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

J Bacteriol. 2005 Nov;187(22):7696-702. doi: 10.1128/JB.187.22.7696-7702.2005.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biodegradation, Environmental
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Genetic Complementation Test
  • Mixed Function Oxygenases / chemistry
  • Mixed Function Oxygenases / genetics*
  • Mixed Function Oxygenases / isolation & purification
  • Mixed Function Oxygenases / metabolism*
  • Molecular Weight
  • Mutagenesis, Insertional
  • NAD / metabolism
  • NADP / metabolism
  • Protein Subunits
  • Pseudomonas alcaligenes / enzymology*
  • Pseudomonas alcaligenes / genetics
  • Xylenes / metabolism*

Substances

  • Protein Subunits
  • Xylenes
  • NAD
  • NADP
  • Mixed Function Oxygenases
  • 3-hydroxybenzoate-6-hydroxylase
  • 3,5-xylenol
  • 2,5-xylenol