Characterization of the active site of S. aureus monofunctional glycosyltransferase (Mtg) by site-directed mutation and structural analysis of the protein complexed with moenomycin

Abstract

Bacterial cell wall transglycosylases (TGs) and transpeptidases (TPs) are ideal drug targets due to their essentiality, accessibility and lack of mammalian homologs. Although antibacterial therapy using the β-lactam family of TP inhibitors has been successful for decades, potent TG inhibitors which are suitable for development into antibiotics for human use have yet to be identified. We sought to further understand the molecular interactions required to inhibit bacterial transglycosylation by characterizing the active site of Staphylococcus aureus (Sa) monofunctional transglycosylase (Mtg). Ten mutants were tested for their ability to polymerize Lipid II and to crystallize in the presence of moenomycin. Five of six putative active site mutants (E100Q, D101N, Q136E, E156T, and Y176F) were found to be catalytically inactive whereas a F104Y mutation did not affect activity. Four mutants generated to enhance crystal formation (F143T, V154T, L157T, and F158T) also retained activity. Here we also report the crystal structure of Sa Mtg E100Q mutant in complex with the inhibitor moenomycin to 2.1 Å resolution. The co-crystal structure revealed detailed interactions between the protein and inhibitor including portions of the polycarbon tail of moenomycin. The structure also contained an ordered phosphate ion which helped to identify the Lipid II binding site.

Introduction

The synthesis of the bacterial cell wall involves the formation of glycopeptide polymers called peptidoglycans (Rogers et al., 1980). These highly cross-linked species help to stabilize the outer membrane of bacteria and give the cell its shape (Schleifer and Kandler, 1972). The synthesis of peptidoglycan has both intracellular and extracellular steps. During the last intracellular step, UDP-N-acetylglucosamine is converted to Lipid II by the enzyme MurG. Lipid II is then translocated to the extracellular environment where its disaccharide N-acetylmuramic acid N-acetylglucosamine (NAG–NAM) portion is polymerized by transglycosylase enzymes to form linear carbohydrate chains containing exclusively β-(1–4) linkages (van Heijenoort, 2001). These carbohydrate polymers are then cross-linked via their peptide portions by transpeptidase enzymes to form the peptidoglycans. In most bacterial species the transglycosylase and transpeptidase enzymatic reactions are primarily carried out by bi-functional enzymes composed of a single polypeptide with two distinct domains (Goffin and Ghuysen, 1998). In Staphylococcus aureus and some other bacterial species, the transglycosylase reaction can also be carried out by a related class of proteins, monofunctional transglycosylases (Coutinho et al., 2003, Terrak and Nguyen-Disteche, 2006, Wang et al., 2001).

Enzymes involved in cell wall biosynthesis have long been the target of antibiotic drug discovery efforts due to their essential function in bacteria and lack of human homologs (Breukink et al., 2003). The use of β-lactams, which irreversibly inhibit the transpeptidase enzymatic activity, to treat bacterial infections has been successful for decades, although significant rates of resistance to this class of inhibitors has emerged (van Heijenoort and Gutmann, 2000). As a result, there is a renewed focus on developing inhibitors of the transglycosylation step (Halliday et al., 2005). We sought to further understand the molecular interactions required to inhibit bacterial transglycosylation by characterizing the active site of the S. aureus (Sa) monofunctional transglycosylase (Mtg) by site-directed mutagenesis experiments in concert with crystallographic analysis, as described below.