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.

FIG. 1.

Gentisate pathway for the degradation of 2,5-xylenol, 3,5-xylenol and m-cresol by P. alcaligenes P25X. Compounds (when R₁ = H and R₂ = H): I, m-cresol; II, 3-hydroxy-benzylalcohol; III, 3-hydroxy-benzaldehyde; IV, 3-hydroxybenzoate; V, gentisate; VI, maleylpyruvate; VII, maleate; VIII, fumarylpyruvate; IX, fumarate. Compounds (when R₁ = H and R₂ = CH₃): I, 2,5-xylenol; II, 3-hydroxy-4-methylbenzylalcohol; III, 3-hydroxy-4-methylbenzaldehyde; IV, 3-hydroxy-4-methylbenzoate; V, 4-methylgentisate; VI, 5-methylmaleylpyruvate; VII, citraconate. Compounds (when R₁ = CH₃ and R₂ = H): I, 3,5-xylenol; II, 3-hydroxy-5-methylbenzylalcohol; III, 3-hydroxy-5-methylbenzaldehyde; IV, 3-hydroxy-5-methylbenzoate; V, 3-methylgentisate; VI, 6-methylmaleylpyruvate; VII, citraconate.

FIG. 2.

(A) Extracts of E. coli BL21(DE3)/pLysS overexpressing XlnD separated by SDS-10% PAGE. E. coli BL21(DE3)/pLysS cells harboring the following plasmids were harvested and lysed 4 h after IPTG induction: pET28xlnD (lane 1), pET30xlnD (lane 2), pET28a (lane 3), and pET30a (lane 4). Lane M contains protein standards with molecular masses as indicated. Note the overexpressed protein bands of approximately 41 kDa in lane 1 and 43 kDa in lane 2 that correspond to the estimated molecular masses of XlnD and an N-terminal His₆-XlnD fusion protein, respectively. (B and C) Purified recombinant XlnD separated on native 10% PAGE (B) and SDS-10% PAGE (C). The HMW native molecular mass standard from Amersham Biosciences was used for the native PAGE (lane M1) and is comprised of the proteins thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), lactate dehydrogenase (140 kDa), and albumin (66 kDa). For SDS-PAGE, the recombinant prestained standards from Bio-Rad was used as molecular mass standards (lane M2).

FIG. 3.

Growth of P. alcaligenes P25X wild-type and its mutant derivatives in liquid minimal media containing the aromatic substrates 2,5-xylenol (2,5XLN), 3,5-xylenol (3,5XLN), 3-hydroxybenzoate (3HBA), and 3-hydroxy-4-methylbenzoate (3H4MBA) as the sole carbon source as determined by measuring OD₆₀₀ over a period of 96 h. (A) Strain P25X wild type; (B) xlnD mutant G50; (C) G50 harboring the recombinant plasmid pRK415-xlnD in which xlnD is expressed from the lac promoter of the pRK415 vector.