Biochem. J. (2006) 398
(37–43) (Printed in Great Britain)
Structural dissection of the reaction mechanism of cellobiose phosphorylase
Masafumi HIDAKA*†, Motomitsu KITAOKA†, Kiyoshi HAYASHI†, Takayoshi WAKAGI*, Hirofumi SHOUN* and Shinya FUSHINOBU*1
*Department of Biotechnology, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan, and †National Food Research Institute, 2-1-12, Kannondai, Tsukuba, Ibaraki 305-8642, Japan
Cellobiose phosphorylase, a member of the glycoside hydrolase family 94, catalyses the reversible phosphorolysis of cellobiose into a-D-glucose 1-phosphate and D-glucose with inversion of the anomeric configuration. The substrate specificity and reaction mechanism of cellobiose phosphorylase from Cellvibrio gilvus have been investigated in detail. We have determined the crystal structure of the glucose-sulphate and glucose-phosphate complexes of this enzyme at a maximal resolution of 2.0 Å (1 Å=0.1 nm). The phosphate ion is strongly held through several hydrogen bonds, and the configuration appears to be suitable for direct nucleophilic attack to an anomeric centre. Structural features around the sugar-donor and sugar-acceptor sites were consistent with the results of extensive kinetic studies. When we compared this structure with that of homologous chitobiose phosphorylase, we identified key residues for substrate discrimination between glucose and N-acetylglucosamine in both the sugar-donor and sugar-acceptor sites. We found that the active site pocket of cellobiose phosphorylase was covered by an additional loop, indicating that some conformational change is required upon substrate binding. Information on the three-dimensional structure of cellobiose phosphorylase will facilitate engineering of this enzyme, the application of which to practical oligosaccharide synthesis has already been established.
Key words: carbohydrate-active enzyme, cellobiose phosphorylase, chitobiose phosphorylase, glycoside hydrolase family 94, substrate specificity, X-ray crystallography.
Abbreviations used: CBP, cellobiose phosphorylase; CDP, cellodextrin phosphorylase; CgCBP, CBP from Cellvibrio gilvus; CgCBP–SO4, CgCBP structure complexed with sulphate; CgCBP–PO4, CgCBP structure complexed with phosphate; ChBP, chitobiose phosphorylase; CuCBP, CBP from Cellulomonas uda; GH, glycoside hydrolase; Glc 1-P, glucose 1-phosphate; GT, glycosyl transferase; rmsd, rootmean square deviation; VpChBP, ChBP from Vibrio proteolyticus.
1To whom correspondence should be addressed (email asfushi@mail.ecc.u-tokyo.ac.jp).
The atomic co-ordinates and structure factors of CgCBP–SO4 and CgCBP–PO4 have been deposited with the RCSB Protein Data Bank under accession codes 2CQS and 2CQT respectively.
Received 16 February 2006/19 April 2006; accepted 28 April 2006
Published as BJ Immediate Publication 28 April 2006, doi:10.1042/BJ20060274
The Biochemical Society, London ©2006
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