Date Approved

2011

Degree Type

Open Access Thesis

Degree Name

Master of Science (MS)

Department

Chemistry

Committee Member

Dr. Hedeel Evans

Committee Member

Dr. Steven Pernecky

Committee Member

Dr. Deborah Heyl-Clegg

Abstract

Bacteremia refers to bacterial presence in the blood. Bacterial proliferation in the blood requires that the organism adapt its metabolism to available nutrients. Nucleotide precursors that could be used are present at low levels in the blood, and thus the invading bacteria must rely on de novo nucleotide biosynthesis for survival. The dihydroorotase domain is a key enzyme in pyrimidine biosynthesis and a promising drug target. The genes encoding the dihydroorotase (DHOase) and aspartate transcarbamoylase (ATCase) of Bacillus anthracis (B. anthracis) were cloned for expression in Escherichia coli (E. coli). The proteins were purified by affinity chromatography and the enzymatic activity was determined by enzyme assays. The data suggests that a physical and functional interaction exists. The activity of ATCase was increased by about 2 fold in the presence of an equimolar concentration of DHOase. An ATCase-DHOase complex was formed as judged by S-300 gel filtration chromatography and cross-linking methods. Moreover, orotate was found to be an effective inhibitor of the DHOase activity at nanomolar concentrations. These results bring us closer to understanding the structural organization of the pyrimidine pathway in the pathogenic B. anthracis bacterium and provide a lead in the design of drugs selective to the bacteria.

Included in

Chemistry Commons

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