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Structural investigation of enzymes in wall teichoic acid biosynthesis and degradation Li, Franco Ka Kit


The bacterial cell wall is a complex polymeric structure with essential roles in defense, survival, and pathogenesis. Targeting the assembly of cell wall peptidoglycan with β-lactam antibiotics is becoming increasingly ineffective due the development and spread of resistance mechanisms. New therapeutic agents are now urgently needed, and one proposed target for drug development is the biosynthesis of the Gram-positive bacterial cell wall polymer known as wall teichoic acid (WTA). WTA plays critical roles in host cell adherence, immune evasion, and regulation of essential physiological processes such as cell division and peptidoglycan assembly. Importantly, the dysregulation of peptidoglycan synthesis in the absence of WTA was found to resensitize methicillin-resistant Staphylococcus aureus strains to β-lactam antibiotics. In addition, interruptions at certain points of WTA biosynthesis is lethal due to the sequestering of lipid precursors shared with peptidoglycan synthesis. With WTA assembly validated as a drug target, it is essential to characterize the responsible enzymes to guide drug discovery and development. In this thesis, high resolution X-ray crystallographic structures of TarI, TarJ, and TarL, in the assembly of the central ribitol-phosphate polymer of S. aureus WTA, are presented. Our ensemble of TarI and TarJ crystal structures illustrate the mechanism of synthesizing CDP-ribitol, the activated donor substrate for WTA polymerization by TarL. Furthermore, our crystal structure of the uncharacterized TarL N-terminal domain discloses potential roles in protein- and substrate-binding. In addition, crystal structures of LCP enzymes were solved, providing novel insights into substrate-binding and catalysis involved in the attachment of the complete WTA polymer onto peptidoglycan. Lastly, atomic structures of GlpQ, a WTA-degrading enzyme involved in cell wall maintenance, are presented. The structures provide the basis for substrate specificity and support an exolytic mechanism of WTA degradation. Together, these structural and biochemical analyses of WTA biosynthetic and degradative enzymes provide mechanistic understanding of their activities and reveal features that can guide drug discovery and development.

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