HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Seto, N. O. L. Articles by Narang, S. A. Search for Related Content PubMed PubMed Citation Articles by Seto, N. O. L. Articles by Narang, S. A. Social Bookmarking                      What's this?

Volume 272, Number 22, Issue of May 30, 1997 pp. 14133-14138
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Sequential Interchange of Four Amino Acids from Blood Group B to Blood Group A Glycosyltransferase Boosts Catalytic Activity and Progressively Modifies Substrate Recognition in Human Recombinant Enzymes

(Received for publication, February 4, 1997, and in revised form, March 19, 1997)

Nina O. L. Seto , Monica M. Palcic ^¶ , Catherine A. Compston ^¶ , Hong Li ^¶ , David R. Bundle ^¶ and Saran A. Narang

From the  Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada K1A 0R6 and the ^¶ Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2

The human blood group A and B glycosyltransferase enzymes are highly homologous and the alteration of four critical amino acid residues (Arg-176  Gly, Gly-235  Ser, Leu-266  Met, and Gly-268  Ala) is sufficient to change the enzyme specificity from a blood group A to a blood group B glycosyltransferase. To carry out a systematic study, a synthetic gene strategy was employed to obtain their genes and to allow facile mutagenesis. Soluble forms of a recombinant glycosyltransferase A and a set of hybrid glycosyltransferase A and B mutants were expressed in Escherichia coli in high yields, which allowed them to be kinetically characterized extensively for the first time. A functional hybrid A/B mutant enzyme was able to catalyze both A and B reactions, with the k_cat being 5-fold higher for the A donor. Surprisingly, even a single amino acid replacement in glycosyltransferase A with the corresponding residue from glycosyltransferase B (Arg-176  Gly) produced enzymes with glycosyltransferase A activity only, but with very large (11-fold) increases in the k_cat and increased specificity. The increases observed in k_cat are among the largest obtained for a single amino acid change and are advantageous for the preparative scale synthesis of blood group antigens.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				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]

				T. Nakahara, O. Hindsgaul, M. M. Palcic, and S.-I. Nishimura Computational design and experimental evaluation of glycosyltransferase mutants: engineering of a blood type B galactosyltransferase with enhanced glucosyltransferase activity Protein Eng. Des. Sel., December 1, 2006; 19(12): 571 - 578. [Abstract] [Full Text] [PDF]

				H. J. Lee, C. H. Barry, S. N. Borisova, N. O. L. Seto, R. B. Zheng, A. Blancher, S. V. Evans, and M. M. Palcic Structural Basis for the Inactivity of Human Blood Group O2 Glycosyltransferase J. Biol. Chem., January 7, 2005; 280(1): 525 - 529. [Abstract] [Full Text] [PDF]

				H. Heissigerova, C. Breton, J. Moravcova, and A. Imberty Molecular modeling of glycosyltransferases involved in the biosynthesis of blood group A, blood group B, Forssman, and iGb3 antigens and their interaction with substrates Glycobiology, May 1, 2003; 13(5): 377 - 386. [Abstract] [Full Text] [PDF]

				S. L. Marcus, R. Polakowski, N. O. L. Seto, E. Leinala, S. Borisova, A. Blancher, F. Roubinet, S. V. Evans, and M. M. Palcic A Single Point Mutation Reverses the Donor Specificity of Human Blood Group B-synthesizing Galactosyltransferase J. Biol. Chem., March 28, 2003; 278(14): 12403 - 12405. [Abstract] [Full Text] [PDF]

				B. D. Lazarus, J. Milland, P. A. Ramsland, E. Mouhtouris, and M. S. Sandrin Histidine 271 has a functional role in pig {alpha}-1,3galactosyltransferase enzyme activity Glycobiology, December 1, 2002; 12(12): 793 - 802. [Abstract] [Full Text] [PDF]

				E. Boix, Y. Zhang, G. J. Swaminathan, K. Brew, and K. R. Acharya Structural Basis of Ordered Binding of Donor and Acceptor Substrates to the Retaining Glycosyltransferase, alpha -1,3-Galactosyltransferase J. Biol. Chem., July 26, 2002; 277(31): 28310 - 28318. [Abstract] [Full Text] [PDF]

				S. Shetterly, I. Tom, T.-Y. Yen, R. Joshi, L. Lee, P. G. Wang, and B. A. Macher {alpha}1,3 galactosyltransferase: new sequences and characterization of conserved cysteine residues Glycobiology, August 1, 2001; 11(8): 645 - 653. [Abstract] [Full Text] [PDF]

				L. Vo, S. Lee, M. C. Marcinko, E. H. Holmes, and B. A. Macher Human alpha 1,3/4-Fucosyltransferases. II. A SINGLE AMINO ACID AT THE COOH TERMINUS OF FucT III AND V ALTERS THEIR KINETIC PROPERTIES J. Biol. Chem., September 25, 1998; 273(39): 25250 - 25255. [Abstract] [Full Text] [PDF]