations using ANS (8-anilino- 1-naphthalenesulfonate) as an exogenous fluorophore. UDP-MurNAc binding was competitive with ANS and phosphate, the second product of MurA, and it inhibited MurA. The inhibition patterns were somewhat ambiguous, likely being competitive with the substrate PEP (phosphoenolpyruvate) and either competitive or noncompetitive with respect to the substrate UDP-GlcNAc (UDP-N-acetylglucosamine). These results indicate a possible role for UDP-MurNAc in regulating the biosynthesis of nucleotide precursors of peptidoglycan through feedback inhibition. Previous studies indicated that UDP-MurNAc binding to MurA was not tight enough to be physiologically relevant; however, this was likely an artifact of the assay conditions. The cytoplasmic phase of peptidoglycan biosynthesis has been studied extensively, but significant questions remain about its regulation (1-4). The first committed step of peptidoglycan biosynthesis is catalyzed by MurA (EP-UDP- GlcNAc synthase) 1 (2). It transfers the enolpyruvyl moiety of phosphoenolpyruvate (PEP) to UDP-N-acetylglucosamine (UDP-GlcNAc), forming enolpyruvyl-UDP-N-acetylglu- cosamine (EP-UDP-GlcNAc) and inorganic phosphate (P i ) (Figure 1). MurA is an antimicrobial target, being irreversibly inhibited by the antibiotic fosfomycin (5, 6). Its mechanism (7-14) and structure (6, 15) have been characterized extensively. In the next step, MurB reduces EP-UDP-GlcNAc to UDP- N-acetylmuramic acid (UDP-MurNAc). Six more cytoplas- mic steps complete synthesis of UDP-MurNAc-pentapeptide (16). An undecaprenyl pyrophosphate carrier lipid and UDP- GlcNAc are added at the cell membrane, followed by transfer to a final acceptor in the expanding peptidoglycan wall. Peptidoglycan synthesis is tightly regulated (16). The mechanism of regulating synthesis of nucleotide precursors of peptidoglycan, including UDP-MurNAc-pentapeptide, appears to involve controlling the entry of precursors into the biosynthetic pathway. It is not clear how this is achieved, but feedback inhibition by a variety of peptidoglycan precursors has been suggested (1, 4). Regulation does not appear to be achieved by controlling enzyme levels, as they appear to be constitutively expressed and are relatively insensitive to a wide variety of conditions (17). For example, MurA levels in fosfomycin-treated Salmonella typhimurium were unaffected by the antibiotic over a range of concentra- tions (18). EP-UDP-GlcNAc concentrations are low, ca. 2 μM(19), and remarkably constant under a wide variety of conditions in wild-type strains, implying tight regulation ( 17). We report here that purified recombinant MurA contained significant amounts of bound UDP-MurNAc after purifica- tion, suggesting a possible physiological role for this interaction. We have shown that UDP-MurNAc is a MurA inhibitor. MATERIALS AND METHODS General. Fosfomycin, PEP, and UDP-GlcNAc were pur- chased from Sigma. [ 33 P]PEP was prepared from [γ- 33 P]ATP as described previously (20, 21). MurB. The strain expressing His 6 -tagged MurB was the generous gift of Dr. Martin Pavelka (University of Roches- † This work was supported by the Canadian Institutes of Health Research, as well as by a graduate scholarship from the Natural Sciences and Engineering Research Council of Canada (to B.B.). * To whom correspondence should be addressed. Telephone: (905) 525-9140 ext 23479. Fax: (905) 522-2509. E-mail: berti@mcmaster.ca. ‡ Department of Chemistry, McMaster University. § Present address: Department of Chemistry, Yarmouk University, Irbid, Jordan. | Department of Biochemistry, McMaster University. 1 Abbreviations: ANS, 8-anilino-1-naphthalenesulfonate; DTT, dithio- threitol; EP-UDP-GlcNAc, enolpyruvyluridine diphospho N-acetylglu- cosamine; Kd,X, equilibrium dissociation constant of compound X binding with MurA; MurA, EP-UDP-GlcNAc synthase; MurB, EP- UDP-GlcNAc reductase; PEP, phosphoenolpyruvate; P i, inorganic phosphate; TOCSY, total correlation spectroscopy; TLC, thin-layer chromatography; UDP-GlcNAc, uridine diphospho N-acetylglu- cosamine; UDP-MurNAc, uridine diphospho N-acetylmuramic acid; UDP-MurNAc-pentapeptide, UDP-N-acetylmuramyl-L-Ala-D-Glu-meso- diaminopimelic acid-D-Ala-D-Ala. 4011 Biochemistry 2005, 44, 4011-4017 10.1021/bi047704w CCC: $30.25 © 2005 American Chemical Society Published on Web 02/16/2005