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

J. Biol. Chem., Vol. 282, Issue 49, 35954-35963, December 7, 2007

This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: 282/49/35954    most recent M703921200v1 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 Google Scholar Google Scholar Articles by Geders, T. W. Articles by Smith, J. L. Search for Related Content PubMed PubMed Citation Articles by Geders, T. W. Articles by Smith, J. L. Social Bookmarking                      What's this?

Crystal Structure of the ECH₂ Catalytic Domain of CurF from Lyngbya majuscula

INSIGHTS INTO A DECARBOXYLASE INVOLVED IN POLYKETIDE CHAIN β-BRANCHING^*

Todd W. Geders¹, Liangcai Gu^¶1, Jonathan C. Mowers², Haichuan Liu^||, William H. Gerwick^**, Kristina Håkansson^||, David H. Sherman^¶3, and Janet L. Smith⁴

From the Life Sciences Institute, Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109, the Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907, the ^¶Life Sciences Institute, Departments of Medicinal Chemistry, Chemistry, Microbiology and Immunology, University of Michigan, Ann Arbor, Michigan 48109, the ^||Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, and the ^**Scripps Institution of Oceanography, University of California at San Diego, La Jolla, California 92093

Curacin A is a mixed polyketide/nonribosomal peptide possessing anti-mitotic and anti-proliferative activity. In the biosynthesis of curacin A, the N-terminal domain of the CurF multifunctional protein catalyzes decarboxylation of 3-methylglutaconyl-acyl carrier protein (ACP) to 3-methylcrotonyl-ACP, the postulated precursor of the cyclopropane ring of curacin A. This decarboxylase is encoded within an "HCS cassette" that is used by several other polyketide biosynthetic systems to generate chemical diversity by introduction of a β-branch functional group to the natural product. The crystal structure of the CurF N-terminal ECH₂ domain establishes that the protein is a crotonase superfamily member. Ala⁷⁸ and Gly¹¹⁸ form an oxyanion hole in the active site that includes only three polar side chains as potential catalytic residues. Site-directed mutagenesis and a biochemical assay established critical functions for His²⁴⁰ and Lys⁸⁶, whereas Tyr⁸² was nonessential. A decarboxylation mechanism is proposed in which His²⁴⁰ serves to stabilize the substrate carboxylate and Lys⁸⁶ donates a proton to C-4 of the acyl-ACP enolate intermediate to form the ² unsaturated isopentenoyl-ACP product. The CurF ECH₂ domain showed a 20-fold selectivity for ACP-over CoA-linked substrates. Specificity for ACP-linked substrates has not been reported for any other crotonase superfamily decarboxylase. Tyr⁷³ may select against CoA-linked substrates by blocking a contact of Arg³⁸ with the CoA adenosine 5'-phosphate.

Received for publication, May 14, 2007 , and in revised form, September 14, 2007.

The atomic coordinates and structure factors (code 2Q2X, 2Q34, and 2Q35) 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 part by National Institutes of Health Grants DK42303 (to J. L. S.) and GM076477 and the J. G. Searle Professorship (to D. H. S.). This work was also supported in part by a graduate fellowship (to L. G.) from Eli Lilly & Co. 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 Figs. S1-S3.

¹ These authors contributed equally to this work.

² Supported by the Medical Scientist Training Program at the University of Michigan.

³ To whom correspondence may be addressed: Life Sciences Institute, University of Michigan, 210 Washtenaw Ave., Ann Arbor, MI 48109. Tel.: 734-615-9907; Fax: 734-615-3641; E-mail: davidhs{at}umich.edu.

⁴ To whom correspondence may be addressed: Life Sciences Institute, University of Michigan, 210 Washtenaw Ave., Ann Arbor, MI 48109. Tel.: 734-615-9564; Fax: 734-763-6492; E-mail: JanetSmith{at}umich.edu.

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