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Cellodextrin phosphorylase from Ruminiclostridium thermocellum: X-ray crystal structure and substrate specificity
Ellis C O'Neill1, Giulia Pergolizzi1, Clare E M Stevenson1
1Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK.
Carbohydrate Research
|August 2, 2017
Summary
This study characterizes cellodextrin phosphorylase (CDP), a GH94 enzyme, exploring its substrate specificity and revealing its first X-ray crystal structure. Findings illuminate structural differences between CDP and similar enzymes, explaining substrate discrimination.
Area of Science:
- Biochemistry and Structural Biology
- Enzymology
- Glycoside Hydrolases
Background:
- Cellodextrin phosphorylase (CDP, EC 2.4.1.49) is a GH94 enzyme producing oligosaccharides from glucans and glucose phosphate.
- CDP is less characterized than cellobiose phosphorylase (CBP), highlighting a knowledge gap in understanding its biochemical function.
- Investigating CDP's substrate specificity and structure is crucial for enzyme mechanism elucidation.
Purpose of the Study:
- To investigate the donor and acceptor substrate specificity of recombinant CDP from Ruminiclostridium thermocellum.
- To characterize a novel glucosamine addition product formed with a non-natural donor.
- To determine the first X-ray crystal structure of CDP and compare it with related enzymes.
Main Methods:
- Biochemical assays to determine substrate specificity of recombinant CDP.
- Isolation and characterization of enzymatic products, including novel adducts.
- X-ray crystallography to determine the three-dimensional structure of CDP.
Main Results:
- Recombinant CDP exhibits specific donor and acceptor substrate preferences.
- A unique glucosamine addition product was identified using α-D-glucosamine 1-phosphate.
- The first X-ray crystal structure of CDP was determined, revealing key structural features for substrate discrimination.
Conclusions:
- The study elucidates the biochemical properties and substrate specificity of CDP.
- Structural comparisons highlight critical differences enabling CDP to process oligosaccharides versus monosaccharides.
- Findings provide insights into the structural basis for enzyme specificity in the GH94 family.
