Structural and biochemical characterization of proteins involved in peptidoglycan synthesis in gram-positive bacteria

Abstract

The peptidoglycan layer is a major component of the cell wall in gram-positive bacteria. It consists of glycan chains and peptide stems containing both L- and D-amino acids. This layer influences cell shape, motility, division, virulence, and pathogenicity, enabling bacterial adaptation to diverse environments.

Peptidoglycan synthesis involves two classes of enzymes:

Enzymes that synthesize precursors.

Penicillin Binding Proteins (PBPs) that cross-link the matrix via transglycosylase, transpeptidase, and carboxypeptidase activities.

This thesis focuses on PBPs and other enzymes involved in the biosynthesis of L-lysine, L-homoserine, and L-threonine-amino acids essential for peptidoglycan cross-links.

Chapter Summaries Chapter 1 - Introduction

Overview of bacterial cell wall constituents and peptidoglycan synthesis.

Genetic and biochemical studies in Staphylococcus aureus.

Role of enzymes in physiology, virulence, and antibiotic resistance.

Chapter 2 - Structure-Function Analysis of PBP4

Structural and functional studies of S. aureus PBP4.

Discovery that modulation of PBP4 activity influences resistance to vancomycin.

Discussion of PBPs in antimicrobial resistance.

Chapter 3 - Detection of PBP2a (MRSA Biomarker)

PBP2a, encoded by mecA, is a hallmark of Methicillin-Resistant S. aureus (MRSA).

Development of a novel electrochemical sensor using titania nanotubes.

Achieved ten-fold higher sensitivity compared to ELISA, with rapid and reusable detection potential.

Chapter 4 - Homoserine Dehydrogenase (HSD)

Structural studies revealed mechanisms of hydride transfer and pH sensitivity.

Insights into substrate recruitment, half-site reactivity, and ACT domain regulation.

Chapter 5 - Dihydrodipicolinate Reductase (DHDPR)

Structure revealed nicotinamide cofactor specificity.

Lys35 identified as critical for nucleotide binding.

Mutational analysis confirmed structural predictions.

Domain orientation studies highlighted dynamic flexibility.

Chapter 6 - Summary and Conclusions

Consolidates findings on PBPs and amino acid biosynthetic enzymes.

Highlights complexity of enzyme function in peptidoglycan synthesis.

Suggests strategies to modulate enzyme activity to combat multidrug-resistant S. aureus.

Appendices

Appendix I: B. subtilis two-component signaling system.

Appendix II: Structural characterization of M. tuberculosis Dihydrodipicolinate Synthase (DHDPS) with inhibitor.

Appendix III: Detailed experimental protocols.