Characterization of a putative monooxygenase involved in NAD catabolism

Theodore Carlton Moore, III, The College of Wooster


Recent increases in efficiency and decreases in the cost of DNA sequencing have lead to a multitude of published genomes. However, only a fraction of the protein-coding sequences in these genomes have been annotated, and there is often no definitive biochemical data to confirm the predictions. This study investigates the annotated, yet uncharacterized, pathway of NAD catabolism in Bordetella bronchiseptica. Specifically, putative monooxygenase [Bordetella bronchispetica] (NP_888323.1, designated BbMO) was demonstrated to have significant sequence homology to the protein 6-hydroxylase 3-monooxygenase (6H3M) in Pseudomonas fluorescens Tn5. This protein was shown to catalyze the decarboxylation and hydroxylation of 6-hydroxynicotinic acid, a metabolite of NAD. BbMO was hypothesized to have an identical function in B. bronchiseptica. Expression of BbMO was attempted with a plasmid using an N-terminal his tag and a periplasmic locator. The expression was weak and purification using nickel-ion affinity chromatography was unsuccessful. Induction conditions were varied in an attempt to optimize expression. A method was developed to separate substrates and cofactors using high performance liquid chromatography (HPLC). Both cell free lysate and solubilized membrane fractions containing the unpurified enzyme were assayed with 6-hydroxynicotinic acid on HPLC. There was no change in 6-hydroxynicotinic acid concentration between induced and uninduced samples, indicating that either 6-hydroxynicotinic acid is not a preferred substrate or that activity of BbMO was lost at some point. The cloning of BbMO into a non-periplasmic vector was initiated in an attempt to improve the expression of BbMO.


© Copyright 2009 Theodore Carlton Moore, III