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Mechanistic studies of 2,5‐dihydroxypyridine 5,6‐dioxygenase (NicX) from Bordetella bronchiseptica
Author(s) -
Henke Matthew T,
Snider Mark J
Publication year - 2011
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.25.1_supplement.714.2
Subject(s) - bordetella bronchiseptica , chemistry , bordetella , microbiology and biotechnology , biology , bacteria , genetics , bordetella pertussis
The genes coding the enzymes of aerobic catabolism of nicotinic acid in the genus Bordetella , the bacterial pathogen responsible for certain respiratory diseases in mammals, have recently been discerned. A key step in this 6‐step mechanism of nicotinic acid degradation to fumaric acid is the oxidative ring‐opening of 2,5‐dihydroxypyridine to N‐formylmaleamic acid, which is catalyzed by NicX – an Fe 2+ dependent extradiol dioxygenase (EC 1.13.11.9). Recent protein modeling studies predict His265, His318 and D320 coordinate the active site Fe 2+ required for molecular oxygen insertion in the substrate. Here we demonstrate, by analysis of the resulting products of the enzymatic reaction by mass spectrometry and proton NMR, that gene BB1781 in Bordetella bronchiseptica codes for a functional NicX. Kinetic analysis of this reaction by following changes in the UV spectrum of the substrate with time for both wt NicX and NicX variants H265A, H318A and D320A will be done to experimentally examine the hypothesized functional role of these conserved residues. This work was supported by a grant to The College of Wooster from the Howard Hughes Medical Institute through the Undergraduate Science Education Program.