Skip Navigation

Protein Engineering Design and Selection 2006 19(12):571-578; doi:10.1093/protein/gzl046
This Article
Right arrow Full Text Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (1)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Nakahara, T.
Right arrow Articles by Nishimura, S.-I.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nakahara, T.
Right arrow Articles by Nishimura, S.-I.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Computational design and experimental evaluation of glycosyltransferase mutants: engineering of a blood type B galactosyltransferase with enhanced glucosyltransferase activity

Taku Nakahara1, Ole Hindsgaul2, Monica M. Palcic2 and Shin-Ichiro Nishimura1,3,4

1 Laboratory of Advanced Chemical Biology, Graduate School of Advanced Life Science, Frontier Research Center for the Post-Genome Science and Technology, Hokkaido University Sapporo 001-0021, Japan 2 Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500 Copenhagen Valby Denmark 3 Drug-Seeds Discovery Research Laboratory, National Institute of Advanced Industrial Science and Technology (AIST) Sapporo 062-8517, Japan.

4To whom correspondence should be addressed. E-mail: shin{at}glyco.sci.hokudai.ac.jp

Glycosyltransferases are an enormous and diverse class of enzyme encompassing 1% of all sequenced genomes. They catalyze the transfer of a monosaccharide from an activated donor such as a sugar-nucleotide to an acceptor molecule. Though the primary sequences of glycosyltransferases have little homology, X-ray structural studies on glycosyltransferases have revealed that there are two main folds and that the orientation of the sugar donors with respect to the folds is highly conserved. It seems that glycosyltransferases have evolved diversified specificities toward donor sugars by changing the amino acids around the monosaccharide moiety without altering the orientation of the nucleotide moiety. In this study, we designed new glycosyltransferases with altered donor specificities by use of a novel empirical model called the Epimer Propensity Index (EPI). The EPI was constructed using 221 carbohydrate–protein complex structures in the Protein Data Bank with either galactose or glucose in the complex. The blood type B synthesizing glycosyltransferase GTB, a galactosyltransferase was our target enzyme. Two GTB mutants designed to exhibit enhanced glucosyltransferase activity were cloned, expressed and characterized experimentally. The predicted GTB mutants, Ser185Asn and Ser185Cys, exhibited 4.3- and 4.8-fold elevations in kcat/Km for UDP-Glc relative to that of wild-type enzyme.

Keywords: carbohydrate–protein complex/computational design/glycosyltransferase

Received July 6, 2006; revised October 3, 2006; accepted October 10, 2006.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
J. A. Alfaro, R. B. Zheng, M. Persson, J. A. Letts, R. Polakowski, Y. Bai, S. N. Borisova, N. O. L. Seto, T. L. Lowary, M. M. Palcic, et al.
ABO(H) Blood Group A and B Glycosyltransferases Recognize Substrate via Specific Conformational Changes
J. Biol. Chem., April 11, 2008; 283(15): 10097 - 10108.
[Abstract] [Full Text] [PDF]


Disclaimer: Please note that abstracts for content published before 1996 were created through digital scanning and may therefore not exactly replicate the text of the original print issues. All efforts have been made to ensure accuracy, but the Publisher will not be held responsible for any remaining inaccuracies. If you require any further clarification, please contact our Customer Services Department.