HbzF catalyzes direct hydrolysis of maleylpyruvate in the gentisate pathway of Pseudomonas alcaligenes NCIMB 9867

Abstract

HbzF from Pseudomonas alcaligenes NCIMB 9867 was purified to homogeneity as a His-tagged protein and likely a dimer by SDS-PAGE and gel filtration. This protein was demonstrated to be a novel maleylpyruvate hydrolase, catalyzing direct hydrolysis of maleylpyruvate to maleate and pyruvate, and belongs to the fumarylacetoacetate hydrolase superfamily. This study reveals the genetic determinate for the direct maleylpyruvate hydrolysis in the gentisate pathway, complementary to the well-studied maleylpyruvate isomerization route.

Fig 1

Two alternative routes for maleylpyruvate catabolism in the gentisate pathway. GDO, gentisate 1,2-dioxygenase; MPH, maleylpyruvate hydrolase (HbzF in this study); MPI, maleylpyruvate isomerase; and FPH, fumarylpyruvate hydrolase.

Fig 2

Spectrophotometric changes during the transformation of maleylpyruvate catalyzed by cell extracts of E. coli BL21(DE3)[pET22b-hbzF] expressing HbzF.

Fig 3

SDS-PAGE of purified HbzF-His₆. Lane 1, molecular mass standards (molecular masses are indicated on the left in kDa); lane 2, 5 μg purified HbzF-His₆.

Fig 4

Agarose gel electrophoresis of RT-PCR analysis of total RNA obtained from 3-hydroxybenzoate-induced cells of P. alcaligenes NCIMB 9867. Primers (FE-RT-F and FE-RT-R) used in this experiment are indicated by small horizontal arrows, with a PCR product of 459 bp. Lane M, DNA marker; lane RT⁻, product of PCR carried out without RT; and lane RT⁺, product of PCR carried out with RT.

Fig 5

Phylogenetic relationship analysis of HbzF with other members of the FAA hydrolase superfamily and maleylpyruvate isomerases by generating a distance neighbor-joining tree. Accession numbers of the proteins are listed on the right. The bootstrap confidence limits (expressed as percentages) based on 10,000 resampled data sets are indicated at the nodes. HbzF is indicated by a star.