Structural and enzymatic characterization of bacterial cell wall enzymes: focus on penicillin-binding proteins

Abstract

The research presented in this dissertation was aimed at understanding several structural and biochemical features underlying the bacterial cell wall biosynthetic process using statistical, biochemical and analytical tools. Penicillin-binding proteins (PBPs) are ubiquitous and essential enzymes necessary for cell wall biosynthesis. PBPs are broadly classified as low molecular mass (LMM) and high molecular mass (HMM) based on their molecular weight. Although all PBPs share a set of highly conserved active site residues, different PBPs have different propensities for catalyzing reactions. To understand the structural differences among PBPs, a global catalytic residue comparison was performed for all the PBPs available in the protein data bank. This dataset was analyzed using univariate and multivariate statistical methods, revealing several interesting relationships such as: (1) Distribution of the dihedral angle for the SXXK-motif Lys side chain was bimodal, and strongly correlated with HMM/transpeptidase vs LMM/hydrolase activity; (2) Distance between the SXXK-motif Lys-NZ atom and the Lys/His-nitrogen atom of the (K/H)T(S)G-motif was highly conserved. Unlike LMM PBPs, HMM PBPs when purified, give undetectable or weak enzyme activity that has impeded efforts to develop new inhibitors. In the next section of this dissertation, we described a fluorescently detected microtiter plate-based assay for HMM PBPs. Purified PBPs were immobilized onto microtiter plate wells and labeled with biotinylated-ampicillin (Bio-Amp). Treatment of Bio-Amp-labeled PBPs with a streptavidin-horseradish peroxidase conjugate followed by a fluorogenic HRP substrate allowed the detection of PBPs. The HMM PBP assay was extended to penicillin-binding protein 2a, the molecular determinant of high-level β-lactam resistance in methicillin-resistant Staphylococcus aureus using Bio-Amp and biotinylated-cephalexin as tagging reagents. This assay was then demonstrated for use in competition assays for screening and characterizing both β-lactam and non β-lactam inhibitors. The cytoplasmic steps of cell wall biosynthesis involve a series of UDP-linked intermediates whose synthesis is catalyzed by Mur enzymes. The final section of the dissertation deals with developing an LC-MS/MS detection and quantification of UDP-peptidoglycan intermediates in the bacterial cell wall biosynthetic pathway. The assay was validated and then used to quantitate the in vivo levels of UDP-intermediates in S. aureus treated with different antibiotics acting at various stages of cell wall biosynthesis.