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J. Biol. Chem., Vol. 281, Issue 40, 29660-29668, October 6, 2006

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A Dedicated Phosphopantetheinyl Transferase for the Fredericamycin Polyketide Synthase from Streptomyces griseus^*

Yong Huang, Evelyn Wendt-Pienkowski, and Ben Shen^¶1

From the Division of Pharmaceutical Sciences, the University of Wisconsin National Cooperative Drug Discovery Group, and the ^¶Department of Chemistry, University of Wisconsin, Madison, Wisconsin 53705

Polyketide synthases cannot be functional unless their apo-acyl carrier proteins (apo-ACPs) are post-translationally modified by covalent attachment of the 4'-phosphopantetheine group to the highly conserved serine residue, and this reaction is catalyzed by phosphopantetheinyl transferases (PPTases). Cloning and sequence analysis of the 33-kb fredericamycin (FDM) biosynthetic gene cluster from Streptomyces griseus revealed fdmW, whose deduced gene product showed significant sequence homology to known PPTases. Biochemical characterization of FdmW in vitro confirmed that it is a PPTase. Inactivation of fdmW resulted in 93% reduction of FDM production, and complementation of the fdmW::aac (3)IV mutant by expressing fdmW in trans restored FDM production to a level comparable with that of the wild-type strain. Although FdmW can phosphopantetheinylate various ACPs, it prefers its cognate substrate, the FdmH ACP, with a K_m of 5.8 µM and a k_cat/K_m of 8.1 µM^–1·min^–1, to heterologous ACPs, such as the TcmM ACP with a K_m of 1.0 x 10² µM and a k_cat /K_m of 0.6 µM^–1·min^–1. These findings suggest that FdmW is specific for FDM biosynthesis. FdmW therefore represents the first holo-ACP synthase-type PPTase identified from an aromatic polyketide biosynthetic gene cluster.

Received for publication, May 22, 2006 , and in revised form, August 2, 2006.

^* This work is supported in part by National Institutes of Health Grants CA35381 and CA113297. 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.

¹ Recipient of National Institutes of Health Independent Scientist Award AI51687. To whom correspondence should be addressed: Division of Pharmaceutical Sciences, School of Pharmacy, University of Wisconsin-Madison, 777 Highland Ave., Madison, WI 53705. Tel.: 608-263-2673; Fax: 608-262-5345; E-mail: bshen{at}pharmacy.wisc.edu.

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