The BH1999 Protein of Bacillus halodurans C-125 Is Gentisyl-Coenzyme A Thioesterase

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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 kcat/Km of 1.6 × 106 M−1 s−1 and the hydrolysis of 3-hydroxybenzoyl-CoA with a kcat/Km of 3.0 × 105 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.