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A dedicated thioesterase of the Hotdog-fold family is required for the biosynthesis of the naphthoquinone ring of vitamin K1.

ABSTRACT: Phylloquinone (vitamin K(1)) is a bipartite molecule that consists of a naphthoquinone ring attached to a phytyl side chain. The coupling of these 2 moieties depends on the hydrolysis of the CoA thioester of 1,4-dihydroxy-2-naphthoate (DHNA), which forms the naphthalenoid backbone. It is not known whether such a hydrolysis is enzymatic or chemical. In this study, comparative genomic analyses identified orthologous genes of unknown function that in most species of cyanobacteria cluster with predicted phylloquinone biosynthetic genes. The encoded approximately 16-kDa proteins display homology with some Hotdog domain-containing CoA thioesterases that are involved in the catabolism of 4-hydroxybenzoyl-CoA and gentisyl-CoA (2,5-dihydroxybenzoyl-CoA) in certain soil-dwelling bacteria. The Synechocystis ortholog, encoded by gene slr0204, was expressed as a recombinant protein and was found to form DHNA as reaction product. Unlike its homologs in the Hotdog domain family, Slr0204 showed strict substrate specificity. The Synechocystis slr0204 knockout was devoid of DHNA-CoA thioesterease activity and accumulated DHNA-CoA. As a result, knockout cells contained 13-fold less phylloquinone than their wild-type counterparts and displayed the typical photosensitivity to high light associated to phylloquinone deficiency in cyanobacteria.

SUBMITTER: Widhalm JR

PROVIDER: S-EPMC2660889 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The hotdog-fold enzyme 4-hydroxybenzoyl-coenzyme A (4-HB-CoA) thioesterase from Arthrobacter sp. strain AU catalyzes the hydrolysis of 4-HB-CoA to form 4-hydroxybenzoate (4-HB) and coenzyme A (CoA) in the final step of the 4-chlorobenzoate dehalogenation pathway. Guided by the published X-ray structures of the liganded enzyme (Thoden, J. B., Zhuang, Z., Dunaway-Mariano, D., and Holden H. M. (2003) J. Biol. Chem. 278, 43709-43716), a series of site-directed mutants were prepared for testing the roles of active site residues in substrate binding and catalysis. The mutant thioesterases were subjected to X-ray structure determination to confirm retention of the native fold, and in some cases, to reveal changes in the active site configuration. In parallel, the wild-type and mutant thioesterases were subjected to transient and steady-state kinetic analysis, and to (18)O-solvent labeling experiments. Evidence is provided that suggests that Glu73 functions in nucleophilic catalysis, that Gly65 and Gln58 contribute to transition-state stabilization via hydrogen bond formation with the thioester moiety and that Thr77 orients the water nucleophile for attack at the 4-hydroxybenzoyl carbon of the enzyme-anhydride intermediate. The replacement of Glu73 with Asp was shown to switch the function of the carboxylate residue from nucleophilic catalysis to base catalysis and thus, the reaction from a two-step process involving a covalent enzyme intermediate to a single-step hydrolysis reaction. The E73D/T77A double mutant regained most of the catalytic efficiency lost in the E73D single mutant. The results from (31)P NMR experiments indicate that the substrate nucleotide unit is bound to the enzyme surface. Kinetic analysis of site-directed mutants was carried out to determine the contributions made by Arg102, Arg150, Ser120, and Thr121 in binding the nucleotide unit. Lastly, we show by kinetic and X-ray analyses of Asp31, His64, and Glu78 site-directed mutants that these three active site residues are important for productive binding of the substrate 4-hydroxybenzoyl ring.

Project description:The 4-hydroxybenzoyl-CoA (4-HB-CoA) thioesterase from Pseudomonas sp. strain CBS3 catalyzes the final step of the 4-chlorobenzoate degradation pathway, which is the hydrolysis of 4-HB-CoA to coenzyme A (CoA) and 4-hydroxybenzoate (4-HB). In previous work, X-ray structural analysis of the substrate-bound thioesterase provided evidence of the role of an active site Asp17 in nucleophilic catalysis [Thoden, J. B., Holden, H. M., Zhuang, Z., and Dunaway-Mariano, D. (2002) X-ray crystallographic analyses of inhibitor and substrate complexes of wild-type and mutant 4-hydroxybenzoyl-CoA thioesterase. J. Biol. Chem. 277, 27468-27476]. In the study presented here, kinetic techniques were used to test the catalytic mechanism that was suggested by the X-ray structural data. The time course for the multiple-turnover reaction of 50 ?M [(14)C]-4-HB-CoA catalyzed by 10 ?M thioesterase supported a two-step pathway in which the second step is rate-limiting. Steady-state product inhibition studies revealed that binding of CoA (K(is) = 250 ± 70 ?M; K(ii) = 900 ± 300 ?M) and 4-HB (K(is) = 1.2 ± 0.2 mM) is weak, suggesting that product release is not rate-limiting. A substantial D(2)O solvent kinetic isotope effect (3.8) on the steady-state k(cat) value (18 s(-1)) provided evidence that a chemical step involving proton transfer is the rate-limiting step. Taken together, the kinetic results support a two-chemical pathway. The microscopic rate constants governing the formation and consumption of the putative aspartyl 17-(4-hydroxybenzoyl)anhydride intermediate were determined by simulation-based fitting of a kinetic model to time courses for the substrate binding reaction (5.0 ?M 4-HB-CoA and 0.54 ?M thioesterase), single-turnover reaction (5 ?M [(14)C]-4-HB-CoA catalyzed by 50 ?M thioesterase), steady-state reaction (5.2 ?M 4-HB-CoA catalyzed by 0.003 ?M thioesterase), and transient-state multiple-turnover reaction (50 ?M [(14)C]-4-HB-CoA catalyzed by 10 ?M thioesterase). Together with the results obtained from solvent (18)O labeling experiments, the findings are interpreted as evidence of the formation of an aspartyl 17-(4-hydroxybenzoyl)anhydride intermediate that undergoes rate-limiting hydrolytic cleavage at the hydroxybenzoyl carbonyl carbon atom.

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A dedicated thioesterase of the Hotdog-fold family is required for the biosynthesis of the naphthoquinone ring of vitamin K1.

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