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

J. Biol. Chem., Vol. 283, Issue 16, 10727-10734, April 18, 2008

This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: 283/16/10727    most recent M709287200v1 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 Kelly, R. M. Articles by Dijkhuizen, L. Search for Related Content PubMed PubMed Citation Articles by Kelly, R. M. Articles by Dijkhuizen, L. Social Bookmarking                      What's this?

Evolution toward Small Molecule Inhibitor Resistance Affects Native Enzyme Function and Stability, Generating Acarbose-insensitive Cyclodextrin Glucanotransferase Variants^*

From the Microbial Physiology, Groningen Biomolecular Sciences and Biotechnology Institute, Centre for Carbohydrate Bioprocessing, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands

Small molecule inhibitors play an essential role in the selective inhibition of enzymes associated with human infection and metabolic disorders. Targeted enzymes may evolve toward inhibitor resistance through selective incorporation of mutations. Acquisition of insensitivity may, however, result in profound devolution of native enzyme function and stability. We therefore investigated the consequential effects on native function and stability by evolving a cyclodextrin glucanotransferase (CGTase) enzyme toward insensitivity to the small molecule inhibitor of the protein, acarbose. Error-prone PCR mutagenesis was applied to search the sequence space of CGTase for acarbose-insensitive variants. Our results show that all selected mutations were localized around the active site of the enzyme, and in particular, at the acceptor substrate binding sites, highlighting the regions importance in acarbose inhibition. Single mutations conferring increased resistance, K232E, F283L, and A230V, raised IC₅₀ values for acarbose between 3,500- and 6,700-fold when compared with wild-type CGTase but at a significant cost to catalytic efficiency. In addition, the thermostability of these variants was significantly lowered. These results reveal not only the relative ease by which resistance may be acquired to small molecule inhibitors but also the considerable cost incurred to native enzyme function and stability, highlighting the subsequent constraints in the further evolutionary potential of inhibitor-resistant variants.

Received for publication, November 12, 2007 , and in revised form, January 22, 2008.

^* This work was supported by grants from the Netherlands Organization for Scientific Research (to H. L.). 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 a supplemental table.

¹ To whom correspondence should be addressed. Tel.: 31-50-3632150; Fax: 31-50-3632154; E-mail: L.Dijkhuizen{at}rug.nl.

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