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

J. Biol. Chem., Vol. 282, Issue 35, 25717-25725, August 31, 2007

This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: 282/35/25717    most recent M703437200v1 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 HighWire Citing Articles via Google Scholar Google Scholar Articles by Zhang, W. Articles by Tang, Y. Search for Related Content PubMed PubMed Citation Articles by Zhang, W. Articles by Tang, Y. Social Bookmarking                      What's this?

Investigation of Early Tailoring Reactions in the Oxytetracycline Biosynthetic Pathway^*

Wenjun Zhang, Kenji Watanabe, Clay C. C. Wang, and Yi Tang¹

From the Department of Chemical and Biomolecular Engineering University of California, Los Angeles, California 90095 and Department of Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles California 90089

Tetracyclines are aromatic polyketides biosynthesized by bacterial type II polyketide synthases. The amidated tetracycline backbone is biosynthesized by the minimal polyketide synthases and an amidotransferase homologue OxyD. Biosynthesis of the key intermediate 6-methylpretetramid requires two early tailoring steps, which are cyclization of the linearly fused tetracyclic scaffold and regioselective C-methylation of the aglycon. Using a heterologous host (CH999)/vector pair, we identified the minimum set of enzymes from the oxytetracycline biosynthetic pathway that is required to afford 6-methylpretetramid in vivo. Only two cyclases (OxyK and OxyN) are necessary to completely cyclize and aromatize the amidated tetracyclic aglycon. Formation of the last ring via C-1/C-18 aldol condensation does not require a dedicated fourth-ring cyclase, in contrast to the biosynthetic mechanism of other tetracyclic aromatic polyketides, such as daunorubicin and tetracenomycin. Acetyl-derived polyketides do not undergo spontaneous fourth-ring cyclization and form only anthracene carboxylic acids as demonstrated both in vivo and in vitro. OxyF was identified to be the C-6 C-methyltransferase that regioselectively methylates pretetramid to yield 6-methylpretetramid. Reconstitution of 6-methylpretetramid in a heterologous host sets the stage for a more systematic investigation of additional tetracycline downstream tailoring enzymes and is a key step toward the engineered biosynthesis of tetracycline analogs.

Received for publication, April 25, 2007 , and in revised form, June 26, 2007.

^* This work was supported by a UCLA startup grant, University of California Cancer Research Coordinating Committee funds, National Science Foundation Chemical, Bioengineering, Environmental, and Transport Systems Grant 0545860, NIGMS, National Institutes of Health Grant RO1-GM75857 (to C. C. C. W.), and American Cancer Society Grant RSG-06-010-01-CDD (to C. C. C. W.). 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 Fig. S1 and Tables S1-S4.

¹ To whom correspondence should be addressed: Dept. of Chemical and Biomolecular Engineering, University of California Los Angeles, Los Angeles, CA 90095. Tel.: 810-825-0375; E-mail: yitang{at}ucla.edu.

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

This article has been cited by other articles:

				E. Szewczyk, Y.-M. Chiang, C. E. Oakley, A. D. Davidson, C. C. C. Wang, and B. R. Oakley Identification and Characterization of the Asperthecin Gene Cluster of Aspergillus nidulans Appl. Envir. Microbiol., December 15, 2008; 74(24): 7607 - 7612. [Abstract] [Full Text] [PDF]