Journal
CARBOHYDRATE RESEARCH
Volume 379, Issue -, Pages 21-25Publisher
ELSEVIER SCI LTD
DOI: 10.1016/j.carres.2013.06.010
Keywords
Cellobiose phosphorylase; Glycoside hydrolase family 94; Oligosaccharide synthesis; Site-directed mutagenesis; Acceptor specificity
Funding
- Grants-in-Aid for Scientific Research [24580134, 24780091] Funding Source: KAKEN
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Cellobiose phosphorylase (EC 2.4.1.20, CBP) catalyzes the reversible phosphorolysis of cellobiose to alpha-D-glucose 1-phosphate (Glc1P) and D-glucose. Cys485, Tyr648, and Glu653 of CBP from Ruminococcus albus, situated at the +1 subsite, were mutated to modulate acceptor specificity. C485A, Y648F, and Y648V were active enough for analysis. Their acceptor specificities were compared with the wild type based on the apparent kinetic parameters determined in the presence of 10 mM Glc1P. C485A showed higher preference for D-glucosamine than the wild type. Apparent k(cat)/K-m values of Y648F for D-mannose and 2-deoxy-D-glucose were 8.2- and 4.0-fold higher than those of the wild type, respectively. Y648V had synthetic activity toward N-acetyl-D-glucosamine, while the other variants did not. The oligosaccharide production in the presence of the same concentrations of wild type and each mutant was compared. C485A produced 4-O-beta-D-glucopyranosyl-D-glucosamine from 10 mM Glc1P and D-glucosamine at a rate similar to the wild type. Y648F and Y648V produced 4-O-beta-D-glucopyranosyl-D-mannose and 4-O-beta-D-glucopyranosyl-N-acetyl-D-glucosamine much more rapidly than the wild type when D-mannose and N-acetyl-D-glucosamine were used as acceptors, respectively. After a 4 h reaction, the amounts of 4-O-beta-D-glucopyranosyl-D-mannose and 4-O-beta-D-glucopyranosyl-N-acetyl-D-glucosamine produced by Y648F and Y648V were 5.9- and 12-fold higher than the wild type, respectively. (C) 2013 Elsevier Ltd. All rights reserved.
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