Dataset Information

Functional annotation and three-dimensional structure of an incorrectly annotated dihydroorotase from cog3964 in the amidohydrolase superfamily.

ABSTRACT: The substrate specificities of two incorrectly annotated enzymes belonging to cog3964 from the amidohydrolase superfamily were determined. This group of enzymes are currently misannotated as either dihydroorotases or adenine deaminases. Atu3266 from Agrobacterium tumefaciens C58 and Oant2987 from Ochrobactrum anthropi ATCC 49188 were found to catalyze the hydrolysis of acetyl-(R)-mandelate and similar esters with values of k(cat)/K(m) that exceed 10(5) M(-1) s(-1). These enzymes do not catalyze the deamination of adenine or the hydrolysis of dihydroorotate. Atu3266 was crystallized and the structure determined to a resolution of 2.62 Å. The protein folds as a distorted (?/?)(8) barrel and binds two zincs in the active site. The substrate profile was determined via a combination of computational docking to the three-dimensional structure of Atu3266 and screening of a highly focused library of potential substrates. The initial weak hit was the hydrolysis of N-acetyl-D-serine (k(cat)/K(m) = 4 M(-1) s(-1)). This was followed by the progressive identification of acetyl-(R)-glycerate (k(cat)/K(m) = 4 × 10(2) M(-1) s(-1)), acetyl glycolate (k(cat)/K(m) = 1.3 × 10(4) M(-1) s(-1)), and ultimately acetyl-(R)-mandelate (k(cat)/K(m) = 2.8 × 10(5) M(-1) s(-1)).

SUBMITTER: Ornelas A

PROVIDER: S-EPMC3542638 | biostudies-literature | 2013 Jan

REPOSITORIES: biostudies-literature

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Functional annotation and three-dimensional structure of an incorrectly annotated dihydroorotase from cog3964 in the amidohydrolase superfamily.

Ornelas Argentina A Korczynska Magdalena M Ragumani Sugadev S Kumaran Desigan D Narindoshvili Tamari T Shoichet Brian K BK Swaminathan Subramanyam S Raushel Frank M FM

Biochemistry 20121220 1

The substrate specificities of two incorrectly annotated enzymes belonging to cog3964 from the amidohydrolase superfamily were determined. This group of enzymes are currently misannotated as either dihydroorotases or adenine deaminases. Atu3266 from Agrobacterium tumefaciens C58 and Oant2987 from Ochrobactrum anthropi ATCC 49188 were found to catalyze the hydrolysis of acetyl-(R)-mandelate and similar esters with values of k(cat)/K(m) that exceed 10(5) M(-1) s(-1). These enzymes do not catalyze ...[more]

PMID: 23214420

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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 annotation and three-dimensional structure of an incorrectly annotated dihydroorotase from cog3964 in the amidohydrolase superfamily.

Publications

Functional annotation and three-dimensional structure of an incorrectly annotated dihydroorotase from cog3964 in the amidohydrolase superfamily.

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