Dataset Information

The active site dynamics of 4-chlorobenzoyl-CoA dehalogenase.

ABSTRACT: A molecular dynamics study was performed to compare the differences in the active-site dynamics of the wild-type and W137F mutant enzymes of 4-chlorobenzoyl-CoA dehalogenase. Only in the wild-type simulation are conformations formed between the catalytic Asp-145 and 4-chlorobenzoyl-CoA, which resemble the ab initio calculated gas-phase transition-state geometry. In the W137F simulation, the hydrogen bond formed between His-90 and Asp-145 persisted throughout the simulation, causing the carboxylate of Asp-145 to be distant from the benzoyl ring of 4-chlorobenzoyl-CoA. In both simulations, water molecules were able to diffuse into the active site of the enzymes. The trajectories provide insight into the routes that water may use to get into position for the hydrolysis portion of the dehalogenation reaction. In both simulations, the water molecule entering the active site forms a hydrogen bond with Asp-145.

SUBMITTER: Lau EY

PROVIDER: S-EPMC55486 | biostudies-literature | 2001 Aug

REPOSITORIES: biostudies-literature

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The active site dynamics of 4-chlorobenzoyl-CoA dehalogenase.

Lau E Y EY Bruice T C TC

Proceedings of the National Academy of Sciences of the United States of America 20010807 17

A molecular dynamics study was performed to compare the differences in the active-site dynamics of the wild-type and W137F mutant enzymes of 4-chlorobenzoyl-CoA dehalogenase. Only in the wild-type simulation are conformations formed between the catalytic Asp-145 and 4-chlorobenzoyl-CoA, which resemble the ab initio calculated gas-phase transition-state geometry. In the W137F simulation, the hydrogen bond formed between His-90 and Asp-145 persisted throughout the simulation, causing the carboxyla ...[more]

PMID: 11493680

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Project description:cis-3-Chloroacrylic acid dehalogenase (cis-CaaD) from Pseudomonas pavonaceae 170 and a homologue from Corynebacterium glutamicum designated Cg10062 are 34% identical in sequence (54% similar). The former catalyzes a key step in a bacterial catabolic pathway for the nematocide 1,3-dichloropropene, whereas the latter has no known biological activity. Although Cg10062 has the six active site residues (Pro-1, His-28, Arg-70, Arg-73, Tyr-103, and Glu-114) that are critical for cis-CaaD activity, it shows only a low level cis-CaaD activity and lacks the specificity of cis-CaaD: Cg10062 processes both isomers of 3-chloroacrylate with a preference for the cis isomer. The basis for these differences is unknown, but a comparison of the crystal structures of the enzymes covalently modified by an adduct resulting from their incubation with the same inhibitor offers a possible explanation. A six-residue active site loop in cis-CaaD shows a conformation strikingly different from that observed in Cg10062: the loop closes down on the active site of cis-CaaD, but not on that of Cg10062. To examine what this loop might contribute to cis-CaaD catalysis and specificity, the residues were changed individually to those found in Cg10062. Subsequent kinetic and mechanistic analysis suggests that the T34A mutant of cis-CaaD is more Cg10062-like. The mutant enzyme shows a 4-fold increase in Km (using cis-3-bromoacrylate), but not to the degree observed for Cg10062 (687-fold). The mutation also causes a 4-fold decrease in the burst rate (compared to that of wild-type cis-CaaD), whereas Cg10062 shows no burst rate. More telling is the reaction of the T34A mutant of cis-CaaD with the alternate substrate, 2,3-butadienoate. In the presence of NaBH4 and the allene, cis-CaaD is completely inactivated after one turnover because of the covalent modification of Pro-1. The same experiment with Cg10062 does not result in the covalent modification of Pro-1. The different outcomes are attributed to covalent catalysis (using Pro-1) followed by hydrolysis of the enamine or imine tautomer in cis-CaaD versus direct hydration of the allene to yield acetoacetate in the case of Cg10062. The T34A mutant shows partial inactivation, requiring five turnovers of the substrate per monomer, which suggests that the direct hydration route is favored 80% of the time. However, the mutation does not alter the stereochemistry at C-2 of [2-D]acetoacetate when the reaction is conducted in D2O. Both cis-CaaD and the T34 mutant generate (2R)-[2-D]acetoacetate, whereas Cg10062 generates mostly the 2S isomer. The combined observations are consistent with a role for the loop region in cis-CaaD specificity and catalysis, but the precise role remains to be determined.

Dataset Information

The active site dynamics of 4-chlorobenzoyl-CoA dehalogenase.

Publications

The active site dynamics of 4-chlorobenzoyl-CoA dehalogenase.

Similar Datasets

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