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Biochemical and Structural Characterization of the Carbohydrate Transport Substrate-binding-protein SP0092
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Structural and mutational analysis of substrate recognition in kojibiose phosphorylase
Satoshi Okada1, Takuo Yamamoto, Hikaru Watanabe
1Department of Biotechnology, University of Tokyo, Japan.
The FEBS Journal
|November 22, 2013
Summary
Structural insights into glycoside hydrolase family 65 enzymes reveal key residues for kojibiose recognition. This study provides the first structural data for kojibiose phosphorylase, aiding in the synthesis of alpha-glucosyl oligosaccharides.
Area of Science:
- Enzymology
- Structural Biology
- Carbohydrate Chemistry
Background:
- Glycoside hydrolase (GH) family 65 enzymes, specifically phosphorylases, act on various alpha-glucosyl oligosaccharides.
- These enzymes can catalyze reverse reactions, enabling the synthesis of alpha-glucosyl oligosaccharides.
Purpose of the Study:
- To determine the crystal structures of kojibiose phosphorylase from Caldicellulosiruptor saccharolyticus.
- To gain structural insights into substrate recognition mechanisms within GH family 65.
Main Methods:
- X-ray crystallography was used to determine the structures of kojibiose phosphorylase in complex with glucose/phosphate and kojibiose/sulfate.
- Site-directed mutagenesis was employed to create variants of key active site residues.
- Enzyme activity assays were performed on wild-type and mutant enzymes using various substrates.
Main Results:
- The crystal structures revealed the first structural insights into substrate recognition for a GH family 65 enzyme.
- A significantly longer loop 3 region in the active site was observed compared to other enzymes.
- Residues Trp391, Glu392, and Thr417 were identified as crucial for kojibiose recognition, with Glu392 being particularly important.
Conclusions:
- The study elucidates the structural basis for kojibiose recognition by kojibiose phosphorylase.
- Specific residues, Trp391 and Glu392, are essential for the enzyme's kojibiose activity.
- These findings contribute to understanding GH family 65 mechanisms and potential applications in oligosaccharide synthesis.
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