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Title	Developing new drug targets in Neisseria gonorrhoeae: functional and structural studies of AmiC, an N-acetylmuramyl-L-alanine amidase required for bacterial cell separation
Author	Robertson, Rosanna Morgan
Date	2011
Extent	vi, 195 leaves : ill. 29 cm
Subject.MeSH	Academic Dissertations -- South Carolina.
Collection	MUSC Theses and Dissertations
Contributing Institution	Waring Historical Library (MUSC)
Digitization Specifications	CanoScan 8400, Archival files are tiffs.
Date Digital	2015
Rights	Copyright is held by the author.
Language	English
SC County	Charleston County (S.C.)
Type	Text
Format	application/pdf
Media Type	Manuscripts
Resource Identifier	mtd2011_robertson_rm
Abstract	For decades, ~-lactam antibiotics have been used to treat bacterial infections, but their efficacy is threatened by antibiotic resistance. ~-lactams target the synthesis of peptidoglycan, an essential component of bacterial cells walls, but many other enzymes involved in peptidoglycan biosynthesis represent potential, unexplored targets. One such enzyme is N-acetylmuramyl-L-alanine amidase (AmiC) from N. gonorrhoeae. Deletion of AmiC in N. gonorrhoeae results in improper cell separation during division and Increases susceptibility to antimicrobial agents. The goals of this project were to characterize AmiC through structural and biochemical studies, and to develop a substrate amenable for high-throughput screening to identify novel inhibitors of the enzyme. Toward this goal, we purified full-length AmiC, as well as constructs of the catalytic Cterminal domain and the N-terminal domain, whose function is unknown. We showed that the C-terminal domain is active independently of the N-terminal domain and that it requires zinc for catalytic activity. Other metals like magnesium, calcium and nickel can substitute the zinc ion in vitro. Mutation of residues predicted to coordinate the zinc [H214, E229, H282] or to participate in catalysis [E390] did not abolish enzyme activity, but did lower the stability of th~ protein. We also established that AmiC preferentially cleaves peptidoglycan-like compounds that contain at least three amino acids in the peptide stem but does not require N-acetylglucosamine for cleavage, suggesting it may act after cleavage of the glycan strand. Toward the goal of discovering inhibitors of ArniC, we designed and synthesized a series of FRET -compounds. One was successfully cleaved by ArniC but, due to low quenching efficiency, would not be suitable for highthroughput screening. Although it was hypothesized that the N-terminal domain of ArniC functions as a peptidoglycan targeting domain, activity assays revealed the surprising finding that the N-terminal domain, alone, exhibits peptidoglycan hydrolase activity and specifically is a glycosidase that cleaves the glycan strand. Crystals of the Nterminal domain that diffract to 2 A were obtained and will facilitate a more detailed investigation of this activity. Overall, these studies demonstrate that AmiC possesses glycosidase activity in addition to its amidase function and, thus, is a bifunctional autolysin.

Developing new drug targets in Neisseria gonorrhoeae: functional and structural studies of AmiC, an N-acetylmuramyl-L-alanine amidase required for bacterial cell separation

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