Mechanistic Studies on N-Acetylmuramic Acid 6-Phosphate Hydrolase (MurQ): An Etherase Involved in Peptidoglycan Recycling † Timin Hadi, ‡ Ulrike Dahl, § Christoph Mayer, § and Martin E. Tanner* ,‡ Department of Chemistry, UniVersity of British Columbia, VancouVer, British Columbia, Canada V6T 1Z1, and Fachbereich Biologie, Molekulare Mikrobiologie, UniVersita ¨t Konstanz, UniVersita ¨tsstrasse 10, 78457 Konstanz, Germany ReceiVed August 1, 2008; ReVised Manuscript ReceiVed August 29, 2008 ABSTRACT: Peptidoglycan recycling is a process in which bacteria import cell wall degradation products and incorporate them back into either peptidoglycan biosynthesis or basic metabolic pathways. The enzyme MurQ is an N-acetylmuramic acid 6-phosphate (MurNAc 6-phosphate) hydrolase (or etherase) that hydrolyzes the lactyl side chain from MurNAc 6-phosphate and generates GlcNAc 6-phosphate. This study supports a mechanism involving the syn elimination of lactate to give an R,-unsaturated aldehyde with (E)-stereochemistry, followed by the syn addition of water to give product. The observation of both a kinetic isotope effect slowing the reaction of [2- 2 H]MurNAc 6-phosphate and the incorporation of solvent- derived deuterium into C2 of the product indicates that the C2-H bond is cleaved during catalysis. The observation that the solvent-derived 18 O isotope is incorporated into the C3 position of the product, but not the C1 position, provides evidence of the cleavage of the C3-O bond and argues against imine formation. The finding that 3-chloro-3-deoxy-GlcNAc 6-phosphate serves as an alternate substrate is also consistent with an elimination-addition mechanism. Upon extended incubations of MurQ with GlcNAc 6-phosphate, the R,-unsaturated aldehydic intermediate accumulates in solution, and 1 H NMR analysis indicates it exists as the ring-closed form of the (E)-alkene. A structural model is developed for the Escherichia coli MurQ and is compared to that of the structural homologue glucosamine-6-phosphate synthase. Putative active site acid/base residues are probed by mutagenesis, and Glu83 and Glu114 are found to be crucial for catalysis. The Glu83Ala mutant is essentially inactive as an etherase yet is capable of exchanging the C2 proton of substrate with solvent-derived deuterium. This suggests that Glu83 may function as the acidic residue that protonates the departing lactate. Peptidoglycan is a key structural component of the bacterial cell wall and is required to protect the bacteria from lysis due to osmotic pressure (1, 2). During bacterial growth, peptidoglycan biosynthesis and peptidoglycan breakdown make up a dynamic process and must be carefully balanced to meet the needs of the organism (3). In certain bacteria, such as Escherichia coli, a complex system of enzymes and proteins has developed that is responsible for importing and recycling cell wall components formed during peptidoglycan breakdown (4). These components may be reused in further peptidoglycan biosynthesis or shuttled into basic metabolic pathways. The peptidoglycan structure is defined by polysac- charide chains comprised of alternating residues of N- acetylglucosamine (GlcNAc) 1 and its 3-O-lactyl ether de- rivative, N-acetylmuramic acid (MurNAc) (Figure 1). These polysaccharide chains are cross-linked via peptide bridges attached to the MurNAc residues. During cell wall degrada- tion and recycling, lytic transglycosylases and endopeptidases † This research was supported by the Natural Sciences and Engineer- ing Research Council of Canada (NSERC) (M.E.T.) and the Deutsche Forschungsgemeinschaft (Heisenberg fellowship and Grant MA2436 to C.M.). * To whom correspondence should be addressed. Phone: (604) 822- 9453. Fax: (604) 822-2847. E-mail: mtanner@chem.ubc.ca. ‡ University of British Columbia. § Universita ¨t Konstanz. 1 Abbreviations: GlcNAc, N-acetylglucosamine; GlmS, glucosamine 6-phosphate synthase; INT, p-iodonitrotetrazolium violet; KIE, kinetic isotope effect; MurNAc, N-acetylmuramic acid; 6P, 6-phosphate; SIS, sugar isomerase; Trien, triethanolamine. FIGURE 1: Formation of anhydroMurNAc during peptidoglycan recycling and its conversion into GlcNAc 6-phosphate and lactate by the enzymes AnmK and MurQ. Biochemistry 2008, 47, 11547–11558 11547 10.1021/bi8014532 CCC: $40.75  2008 American Chemical Society Published on Web 10/07/2008