Efficient chemoenzymatic oligosaccharide synthesis by reverse phosphorolysis using cellobiose phosphorylase and cellodextrin phosphorylase from Clostridium thermocellum

• Published in: Biochimie 2010-12, Vol.92 (12), p.1818-1826
• Main Authors: Nakai, Hiroyuki, Hachem, Maher Abou, Petersen, Bent O., Westphal, Yvonne, Mannerstedt, Karin, Baumann, Martin J., Dilokpimol, Adiphol, Schols, Henk A., Duus, Jens Ø., Svensson, Birte
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Binding Sites - genetics; Biocatalysis; Cellobiose - biosynthesis; Cellobiose - chemistry; cellulomonas-uda; Cellulose - analogs & derivatives; Cellulose - biosynthesis; Cellulose - chemistry; cellvibrio-gilvus; chitobiose phosphorylase; Chromatography, High Pressure Liquid; Clostridium thermocellum - enzymology; Clostridium thermocellum - genetics; Clostridium thermocellum - metabolism; d-glucose; Dextrins - biosynthesis; Dextrins - chemistry; Enzyme Stability; escherichia-coli; Glucosyltransferases - chemistry; Glucosyltransferases - genetics; Glucosyltransferases - metabolism; Glycoside hydrolase family 94; Hydrogen-Ion Concentration; Inverting glycosynthase reaction; maltose phosphorylase; Models, Molecular; Molecular Sequence Data; Molecular Structure; Non-natural oligosaccharides; Oligosaccharides - biosynthesis; Oligosaccharides - chemistry; Protein Structure, Tertiary; reaction-mechanism; Regioselectivity; ruminococcus-flavefaciens; Sequence Homology, Amino Acid; Stereoisomerism; Temperature; thermotoga-maritima; vibrio-proteolyticus; α- d-Glucose 1-phosphate; α- d-Glucosyl 1-fluoride
• ISSN: 0300-9084; 1638-6183
• DOI: 10.1016/j.biochi.2010.07.013

Inverting cellobiose phosphorylase (CtCBP) and cellodextrin phosphorylase (CtCDP) from Clostridium thermocellum ATCC27405 of glycoside hydrolase family 94 catalysed reverse phosphorolysis to produce cellobiose and cellodextrins in 57% and 48% yield from α- d-glucose 1-phosphate as donor with glucose and cellobiose as acceptor, respectively. Use of α- d-glucosyl 1-fluoride as donor increased product yields to 98% for CtCBP and 68% for CtCDP. CtCBP showed broad acceptor specificity forming β-glucosyl disaccharides with β-(1→4)- regioselectivity from five monosaccharides as well as branched β-glucosyl trisaccharides with β-(1→4)-regioselectivity from three (1→6)-linked disaccharides. CtCDP showed strict β-(1→4)-regioselectivity and catalysed linear chain extension of the three β-linked glucosyl disaccharides, cellobiose, sophorose, and laminaribiose, whereas 12 tested monosaccharides were not acceptors. Structure analysis by NMR and ESI-MS confirmed two β-glucosyl oligosaccharide product series to represent novel compounds, i.e. β- d-glucopyranosyl-[(1→4)-β- d-glucopyranosyl] n-(1→2)- d-glucopyranose, and β- d-glucopyranosyl-[(1→4)-β- d-glucopyranosyl] n-(1→3)- d-glucopyranose ( n = 1–7). Multiple sequence alignment together with a modelled CtCBP structure, obtained using the crystal structure of Cellvibrio gilvus CBP in complex with glucose as a template, indicated differences in the subsite +1 region that elicit the distinct acceptor specificities of CtCBP and CtCDP. Thus Glu636 of CtCBP recognized the C1 hydroxyl of β-glucose at subsite +1, while in CtCDP the presence of Ala800 conferred more space, which allowed accommodation of C1 substituted disaccharide acceptors at the corresponding subsites +1 and +2. Furthermore, CtCBP has a short Glu496-Thr500 loop that permitted the C6 hydroxyl of glucose at subsite +1 to be exposed to solvent, whereas the corresponding longer loop Thr637–Lys648 in CtCDP blocks binding of C6-linked disaccharides as acceptors at subsite +1. High yields in chemoenzymatic synthesis, a novel regioselectivity, and novel oligosaccharides including products of CtCDP catalysed oligosaccharide oligomerisation using α- d-glucosyl 1-fluoride, all together contribute to the formation of an excellent basis for rational engineering of CBP and CDP to produce desired oligosaccharides.