Bacterial lytic transglycosylases: Biochemical characterization and rational design of inhibitors
Lytic transglycosylases cleave the [beta]-(1[right arrow]4) glycosidic bond in the bacterial cell wall heteropolymer, peptidoglycan, between the ' N'-acetylmuramic acid (MurNAc) and 'N'-acetylglucosamine (GlcNAc) residues with the concomitant formation of a 1,6 anhydromuramoyl residue. All lytic transglycosylases characterized to date possess only one catalytic acid/base in the active site. This has led to the proposal that lytic transglycosylases utilize substrate-assisted catalysis in order to stabilize the oxocarbenium ion upon cleavage of the glycosidic bond. This thesis describes the biochemical characterization of the family 3 lytic transglycosylase (Blackburn and Clarke, 2001) MltB from 'Pseudomonas aeruginosa'. An engineered soluble form of the enzyme (termed sMltB) was used in this study. Substrate binding studies by UV difference spectroscopy and hydrogen/deuterium exchange MALDI-MS (SUPREX) demonstrated the structural features of peptidoglycan that are recognized by sMltB. Key features included the C-3 lactyl moeity and 'N'-acetyl group on MurNAc and the associated stem peptide. Using the data obtained from the substrate binding assays, sequence alignments, and 3-dimensional modeling of sMltB, several residues were identified that potentially interacted with the substrate. Site-directed mutagenesis studies of Arg187 and 188, two residues associated with binding the stem peptide in the -1 subsite, generated enzymes whose activity showed deviations from Michaelis-Menten kinetics. Mutagenesis of these residues has brought into question the role of the stem peptide in catalysis. Mutagenesis of Ser216 has provided initial evidence that lytic transglycosylases invoke anchimeric assistance. Initial studies with Asn339 suggest that this residue is involved in excluding water from the active site and in recognition of the non-reducing end of a peptidoglycan strand. Work with the [beta]-hexosaminidase inhibitor NAG-thiazoline has provided evidence that lytic transglycosylases employ substrate-assisted catalysis ' via' mimicry of the oxazoline intermediate. Studies with ' E. coli' have shown that NAG-thiazoline causes morphological changes and alterations in cell surface properties. Inhibition and binding studies have shown that NAG-thiazoline binds sMltB with a KD of 1.07 mM. Attempts to develop more effective inhibitors of lytic transglycosylases have employed the use of 2-methylthiazoline 4-carboxylate as a scaffold. Initial studies with this new scaffold indicate that it has much improved activity over NAG-thiazoline (IC50, 5 [mu]M).