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Functional identification and structure determination of two novel prolidases from cog1228 in the amidohydrolase superfamily .

ABSTRACT: Two uncharacterized enzymes from the amidohydrolase superfamily belonging to cog1228 were cloned, expressed, and purified to homogeneity. The two proteins, Sgx9260c ( gi|44242006 ) and Sgx9260b ( gi|44479596 ), were derived from environmental DNA samples originating from the Sargasso Sea. The catalytic function and substrate profiles for Sgx9260c and Sgx9260b were determined using a comprehensive library of dipeptides and N-acyl derivative of l-amino acids. Sgx9260c catalyzes the hydrolysis of Gly-l-Pro, l-Ala-l-Pro, and N-acyl derivatives of l-Pro. The best substrate identified to date is N-acetyl-l-Pro with a value of k(cat)/K(m) of 3 x 10(5) M(-1) s(-1). Sgx9260b catalyzes the hydrolysis of l-hydrophobic l-Pro dipeptides and N-acyl derivatives of l-Pro. The best substrate identified to date is N-propionyl-l-Pro with a value of k(cat)/K(m) of 1 x 10(5) M(-1) s(-1). Three-dimensional structures of both proteins were determined by X-ray diffraction methods (PDB codes 3MKV and 3FEQ ). These proteins fold as distorted (beta/alpha)(8)-barrels with two divalent cations in the active site. The structure of Sgx9260c was also determined as a complex with the N-methylphosphonate derivative of l-Pro (PDB code 3N2C ). In this structure the phosphonate moiety bridges the binuclear metal center, and one oxygen atom interacts with His-140. The alpha-carboxylate of the inhibitor interacts with Tyr-231. The proline side chain occupies a small substrate binding cavity formed by residues contributed from the loop that follows beta-strand 7 within the (beta/alpha)(8)-barrel. A total of 38 other proteins from cog1228 are predicted to have the same substrate profile based on conservation of the substrate binding residues. The structure of an evolutionarily related protein, Cc2672 from Caulobacter crecentus, was determined as a complex with the N-methylphosphonate derivative of l-arginine (PDB code 3MTW ).

SUBMITTER: Xiang DF

PROVIDER: S-EPMC2914802 | biostudies-literature | 2010 Aug

REPOSITORIES: biostudies-literature

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Functional identification and structure determination of two novel prolidases from cog1228 in the amidohydrolase superfamily .

Xiang Dao Feng DF Patskovsky Yury Y Xu Chengfu C Fedorov Alexander A AA Fedorov Elena V EV Sisco Abby A AA Sauder J Michael JM Burley Stephen K SK Almo Steven C SC Raushel Frank M FM

Biochemistry 20100801 31

Two uncharacterized enzymes from the amidohydrolase superfamily belonging to cog1228 were cloned, expressed, and purified to homogeneity. The two proteins, Sgx9260c ( gi|44242006 ) and Sgx9260b ( gi|44479596 ), were derived from environmental DNA samples originating from the Sargasso Sea. The catalytic function and substrate profiles for Sgx9260c and Sgx9260b were determined using a comprehensive library of dipeptides and N-acyl derivative of l-amino acids. Sgx9260c catalyzes the hydrolysis of G ...[more]

PMID: 20604542

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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

Functional identification and structure determination of two novel prolidases from cog1228 in the amidohydrolase superfamily .

Publications

Functional identification and structure determination of two novel prolidases from cog1228 in the amidohydrolase superfamily .

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