The BH1999 protein of Bacillus halodurans C-125 is gentisyl-coenzyme A thioesterase

Abstract

In this study, we have shown that recombinant BH1999 from Bacillus halodurans catalyzes the hydrolysis of gentisyl coenzyme A (CoA) (2,5-dihydroxybenzoyl-coenzyme A) at physiological pH with a k(cat)/K(m) of 1.6 x 10(6) M(-1) s(-1) and the hydrolysis of 3-hydroxybenzoyl-CoA with a k(cat)/K(m) of 3.0 x 10(5) M(-1) s(-1). All other acyl-CoA thioesters tested had low or no substrate activity. The BH1999 gene is juxtaposed with a gene cluster that contains genes believed to function in gentisate oxidative degradation. It is hypothesized that BH1999 functions as a gentisyl-CoA thioesterase. Gentisyl-CoA thioesterase shares the backbone fold and the use of an active site aspartate residue to mediate catalysis with the 4-hydroxybenzoyl-CoA thioesterase of the hotdog fold enzyme superfamily. A comparative study of these two enzymes showed that they differ greatly in the rate contribution made by the catalytic aspartate, in the pH dependence of catalysis, and in substrate specificity.

FIG. 1.

Organization of the gentisate oxidation pathway gene cluster in B. halodurans. Gene function assignment is tentatively based on the identity of the homologue found in the protein database. SD, Shine-Dalgarno sequence. Promoter- and terminator-like sequences were predicted by the genetic information processing software Genetyx, Mac version 11.0 (Software Development Co. Ltd., Tokyo, Japan).

FIG. 2.

SDS-PAGE gel (18% cross-link; Coomassie blue stained) of (from left to right) the Invitrogen BenchMark protein ladder and wild-type, D16N, D16A, and D31N gentisyl-CoA thioesterases.

FIG. 3.

(A) Kinetic pH rate profile: log k_cat (per second) versus pH rate for wild-type B. halodurans gentisyl-CoA thioesterase measured at 25°C. See the Materials and Methods for details. (B) The stability pH profile: k_cat (per second) of wild-type B. halodurans gentisyl-CoA thioesterase incubated for 2 min in the buffers used in A and then assayed at 25°C in 50 mM CAPSO and CAPS (pH 10)-0.2 M KCl buffer.

FIG. 4.

Pairwise alignment of B. halodurans gentisyl-CoA thioesterase (BH1999) and the Pseudomonas 4-hydroxybenzoyl-CoA (Psthio) thioesterase generated by the 3D-PSSM program. The secondary structure is designated with a block (α helix) or an arrow (β strand). Conserved residues are shaded, and the gentisyl-CoA thioesterase residues Asp16 and Asp31 are labeled.