Nicotinic Acid Degradation in Bordetella bronchiseptica: Mechanistic Studies of the Novel Enzyme NicF

The mechanism of aerobic nicotinic acid degradation in bacteria has not been well characterized. Based on studies in Pseudomonas putida , it is predicted that maleamic acid is converted to maleic acid by the protein product of peg.1774 in Bordetella bronchiseptica ( Bor ). This gene ( nicF ) was cloned using several vectors to enable expression of a C‐terminal His 6 ‐tagged or N‐terminal maltose‐binding protein fusion recombinant enzyme in E. coli . Results of purification by HisTrap nickel affinity chromatography and subsequent kinetic activity using a linked assay with glutamate dehydrogenase will be presented. Catalytic proficiency, defined as (k cat /K M )/k uncat , of NicF will be determined by comparing the enzyme's catalytic efficiency with the rate constant of uncatalyzed maleamic acid hydrolysis, measured by 1 H NMR.