Genetic and biochemical characterization of unknown XlnD and HbzB from Pseudomonas Alcaligenes NCIMB 9867

Abstract

Pseudomonas alcaligenes NCIMB 9867 (strain P25X) produces isofunctional enzymes of the gentisate pathway that enable the degradation of xylenols and cresols. In this study, we investigated the function of xlnD, overexpressed it, purified and characterized the protein XlnD from E. coli BL21. Enzyme assays showed that XlnD was functioning as a 3-hydroxybenzoate 6-hydroxylase. A P25X xlnD knockout mutant, strain G50, was constructed and in this mutant, 6-hydroxylase activity could only be detected when cells were grown in either 3-hydroxybenzoate or gentisate, thus indicating the presence of another 6-hydroxylase (6-hydroxylase II) in strain P25X which was strictly inducible by 3-hydroxybenzoate. Strain G50 lost the ability to grow on 2,5-xylenol, 3,5-xylenol and 3-hydroxy-4-methylbenzoate and this ability was restored when xlnD was provided on a separate pRK415-derived plasmid. Sequence analysis of the gene hbzB indicated that it encodes gentisate 1,2-dioxygenase II. Expression, purification and characterization of gentisate 1,2-dioxygenase II was also reported in this work.