Dataset Information

Discovery of an L-fucono-1,5-lactonase from cog3618 of the amidohydrolase superfamily.

ABSTRACT: A member of the amidohydrolase superfamily, BmulJ_04915 from Burkholderia multivorans, of unknown function was determined to hydrolyze a series of sugar lactones: L-fucono-1,4-lactone, D-arabino-1,4-lactone, L-xylono-1,4-lactone, D-lyxono-1,4-lactone, and L-galactono-1,4-lactone. The highest activity was shown for L-fucono-1,4-lactone with a k(cat) value of 140 s(-1) and a k(cat)/K(m) value of 1.0 × 10(5) M(-1) s(-1) at pH 8.3. The enzymatic product of an adjacent L-fucose dehydrogenase, BmulJ_04919, was shown to be L-fucono-1,5-lactone via nuclear magnetic resonance spectroscopy. L-Fucono-1,5-lactone is unstable and rapidly converts nonenzymatically to L-fucono-1,4-lactone. Because of the chemical instability of L-fucono-1,5-lactone, 4-deoxy-L-fucono-1,5-lactone was enzymatically synthesized from 4-deoxy-L-fucose using L-fucose dehydrogenase. BmulJ_04915 hydrolyzed 4-deoxy-L-fucono-1,5-lactone with a k(cat) value of 990 s(-1) and a k(cat)/K(m) value of 8.0 × 10(6) M(-1) s(-1) at pH 7.1. The protein does not require divalent cations in the active site for catalytic activity. BmulJ_04915 is the second enzyme from cog3618 of the amidohydrolase superfamily that does not require a divalent metal for catalytic activity. BmulJ_04915 is the first enzyme that has been shown to catalyze the hydrolysis of either L-fucono-1,4-lactone or L-fucono-1,5-lactone. The structures of the fuconolactonase and the fucose dehydrogenase were determined by X-ray diffraction methods.

SUBMITTER: Hobbs ME

PROVIDER: S-EPMC3542637 | biostudies-literature | 2013 Jan

REPOSITORIES: biostudies-literature

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Discovery of an L-fucono-1,5-lactonase from cog3618 of the amidohydrolase superfamily.

Hobbs Merlin Eric ME Vetting Matthew M Williams Howard J HJ Narindoshvili Tamari T Kebodeaux Devon M DM Hillerich Brandan B Seidel Ronald D RD Almo Steven C SC Raushel Frank M FM

Biochemistry 20121220 1

A member of the amidohydrolase superfamily, BmulJ_04915 from Burkholderia multivorans, of unknown function was determined to hydrolyze a series of sugar lactones: L-fucono-1,4-lactone, D-arabino-1,4-lactone, L-xylono-1,4-lactone, D-lyxono-1,4-lactone, and L-galactono-1,4-lactone. The highest activity was shown for L-fucono-1,4-lactone with a k(cat) value of 140 s(-1) and a k(cat)/K(m) value of 1.0 × 10(5) M(-1) s(-1) at pH 8.3. The enzymatic product of an adjacent L-fucose dehydrogenase, BmulJ_0 ...[more]

PMID: 23214453

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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.

Project description:The catalytic activities of three members of the amidohydrolase superfamily were discovered using amino acid substrate libraries. Bb3285 from Bordetella bronchiseptica, Gox1177 from Gluconobacter oxidans, and Sco4986 from Streptomyces coelicolor are currently annotated as d-aminoacylases or N-acetyl-d-glutamate deacetylases. These three enzymes are 22-34% identical to one another in amino acid sequence. Substrate libraries containing nearly all combinations of N-formyl-d-Xaa, N-acetyl-d-Xaa, N-succinyl-d-Xaa, and l-Xaa-d-Xaa were used to establish the substrate profiles for these enzymes. It was demonstrated that Bb3285 is restricted to the hydrolysis of N-acyl-substituted derivatives of d-glutamate. The best substrates for this enzyme are N-formyl-d-glutamate (k(cat)/K(m) = 5.8 x 10(6) M(-1) s(-1)), N-acetyl-d-glutamate (k(cat)/K(m) = 5.2 x 10(6) M(-1) s(-1)), and l-methionine-d-glutamate (k(cat)/K(m) = 3.4 x 10(5) M(-1) s(-1)). Gox1177 and Sco4986 preferentially hydrolyze N-acyl-substituted derivatives of hydrophobic d-amino acids. The best substrates for Gox1177 are N-acetyl-d-leucine (k(cat)/K(m) = 3.2 x 10(4) M(-1) s(-1)), N-acetyl-d-tryptophan (k(cat)/K(m) = 4.1 x 10(4) M(-1) s(-1)), and l-tyrosine-d-leucine (k(cat)/K(m) = 1.5 x 10(4) M(-1) s(-1)). A fourth protein, Bb2785 from B. bronchiseptica, did not have d-aminoacylase activity. The best substrates for Sco4986 are N-acetyl-d-phenylalanine and N-acetyl-d-tryptophan. The three-dimensional structures of Bb3285 in the presence of the product acetate or a potent mimic of the tetrahedral intermediate were determined by X-ray diffraction methods. The side chain of the d-glutamate moiety of the inhibitor is ion-paired to Arg-295, while the alpha-carboxylate is ion-paired with Lys-250 and Arg-376. These results have revealed the chemical and structural determinants for substrate specificity in this protein. Bioinformatic analyses of an additional approximately 250 sequences identified as members of this group suggest that there are no simple motifs that allow prediction of substrate specificity for most of these unknowns, highlighting the challenges for computational annotation of some groups of homologous proteins.

Dataset Information

Discovery of an L-fucono-1,5-lactonase from cog3618 of the amidohydrolase superfamily.

Publications

Discovery of an L-fucono-1,5-lactonase from cog3618 of the amidohydrolase superfamily.

Similar Datasets

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