Student Theses and Dissertations

Date of Award

2010

Document Type

Thesis

RU Laboratory

Stebbins Laboratory

Keywords

Bacillus anthracis, Salmonella, UDP-GlcNAc 2-Epimerase, enzymes, Na.4030

Abstract

Structural biology has been an exceptionally powerful tool in the study of bacterial pathogenesis, not only in elucidating the molecular basis of disease as the first step towards the design of therapeutics but also in many cases leading to insights into hostpathogen biology that would not have been possible otherwise (Remaut & Waksman 2004). In this work such an approach has been taken towards the study of the Bacillus anthracis cell-wall enzyme UDP-GlcNAc 2-Epimerase and the Salmonella secreted virulence effector SseI. UDP-GlcNAc 2-Epimerase is an essential B. anthracis enzyme required for formation of the peptidoglycan cell wall precursor UDP-ManAc. We have solved the epimerase’s crystal structure to 1.70 Å, explaining the mechanism of its allosteric regulation, as well as showing the first example of direct contact between an allosteric and catalytic substrate molecule. Arg210 was identified as a critical residue for both binding sites. Furthermore, the structure was used to design an inhibitor that would bind to the conserved bacterial allosteric site and inhibit epimerase activity. After multiple rounds of s iv screening against Gram-positive strains and in in vitro assays a lead compound, Na.4030, was identified for further testing in mouse models of B. anthracis infection. SseI is a Salmonella virulence effector secreted by its second Type III secretion system that is induced once the bacteria has invaded host cells. It has been implicated in having a direct role in establishing systemic infections. We have solved the structure of its catalytic C-terminal domain to 1.70 Å, using structural homology to identify it as a member of the Cysteine Protease Superfamily, having either acyl hydrolase or transferase activity. Furthermore, Cys178, His216, and Asp231 were identified as its catalytic triad. We were not able to show these activities in in vitro assays using small molecule and peptide substrates, but based on co-expression experiments with small Rho GTPases we were able to identify Cdc42 by mass spectrometry as a possible substrate undergoing deamidation at Gln61. The biological relevance of this result during infection is being investigated.

Comments

A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy.

Permanent URL

http://hdl.handle.net/10209/366

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