Cellodextrin phosphorylase from Ruminiclostridium thermocellum: X-ray crystal structure and substrate specificity analysis

Highlights

• Screening of CDP activity against a variety of donors and acceptors.

• GlcN-1-P acts as CDP donor and generates a single turnover product.

• Xyl-1-P and Man-1-P are poor donors but competitive inhibitors of CDP.

• Oligosaccharides with GlcN and Xyl at the non-reducing end are good CDP acceptors.

• The apo structure of CDP and its complex with cellotetraose and Pi have been resolved.

Abstract

The GH94 glycoside hydrolase cellodextrin phosphorylase (CDP, EC 2.4.1.49) produces cellodextrin oligomers from short β-1→4-glucans and α-D-glucose 1-phosphate. Compared to cellobiose phosphorylase (CBP), which produces cellobiose from glucose and α-D-glucose 1-phosphate, CDP is biochemically less well characterised. Herein, we investigate the donor and acceptor substrate specificity of recombinant CDP from Ruminiclostridium thermocellum and we isolate and characterise a glucosamine addition product to the cellobiose acceptor with the non-natural donor α-D-glucosamine 1-phosphate. In addition, we report the first X-ray crystal structure of CDP, along with comparison to the available structures from CBPs and other closely related enzymes, which contributes to understanding of the key structural features necessary to discriminate between monosaccharide (CBP) and oligosaccharide (CDP) acceptor substrates.