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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 alpha-D-glucose 1-phosphate as donor with glucose and cellobiose as acceptor, respectively. Use of alpha-D-glucosyl 1-fluoride as donor increased product yields to 98% for CtCBP and 68% for CtCDP. CtCBP showed broad acceptor specificity forming beta-glucosyl disaccharides with beta-(1-->4)- regioselectivity from five monosaccharides as well as branched beta-glucosyl trisaccharides with beta-(1-->4)-regioselectivity from three (1-->6)-linked disaccharides. CtCDP showed strict beta-(1-->4)-regioselectivity and catalysed linear chain extension of the three beta-linked glucosyl disaccharides, cellobiose, sophorose, and laminaribiose, whereas 12 tested monosaccharides were not acceptors. Structure analysis by NMR and ESI-MS confirmed two beta-glucosyl oligosaccharide product series to represent novel compounds, i.e. beta-D-glucopyranosyl-[(1-->4)-beta-D-glucopyranosyl](n)-(1-->2)-D-gluco pyranose, and beta-D-glucopyranosyl-(1-->4)-beta-D-glucopyranosyl](n)-(1-->3)-D-glucop yranose (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 Cl hydroxyl of beta-glucose at subsite +1, while in CtCDP the presence of Ala800 conferred more space, which allowed accommodation of Cl 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 alpha-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.

Disaccharide phosphorylases are able to catalyze both the synthesis and the breakdown of disaccharides and have thus emerged as attractive platforms for tailor-made sugar synthesis. Cellobiose phosphorylase from Cellulomonas uda (CPCuda) is an enzyme that belongs to glycoside hydrolase family 94 and catalyzes the reversible breakdown of cellobiose [beta-D-glucopyranosyl-(1,4)-D-glucopyranose] to alpha-D-glucose-1-phosphate and D-glucose. Crystals of ligand-free recombinant CPCuda and of its complexes with substrates and reaction products yielded complete X-ray diffraction data sets to high resolution using synchrotron radiation but suffered from significant variability in diffraction quality. In at least one case an intriguing space-group transition from a primitive monoclinic to a primitive orthorhombic lattice was observed during data collection. The structure of CPCuda was determined by maximum-likelihood molecular replacement, thus establishing a starting point for an investigation of the structural and mechanistic determinants of disaccharide phosphorylase activity.