title: Molecular and biochemical characterization of the xlnD-encoded 3-hydroxybenzoate 6-hydroxylase involved in the degradation of 2,5-xylenol via the gentisate pathway in Pseudomonas alcaligenes NCIMB 9867.
abstract: The xlnD gene from Pseudomonas alcaligenes NCIMB 9867 (strain P25X) was shown to encode 3-hydroxybenzoate 6-hydroxylase I, the enzyme that catalyzes the NADH-dependent conversion of 3-hydroxybenzoate to gentisate. Active recombinant XlnD was purified as a hexahistidine fusion protein from Escherichia coli, had an estimated molecular mass of 130 kDa, and is probably a trimeric protein with a subunit mass of 43 kDa. This is in contrast to the monomeric nature of the few 3-hydroxybenzoate 6-hydroxylases that have been characterized thus far. Like other 3-hydroxybenzoate 6-hydroxylases, XlnD could utilize either NADH or NADPH as the electron donor. P25X harbors a second 3-hydroxybenzoate 6-hydroxylase II that was strictly inducible by specific aromatic substrates. However, the degradation of 2,5-xylenol and 3,5-xylenol in strain P25X was found to be dependent on the xlnD-encoded 6-hydroxylase I and not the second, strictly inducible 6-hydroxylase II.
authors.affiliation: Programme in Environmental Microbiology, Department of Microbiology, National University of Singapore.
authors.forename: Xiaoli
authors.initials: X
authors.lastname: Gao
authors.forename: Chew Ling
authors.initials: CL
authors.lastname: Tan
authors.forename: Chew Chieng
authors.initials: CC
authors.lastname: Yeo
authors.forename: Chit Laa
authors.initials: CL
authors.lastname: Poh
mesh_terms: D001673:Biodegradation, Environmental; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005816:Genetic Complementation Test; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D008970:Molecular Weight; D016254:Mutagenesis, Insertional; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D021122:Protein Subunits; D044202:Pseudomonas alcaligenes / Q000201:enzymology* / Q000235:genetics; D014992:Xylenes / Q000378:metabolism*
chemical_list: D021122:Protein Subunits; D014992:Xylenes; D009243:NAD; D009249:NADP; D006899:Mixed Function Oxygenases; C023315:3-hydroxybenzoate-6-hydroxylase; C016834:3,5-xylenol; C048893:2,5-xylenol