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Evolutionary expansion of the amidohydrolase superfamily in bacteria in response to the synthetic compounds molinate and diuron.

ABSTRACT: The amidohydrolase superfamily has remarkable functional diversity, with considerable structural and functional annotation of known sequences. In microbes, the recent evolution of several members of this family to catalyze the breakdown of environmental xenobiotics is not well understood. An evolutionary transition from binuclear to mononuclear metal ion coordination at the active sites of these enzymes could produce large functional changes such as those observed in nature, but there are few clear examples available to support this hypothesis. To investigate the role of binuclear-mononuclear active-site transitions in the evolution of new function in this superfamily, we have characterized two recently evolved enzymes that catalyze the hydrolysis of the synthetic herbicides molinate (MolA) and phenylurea (PuhB). In this work, the crystal structures, mutagenesis, metal ion analysis, and enzyme kinetics of both MolA and PuhB establish that these enzymes utilize a mononuclear active site. However, bioinformatics and structural comparisons reveal that the closest putative ancestor of these enzymes had a binuclear active site, indicating that a binuclear-mononuclear transition has occurred. These proteins may represent examples of evolution modifying the characteristics of existing catalysts to satisfy new requirements, specifically, metal ion rearrangement leading to large leaps in activity that would not otherwise be possible.

SUBMITTER: Sugrue E

PROVIDER: S-EPMC4357960 | biostudies-literature | 2015 Apr

REPOSITORIES: biostudies-literature

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Evolutionary expansion of the amidohydrolase superfamily in bacteria in response to the synthetic compounds molinate and diuron.

Sugrue Elena E Fraser Nicholas J NJ Hopkins Davis H DH Carr Paul D PD Khurana Jeevan L JL Oakeshott John G JG Scott Colin C Jackson Colin J CJ

Applied and environmental microbiology 20150130 7

The amidohydrolase superfamily has remarkable functional diversity, with considerable structural and functional annotation of known sequences. In microbes, the recent evolution of several members of this family to catalyze the breakdown of environmental xenobiotics is not well understood. An evolutionary transition from binuclear to mononuclear metal ion coordination at the active sites of these enzymes could produce large functional changes such as those observed in nature, but there are few cl ...[more]

PMID: 25636851

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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

Evolutionary expansion of the amidohydrolase superfamily in bacteria in response to the synthetic compounds molinate and diuron.

Publications

Evolutionary expansion of the amidohydrolase superfamily in bacteria in response to the synthetic compounds molinate and diuron.

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