title: Molecular and biochemical characterization of 3-hydroxybenzoate 6-hydroxylase from Polaromonas naphthalenivorans CJ2.
abstract: Prior research revealed that Polaromonas naphthalenivorans CJ2 carries and expresses genes encoding the gentisate metabolic pathway for naphthalene. These metabolic genes are split into two clusters, comprising nagRAaGHAbAcAdBFCQEDJI'-orf1-tnpA and nagR2-orf2I''KL (C. O. Jeon, M. Park, H. Ro, W. Park, and E. L. Madsen, Appl. Environ. Microbiol. 72:1086-1095, 2006). BLAST homology searches of sequences in GenBank indicated that the orf2 gene from the small cluster likely encoded a salicylate 5-hydroxylase, presumed to catalyze the conversion of salicylate into gentisate. Here, we report physiological and genetic evidence that orf2 does not encode salicylate 5-hydroxylase. Instead, we have found that orf2 encodes 3-hydroxybenzoate 6-hydroxylase, the enzyme which catalyzes the NADH-dependent conversion of 3-hydroxybenzoate into gentisate. Accordingly, we have renamed orf2 nagX. After expression in Escherichia coli, the NagX enzyme had an approximate molecular mass of 43 kDa, as estimated by gel filtration, and was probably a monomeric protein. The enzyme was able to convert 3-hydroxybenzoate into gentisate without salicylate 5-hydroxylase activity. Like other 3-hydroxybenzoate 6-hydroxylases, NagX utilized both NADH and NADPH as electron donors and exhibited a yellowish color, indicative of a bound flavin adenine dinucleotide. An engineered mutant of P. naphthalenivorans CJ2 defective in nagX failed to grow on 3-hydroxybenzoate but grew normally on naphthalene. These results indicate that the previously described small catabolic cluster in strain CJ2 may be multifunctional and is essential for the degradation of 3-hydroxybenzoate. Because nagX and an adjacent MarR-type regulatory gene are both closely related to homologues in Azoarcus species, this study raises questions about horizontal gene transfer events that contribute to operon evolution.
authors.affiliation: Division of Applied Life Science, EB-NCRC, PMBBRC, Gyeongsang National University, Jinju 660-701, Republic of Korea.
authors.forename: Minjeong
authors.initials: M
authors.lastname: Park
authors.forename: Yeji
authors.initials: Y
authors.lastname: Jeon
authors.forename: Ho Hee
authors.initials: HH
authors.lastname: Jang
authors.forename: Hyun-Su
authors.initials: HS
authors.lastname: Ro
authors.forename: Woojun
authors.initials: W
authors.lastname: Park
authors.forename: Eugene L
authors.initials: EL
authors.lastname: Madsen
authors.forename: Che Ok
authors.initials: CO
authors.lastname: Jeon
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D042621:Comamonadaceae / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D003998:Dicarboxylic Acids / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005650:Fumarates / Q000737:chemistry / Q000378:metabolism; D017353:Gene Deletion; D005841:Gentisates / Q000737:chemistry / Q000378:metabolism; D062385:Hydroxybenzoates / Q000737:chemistry / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism*; D008957:Models, Genetic; D015394:Molecular Structure; D009154:Mutation; D009281:Naphthalenes / Q000737:chemistry / Q000378:metabolism; D010867:Pimelic Acids / Q000737:chemistry / Q000378:metabolism; D011773:Pyruvates / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D003998:Dicarboxylic Acids; D005650:Fumarates; D005841:Gentisates; D062385:Hydroxybenzoates; D009281:Naphthalenes; D010867:Pimelic Acids; D011773:Pyruvates; D011994:Recombinant Proteins; C113957:maleylpyruvic acid; C073688:fumarylpyruvate; C031721:naphthalene; D006899:Mixed Function Oxygenases; C023315:3-hydroxybenzoate-6-hydroxylase