Efficient chemoenzymatic oligosaccharide synthesis by reverse phosphorolysis using cellobiose phosphorylase and cellodextrin phosphorylase from Clostridium thermocellum.
Biochimie
; 92(12): 1818-26, 2010 Dec.
Article
en En
| MEDLINE
| ID: mdl-20678539
ABSTRACT
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 ß-glucosyl disaccharides with ß-(1â4)- regioselectivity from five monosaccharides as well as branched ß-glucosyl trisaccharides with ß-(1â4)-regioselectivity from three (1â6)-linked disaccharides. CtCDP showed strict ß-(1â4)-regioselectivity and catalysed linear chain extension of the three ß-linked glucosyl disaccharides, cellobiose, sophorose, and laminaribiose, whereas 12 tested monosaccharides were not acceptors. Structure analysis by NMR and ESI-MS confirmed two ß-glucosyl oligosaccharide product series to represent novel compounds, i.e. ß-D-glucopyranosyl-[(1â4)-ß-D-glucopyranosyl](n)-(1â2)-D-glucopyranose, and ß-D-glucopyranosyl-[(1â4)-ß-D-glucopyranosyl](n)-(1â3)-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 ß-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.
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Oligosacáridos
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Proteínas Bacterianas
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Clostridium thermocellum
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Glucosiltransferasas
Idioma:
En
Revista:
Biochimie
Año:
2010
Tipo del documento:
Article
País de afiliación:
Dinamarca