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

J. Biol. Chem., Vol. 283, Issue 44, 30064-30072, October 31, 2008

This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: 283/44/30064    most recent M803184200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted 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 Request Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Schneider, S. Articles by Samland, A. K. Search for Related Content PubMed PubMed Citation Articles by Schneider, S. Articles by Samland, A. K. Social Bookmarking                      What's this?

Replacement of a Phenylalanine by a Tyrosine in the Active Site Confers Fructose-6-phosphate Aldolase Activity to the Transaldolase of Escherichia coli and Human Origin^*

Sarah Schneider, Tatyana Sandalova, Gunter Schneider, Georg A. Sprenger, and Anne K. Samland¹

From the Institute of Microbiology, Universität Stuttgart, 70550 Stuttgart, Germany and the Division of Molecular Structural Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, 17177 Stockholm, Sweden

Based on a structure-assisted sequence alignment we designed 11 focused libraries at residues in the active site of transaldolase B from Escherichia coli and screened them for their ability to synthesize fructose 6-phosphate from dihydroxyacetone and glyceraldehyde 3-phosphate using a newly developed color assay. We found one positive variant exhibiting a replacement of Phe¹⁷⁸ to Tyr. This mutant variant is able not only to transfer a dihydroxyacetone moiety from a ketose donor, fructose 6-phosphate, onto an aldehyde acceptor, erythrose 4-phosphate (14 units/mg), but to use it as a substrate directly in an aldolase reaction (7 units/mg). With a single amino acid replacement the fructose-6-phosphate aldolase activity was increased considerably (>70-fold compared with wild-type). Structural studies of the wild-type and mutant protein suggest that this is due to a different H-bond pattern in the active site leading to a destabilization of the Schiff base intermediate. Furthermore, we show that a homologous replacement has a similar effect in the human transaldolase Taldo1 (aldolase activity, 14 units/mg). We also demonstrate that both enzymes TalB and Taldo1 are recognized by the same polyclonal antibody.

Received for publication, April 25, 2008 , and in revised form, June 18, 2008.

The atomic coordinates and structure factors (code 3CWN) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by Deutsche Forschungsgemeinschaft Grant SPP1170/Sp503/4-2 (to G. Sprenger) and a grant from the Swedish Research Council (to G. Schneider). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables S1 and S2 and Figs. S1 and S2.

¹ To whom correspondence should be addressed: Allmandring 31, D-70550 Stuttgart, Germany. Tel.: 49-711-6856-5491; Fax: 49-711-6856-5725; E-mail: anne.samland{at}imb.uni-stuttgart.de.

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