NAD catabolism: Identification of a 6-hydroxynicotinate 3-monooxygenase in bordetella bronchiseptica
Advances in DNA sequencing technology and bioinformatics techniques have led to a multitude of fully sequenced genomes complete with gene annotations. Many of these annotations are founded upon a prediction of the amino acid sequence of the protein coded by each gene and a subsequent homology comparison with a previously characterized protein. Although this method is often useful, it may be unreliable, so complete characterization of a gene requires a physical confirmation of the function of the protein it encodes. Based on its homology to confirmed enzymes and its presence within a cluster of genes with predicted related functions, the nicC gene in Bordetella bronchiseptica was annotated as encoding a 6HNA 3- monooxygenase, and it was hypothesized that this enzyme catalyzed one step in the catabolic metabolism of nicotinic acid. Specifically, we hypothesized that the NicC protein was capable of catalyzing the conversion of 6-hydroxynicotinic acid to 2,5-dihydroxypyridine. In order to confirm this hypothesis, the nicC gene from Bordetella bronchiseptica was cloned and expressed in Escherichia coli BL21 (DE3). The resulting MBP-His6 tag fusion protein was purified by amylose affinity chromatography and His-Trap Ni2+ affinity chromatography. Discontinuous enzyme activity assays were analyzed by HPLC on a reverse-phase C18 column. Originally, no enzyme activity was detected using NADH as a co-substrate in catalysis. However, when NADPH was used as a co-substrate in place of NADH, the reaction solution developed a translucent blue color. Analysis of the blue solution by HPLC revealed an unidentifiable, uniquely eluting molecule. Later analysis of various enzyme activity assays by LCMS/MS detected an ion with an m/z ratio of 112.1, the predicted m/z of 2,5-dihydroxypyridine, indicating that the enzyme is capable of catalysis using both NADH and NADPH as a co-substrate and in the absence of supplementary FAD.
© Copyright 2010 Ryan Wilson Story