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Disaccharide phosphorylases: Structure, catalytic mechanisms and directed evolution.

ABSTRACT: Disaccharide phosphorylases (DSPs) are carbohydrate-active enzymes with outstanding potential for the biocatalytic conversion of common table sugar into products with attractive properties. They are modular enzymes that form active homo-oligomers. From a mechanistic as well as a structural point of view, they are similar to glycoside hydrolases or glycosyltransferases. As the majority of DSPs show strict stereo- and regiospecificities, these enzymes were used to synthesize specific disaccharides. Currently, protein engineering of DSPs is pursued in different laboratories to broaden the donor and acceptor substrate specificities or improve the industrial particularity of naturally existing enzymes, to eventually generate a toolbox of new catalysts for glycoside synthesis. Herein we review the characteristics and classifications of reported DSPs and the glycoside products that they have been used to synthesize.

SUBMITTER: Sun S

PROVIDER: S-EPMC7896129 | biostudies-literature | 2021 Mar

REPOSITORIES: biostudies-literature

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Disaccharide phosphorylases: Structure, catalytic mechanisms and directed evolution.

Sun Shangshang S You Chun C

Synthetic and systems biotechnology 20210213 1

Disaccharide phosphorylases (DSPs) are carbohydrate-active enzymes with outstanding potential for the biocatalytic conversion of common table sugar into products with attractive properties. They are modular enzymes that form active homo-oligomers. From a mechanistic as well as a structural point of view, they are similar to glycoside hydrolases or glycosyltransferases. As the majority of DSPs show strict stereo- and regiospecificities, these enzymes were used to synthesize specific disaccharides ...[more]

PMID: 33665389

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Project description:BackgroundTrehalases have potential applications in several fields, including food additives, insecticide development, and transgenic plant. In the present study, we focused on a trehalase from the marine bacterium Zunongwangia sp., which hydrolyzes trehalose to glucose.ResultsA novel gene, treZ (1590 bp) encoding an α, α-trehalase of 529 amino acids was cloned from Zunongwangia sp., and TreZ was found to have an optimal activity at 50 °C and pH 6. The activity of TreZ was increased by the presence of NaCl, showing the highest activity (136 %) at 1 M NaCl. A variant C4 with improved catalytic activity was obtained by error-prone PCR and followed by a 96-well plate high-throughput screening. The variant C4 with two altered sites (Y227H, and R442G) displayed a 3.3 fold increase in catalytic efficiency (k cat/K m, 1143.40 mmol(-1) s(-1)) compared with the wild type enzyme (265.91 mmol(-1) s(-1)). In order to explore the contribution of the mutations found in variant C4 to the increased catalytic activity, two mutants Y227H and R442G were constructed by site-directed mutagenesis. The results showed that the catalytic efficiencies of Y227H and R442G were 416.78 mmol(-1) s(-1) and 740.97 mmol(-1) s(-1), respectively, indicating that both mutations contributed to the increased catalytic efficiency of variant C4. The structure modeling and substrate docking revealed that the substitution Y227H enlarged the shape of the binding pocket, to improve the binding of the substrate and the release of the products; while the substitution R442G reduced the size of the side chain and decreased the steric hindrance, which contributed to channel the substrate into the active cavity easier and promote the release of the product.ConclusionIn this study, a novel trehalase was cloned, purified, characterized, and engineered. A variant C4 with dramatically improved catalytic activity was obtained by directed evolution, and the mutation sites Y227H and R442G were found to play a significant role in the catalytic efficiency. The overall results provide useful information about the structure and function of trehalase.

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Disaccharide phosphorylases: Structure, catalytic mechanisms and directed evolution.

Publications

Disaccharide phosphorylases: Structure, catalytic mechanisms and directed evolution.

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