TY - JOUR
T1 - Efficient chemoenzymatic oligosaccharide synthesis by reverse phosphorolysis using cellobiose phosphorylase and cellodextrin phosphorylase from Clostridium thermocellum
AU - Nakai, H.
AU - Abou Hachem, M.
AU - Petersen, B.O.
AU - Westphal, Y.
AU - Mannerstedt, K.
AU - Baumann, M.J.
AU - Dilokpimol, A.
AU - Schols, H.A.
AU - Duus, J.O.
AU - Svensson, B.
N1 - ISI:000286295900015
PY - 2010
Y1 - 2010
N2 - 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.
AB - 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.
KW - cellvibrio-gilvus
KW - reaction-mechanism
KW - ruminococcus-flavefaciens
KW - chitobiose phosphorylase
KW - maltose phosphorylase
KW - vibrio-proteolyticus
KW - thermotoga-maritima
KW - escherichia-coli
KW - cellulomonas-uda
KW - d-glucose
U2 - 10.1016/j.biochi.2010.07.013
DO - 10.1016/j.biochi.2010.07.013
M3 - Article
SN - 0300-9084
VL - 92
SP - 1818
EP - 1826
JO - Biochimie
JF - Biochimie
IS - 12
ER -