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Unravelling the relaxed specificity of laminaribiose phosphorylase from Paenibacillus sp. strain YM-1 towards mannose 1-phosphate

DOI: 10.1002/cbic.201800260 DOI Help

Authors: Robert Andrew Field (John Innes Centre) , Sakonwan Kuhaudomlarp (John Innes Centre) , Samuel Walpole (University of East Anglia) , Clare Stevenson (John Innes Centre) , Sergey Nepogodiev (John Innes Centre) , David Lawson (John Innes Centre) , Jesus Angulo (University of East Anglia)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Chembiochem

State: Published (Approved)
Published: June 2018
Diamond Proposal Number(s): 13467

Abstract: Glycoside phosphorylases (GPs) carry out a reversible phosphorolysis of carbohydrates into oligosaccharide acceptors and the corresponding sugar 1‐phosphates. The reversibility of the reaction enables the use of GPs as biocatalysts for carbohydrate synthesis. Glycosyl hydrolase family 94 (GH94), which only comprises GPs, is one of the most studied GP families that have been used as biocatalysts for carbohydrate synthesis, in academic research and in industrial production. Understanding the mechanism of GH94 enzymes is a crucial step towards enzyme engineering to improve and expand the applications of these enzymes in synthesis. In this work with a GH94 laminaribiose phosphorylase from Paenibacillus sp. YM1 (PsLBP), we have demonstrated an enzymatic synthesis of disaccharide 1 using natural acceptor glucose and non‐cognate donor substrate ‐mannose 1‐phosphate (Man1P). To investigate how the enzyme recognizes different sugar 1‐phosphates, we solved the X‐ray crystal structures of PsLBP in complex with Glc1P and Man1P, providing the first molecular detail of the recognition of a non‐cognate donor substrate by GPs, which revealed the importance of hydrogen bonding between the active site residues and hydroxyl groups at C2, C4 and C6 of sugar 1‐phosphates. Furthermore, we used STD NMR to support the crystallographic studies on the sugar 1‐phosphates, as well as to provide further insights into the PsLBP recognition of the acceptors and the disaccharide products.

Journal Keywords: laminaribiose phosphorylase; Enzymatic synthesis; structural biology; glycosyl hydrolase family 94

Diamond Keywords: Enzymes

Subject Areas: Biology and Bio-materials, Chemistry

Instruments: I03-Macromolecular Crystallography , I04-Macromolecular Crystallography

Added On: 07/06/2018 08:59

Discipline Tags:

Biochemistry Catalysis Chemistry Structural biology Life Sciences & Biotech

Technical Tags:

Diffraction Macromolecular Crystallography (MX)