Dataset Information

Structural characterization and function determination of a nonspecific carboxylate esterase from the amidohydrolase superfamily with a promiscuous ability to hydrolyze methylphosphonate esters.

ABSTRACT: The uncharacterized protein Rsp3690 from Rhodobacter sphaeroides is a member of the amidohydrolase superfamily of enzymes. In this investigation the gene for Rsp3690 was expressed in Escherichia coli and purified to homogeneity, and the three-dimensional structure was determined to a resolution of 1.8 Å. The protein folds as a distorted (?/?)8-barrel, and the subunits associate as a homotetramer. The active site is localized to the C-terminal end of the ?-barrel and is highlighted by the formation of a binuclear metal center with two manganese ions that are bridged by Glu-175 and hydroxide. The remaining ligands to the metal center include His-32, His-34, His-207, His-236, and Asp-302. Rsp3690 was shown to catalyze the hydrolysis of a wide variety of carboxylate esters, in addition to organophosphate and organophosphonate esters. The best carboxylate ester substrates identified for Rsp3690 included 2-naphthyl acetate (kcat/Km = 1.0 × 10(5) M(-1) s(-1)), 2-naphthyl propionate (kcat/Km = 1.5 × 10(5) M(-1) s(-1)), 1-naphthyl acetate (kcat/Km = 7.5 × 10(3) M(-1) s(-1)), 4-methylumbelliferyl acetate (kcat/Km = 2.7 × 10(3) M(-1) s(-1)), 4-nitrophenyl acetate (kcat/Km = 2.3 × 10(5) M(-1) s(-1)), and 4-nitrophenyl butyrate (kcat/Km = 8.8 × 10(5) M(-1) s(-1)). The best organophosphonate ester substrates included ethyl 4-nitrophenyl methylphosphonate (kcat/Km = 3.8 × 10(5) M(-1) s(-1)) and isobutyl 4-nitrophenyl methylphosphonate (kcat/Km = 1.1 × 10(4) M(-1) s(-1)). The (SP)-enantiomer of isobutyl 4-nitrophenyl methylphosphonate was hydrolyzed 10 times faster than the less toxic (RP)-enantiomer. The high inherent catalytic activity of Rsp3690 for the hydrolysis of the toxic enantiomer of methylphosphonate esters make this enzyme an attractive target for directed evolution investigations.

SUBMITTER: Xiang DF

PROVIDER: S-EPMC4045322 | biostudies-literature | 2014 Jun

REPOSITORIES: biostudies-literature

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Structural characterization and function determination of a nonspecific carboxylate esterase from the amidohydrolase superfamily with a promiscuous ability to hydrolyze methylphosphonate esters.

Xiang Dao Feng DF Kumaran Desigan D Swaminathan Subramanyam S Raushel Frank M FM

Biochemistry 20140522 21

The uncharacterized protein Rsp3690 from Rhodobacter sphaeroides is a member of the amidohydrolase superfamily of enzymes. In this investigation the gene for Rsp3690 was expressed in Escherichia coli and purified to homogeneity, and the three-dimensional structure was determined to a resolution of 1.8 Å. The protein folds as a distorted (β/α)8-barrel, and the subunits associate as a homotetramer. The active site is localized to the C-terminal end of the β-barrel and is highlighted by the formati ...[more]

PMID: 24832101

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Project description:Two proteins from the amidohydrolase superfamily of enzymes were cloned, expressed, and purified to homogeneity. The first protein, Cc0300, was from Caulobacter crescentus CB-15 (Cc0300), while the second one (Sgx9355e) was derived from an environmental DNA sequence originally isolated from the Sargasso Sea ( gi|44371129 ). The catalytic functions and the substrate profiles for the two enzymes were determined with the aid of combinatorial dipeptide libraries. Both enzymes were shown to catalyze the hydrolysis of l-Xaa-l-Xaa dipeptides in which the amino acid at the N-terminus was relatively unimportant. These enzymes were specific for hydrophobic amino acids at the C-terminus. With Cc0300, substrates terminating in isoleucine, leucine, phenylalanine, tyrosine, valine, methionine, and tryptophan were hydrolyzed. The same specificity was observed with Sgx9355e, but this protein was also able to hydrolyze peptides terminating in threonine. Both enzymes were able to hydrolyze N-acetyl and N-formyl derivatives of the hydrophobic amino acids and tripeptides. The best substrates identified for Cc0300 were l-Ala-l-Leu with k(cat) and k(cat)/K(m) values of 37 s(-1) and 1.1 x 10(5) M(-1) s(-1), respectively, and N-formyl-l-Tyr with k(cat) and k(cat)/K(m) values of 33 s(-1) and 3.9 x 10(5) M(-1) s(-1), respectively. The best substrate identified for Sgx9355e was l-Ala-l-Phe with k(cat) and k(cat)/K(m) values of 0.41 s(-1) and 5.8 x 10(3) M(-1) s(-1). The three-dimensional structure of Sgx9355e was determined to a resolution of 2.33 A with l-methionine bound in the active site. The alpha-carboxylate of the methionine is ion-paired to His-237 and also hydrogen bonded to the backbone amide groups of Val-201 and Leu-202. The alpha-amino group of the bound methionine interacts with Asp-328. The structural determinants for substrate recognition were identified and compared with other enzymes in this superfamily that hydrolyze dipeptides with different specificities.

Dataset Information

Structural characterization and function determination of a nonspecific carboxylate esterase from the amidohydrolase superfamily with a promiscuous ability to hydrolyze methylphosphonate esters.

Publications

Structural characterization and function determination of a nonspecific carboxylate esterase from the amidohydrolase superfamily with a promiscuous ability to hydrolyze methylphosphonate esters.

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