title: Efficient chemoenzymatic oligosaccharide synthesis by reverse phosphorolysis using cellobiose phosphorylase and cellodextrin phosphorylase from Clostridium thermocellum.
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 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.
authors.affiliation: Enzyme and Protein Chemistry, Department of Systems Biology, Technical University of Denmark, Søltofts Plads, Building 224, DK-2800 Kgs. Lyngby, Denmark.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Nakai
authors.forename: Maher Abou
authors.initials: MA
authors.lastname: Hachem
authors.forename: Bent O
authors.initials: BO
authors.lastname: Petersen
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Westphal
authors.forename: Karin
authors.initials: K
authors.lastname: Mannerstedt
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Baumann
authors.forename: Adiphol
authors.initials: A
authors.lastname: Dilokpimol
authors.forename: Henk A
authors.initials: HA
authors.lastname: Schols
authors.forename: Jens Ø
authors.initials: JØ
authors.lastname: Duus
authors.forename: Birte
authors.initials: B
authors.lastname: Svensson
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D055162:Biocatalysis; D002475:Cellobiose / Q000096:biosynthesis / Q000737:chemistry; D002482:Cellulose / Q000031:analogs & derivatives / Q000096:biosynthesis / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D048013:Acetivibrio thermocellus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D003912:Dextrins / Q000096:biosynthesis / Q000737:chemistry; D004795:Enzyme Stability; D005964:Glucosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009844:Oligosaccharides / Q000096:biosynthesis* / Q000737:chemistry; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013237:Stereoisomerism; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D003912:Dextrins; D009844:Oligosaccharides; D002475:Cellobiose; D002482:Cellulose; C068994:cellodextrin; D005964:Glucosyltransferases; C034714:cellobiose phosphorylase; C105024:cellodextrin phosphorylase