| Lytic transglycosylases are involved in peptidoglycan metabolism by cleaving the β-1,4-glycosidic linkage between N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc) and concomitantly forming 1,6-anhydromuramic acid (AnhMurNAc). In Escherichia coli, at least 6 lytic transglycosylases exist which can be classified into three different families based on amino acid homologies. While it is not clear why bacteria contain multiple lytic transglycosylases, the Pseudomonas aeruginosa strain PAO1 genome contains four family 3 isoforms. This thesis is a biochemical investigation of the family 3 isoforms of PAO1 after in vitro expression and purification. One enzyme (MltB) was predicted to contain a N-terminal lipidation signal and behaved like a lipoprotein in vitro while the other three enzymes (SltB1, SltB2 and SltB3) appeared to be soluble forms. All four enzymes, purified as His-tagged fusions, were capable of degrading peptidoglycans which varied in their stem peptides. However, O-acetylation of the C-6 of MurNAc appeared to inhibit activity as predicted by the proposed mechanism of these enzymes. Two HPLC-based enzyme assays were developed and optimized during this study to evaluate the kinetic properties of lytic transglycosylases. Detection of MurNAc by amino acid analysis in the presence of NaBH4 confirmed that AnhMurNAc was product of lyric transglycosylase activity. HPAEC-PAD was able to resolve mixtures of soluble muropeptides released from intact, insoluble peptidoglycan after treatment with lytic transglycosylases. ESI-MS was then used to identify three major peaks and confirm that each corresponded to anhydro-muropeptides. After determining pH and temperature optima and stability parameters by HPAEC-PAD, Michaelis-Menten parameters were determined for sMltB-His6 and SltB1-His6. Using PAO1 peptidoglycan as a substrate, sMltB-His 6 functions in vitro with a catalytic efficiency ( kcat/KM) of 6240 s −1M−1 while SltB-His6 is almost 2.5 times more efficient with a value of 13900 s−1M −1. The recent crystal structure of a family 3 enzyme from E. coli was used to predict logical amino acid replacements in order to identify the putative catalytic acid and substrate-binding domains in sMltB-His 6 and SltBl-His6. Interaction of sMltB-His6 with a competitive inhibitor of N-acetylhexosaminidases, NAG-thiazoline, provided preliminary evidence that lytic transglycosylases use substrate-assisted catalysis in their mechanism of action. |
