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Nicotinic Acid Degradation in Bordetella bronchiseptica: Mechanistic Studies of the Novel Enzyme NicF
Author(s) -
Kincaid Virginia Anne
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.835.7
Subject(s) - chemistry , pseudomonas putida , biochemistry , enzyme , hydrolysis , product inhibition , non competitive inhibition
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.