Studies towards a Solution Structure of the Peptidoglycan Glycosyltransferases

Abstract

Peptidoglycan glycosyltransferases (PGTs) are highly conserved bacterial enzymes that catalyze the polymerization of the lipidic disaccharide, Lipid II, to form individual peptidoglycan (PG) strands which are subsequently cross-linked to form mature PG, the major skeletal component of the bacterial cell wall. Recent advances in the preparation of well-defined PGT substrates have enabled the biochemical characterization of Lipid II polymerization by the PGTs. In the course of these studies, we have observed that a distinctive lag phase in the initial rate of PG synthesis by the PGTs can be abrogated if the enzyme is preincubated with Lipid IV, the shortest PG fragment. The origins of this lag phase are intriguing because the chemical transformation involved in coupling Lipid II to yield Lipid IV is identical to the transformation involved in the synthesis of longer PG fragments from Lipid II. Crystallographic structures of the PGTs with Moenomycin A, an inhibitor that is believed to bind to the same site as Lipid IV, suggest that the PGTs possess flexible regions near the putative active site that can undergo substrate-induced conformational changes to accelerate PG synthesis. However, there is currently no structural evidence on how the PGTs interact with its substrates. The work in this thesis lays the foundation for pursuing a solution structure of a Lipid IV bound PGT complex by Nuclear Magnetic Resonance (NMR) spectroscopy, enabling the study of important enzyme conformational states and structural dynamics involved in PG synthesis. Specifically, Chapter 2 of this thesis presents the biochemical evidence that the preincubation of the PGTs with a Lipid IV derivative, Gal-Lipid IV abrogates the lag phase and accelerates the initial rate of PG synthesis. Chapter 3 presents a robust methodology for obtaining multimilligram quantities of isotope labeled, monodisperse and monomeric SgtB, a PGT from a clinically relevant pathogen, Staphylococcus aureus for solution structural studies. Chapter 4 describes the systematic development of a methodology for producing a well-behaved, stable sample of Moenomycin A bound SgtB for NMR spectroscopy. Chapter 5 delineates the adaptation of the methodology described in Chapter 4 for pursuing the solution structure of Lipid IV bound SgtB.