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dc.contributor.supervisor Mark, Brian (Microbiology) en
dc.contributor.author Balcewich, Misty Dawn
dc.date.accessioned 2010-04-12T14:57:03Z
dc.date.available 2010-04-12T14:57:03Z
dc.date.issued 2010-04-12T14:57:03Z
dc.identifier.citation Balcewich M.D., Stubbs K., He Y., James T., Davies G., Vocadlo D.J., Mark B.L. (2009). Insight into a strategy for attenuating AmpC-mediated-lactam resistance: Structural basis for selective inhibition of the glycoside hydrolase NagZ, Protein Science 18:1541-1551. en
dc.identifier.citation Stubbs K.A., Balcewich M.D., Mark B.L., Vocadlo D.J. (2007) Small molecule inhibitors of a glycoside hydrolase attenuate inducible AmpC-mediated beta-lactam resistance. Journal of Biological Chemistry 282:21382-21391. en
dc.identifier.uri http://hdl.handle.net/1993/3975
dc.description.abstract Inducible chomosomal AmpC β-lactamase (AmpC) is present in many Gram-negative opportunistic human pathogens. Expressed in response to β-lactam antibiotics, AmpC is an enzyme that can deactivate an extended spectrum of β-lactam antibiotics and thereby promote bacterial survival. Inducible chromosomal ampC is associated with ampR, a gene that encodes a LysR-type transcriptional regulator that suppresses ampC expression in the absence of β-lactam exposure. Together, ampR and ampC form a divergent operon with overlapping promoters to which the AmpR protein binds and regulates the transcription of both genes. AmpR induces ampC expression by interacting with 1,6-anhydro-N-acetylmuramyl peptide, an intermediate of peptidoglycan recycling that is generated by a glycoside hydrolase encoded by nagZ. Given the role of NagZ and AmpR in the AmpC induction pathway, the structure and function of these proteins were investigated to understand the molecular basis for how they participate in AmpC production. The crystal structure of NagZ from Vibrio cholerae was determined in complex with the glycoside hydrolase inhibitor PUGNAc (O-(2-Deoxy-2-N-2-ethylbutyryl-D-glucopyranosylidene)amino-N-phenylcarbamate) to 1.8 Å resolution. Since PUGNAc also inhibits functionally related human enzymes, the structure of the enzyme was also determined in complex with the NagZ selective PUGNAc derivatives N-butyryl-PUGNAc (2.3 Å resolution) and N-valeryl-PUGNAc (2.4 Å resolution). These structural studies revealed the molecular basis for how 2-N-acyl derivatives of PUGNAc selectively inhibit the bacterial enzyme NagZ. The effector binding domain of AmpR from Citrobacter Spp. was determined to 1.83 Å resolution and lead to the identification of a putative effector molecule binding site. In vivo functional analysis of site directed mutants of AmpR containing amino acid substitutions at the base of the putative binding pocket verified its role in AmpR function. A protocol was subsequently devised to purify milligram quantities of soluble full-length AmpR. Biochemical and biophysical analysis, including non-denaturing mass spectrometry and small angle X-ray scattering, revealed that the purified full-length protein is tetrameric and specifically binds ampC promoter DNA. In summary, this research provides the basis for the development of small-molecules that could specifically block the activity of these proteins to suppress AmpC β-lactamase production during β-lactam therapy. en
dc.format.extent 44691059 bytes
dc.format.mimetype application/pdf
dc.language.iso en_US
dc.subject antibiotic resistance en
dc.subject protein crystallography en
dc.title Structural and functional studies of proteins involved in the AmpC β-lactamase induction pathway en
dc.degree.discipline Microbiology en
dc.contributor.examiningcommittee Loewen, Peter (Microbiology) Carpenter, Michael (Medical Microbiology) Rose, David (University of Waterloo, Biology) en
dc.degree.level Doctor of Philosophy (Ph.D.) en
dc.description.note May 2010 en


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