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

J. Biol. Chem., Vol. 283, Issue 33, 22628-22636, August 15, 2008

This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: 283/33/22628    most recent M800503200v1 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 Google Scholar Articles by Singh, S. Articles by Thorson, J. S. PubMed PubMed Citation Articles by Singh, S. Articles by Thorson, J. S. Social Bookmarking                      What's this?

Structure and Mechanism of the Rebeccamycin Sugar 4'-O-Methyltransferase RebM^*

Shanteri Singh, Jason G. McCoy^¶, Changsheng Zhang, Craig A. Bingman^¶, George N. Phillips, Jr.^¶1, and Jon S. Thorson²

From the Division of Pharmaceutical Sciences, School of Pharmacy and University of Wisconsin National Drug Discovery Group, University of Wisconsin, Madison, Wisconsin 53705 and ^¶Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706

The 2.65-Å crystal structure of the rebeccamycin 4'-O-methyltransferase RebM in complex with S-adenosyl-L-homocysteine revealed RebM to adopt a typical S-adenosylmethionine-binding fold of small molecule O-methyltransferases (O-MTases) and display a weak dimerization domain unique to MTases. Using this structure as a basis, the RebM substrate binding model implicated a predominance of nonspecific hydrophobic interactions consistent with the reported ability of RebM to methylate a wide range of indolocarbazole surrogates. This model also illuminated the three putative RebM catalytic residues (His^140/141 and Asp¹⁶⁶) subsequently found to be highly conserved among sequence-related natural product O-MTases from GC-rich bacteria. Interrogation of these residues via site-directed mutagenesis in RebM demonstrated His¹⁴⁰ and Asp¹⁶⁶ to be most important for catalysis. This study reveals RebM to be a member of the general acid/base-dependent O-MTases and, as the first crystal structure for a sugar O-MTase, may also present a template toward the future engineering of natural product MTases for combinatorial applications.

Received for publication, January 18, 2008 , and in revised form, March 11, 2008.

The atomic coordinates and structure factors (code 3BUS) 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, in whole or in part, by National Institutes of Health Grants AI52218, CA84374, and U19 CA113297 (to J. S. T.). 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 Table S1 and Fig. S1.

¹ To whom correspondence may be addressed. E-mail: phillips{at}biochem.wisc.edu. ² To whom correspondence may be addressed. E-mail: jsthorson{at}pharmacy.wisc.edu.

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