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J. Biol. Chem., Vol. 281, Issue 25, 17390-17399, June 23, 2006

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Roles of the Active Site Water, Histidine 303, and Phenylalanine 396 in the Catalytic Mechanism of the Elongation Condensing Enzyme of Streptococcus pneumoniae^*

Yong-Mei Zhang, Jason Hurlbert, Stephen W. White, and Charles O. Rock¹

From the Departments of Infectious Diseases and Structural Biology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105

-Ketoacyl-ACP synthases catalyze the condensation steps in fatty acid and polyketide synthesis and are targets for the development of novel antibiotics and anti-obesity and anti-cancer agents. The roles of the active site residues in Streptococcus pneumoniae FabF (-ketoacyl-ACP synthase II; SpFabF) were investigated to clarify the mechanism for this enzyme superfamily. The nucleophilic cysteine of the active site triad was required for acyl-enzyme formation and the overall condensation activity. The two active site histidines in the elongation condensing enzyme have different electronic states and functions. His³³⁷ is essential for condensation activity, and its protonated N stabilizes the negative charge developed on the malonyl thioester carbonyl in the transition state. The N of His³⁰³ accelerated catalysis by deprotonating a structured active site water for nucleophilic attack on the C3 of malonate, releasing bicarbonate. Lys³³² controls the electronic state of His³⁰³ and also plays a critical role in the positioning of His³³⁷. Phe³⁹⁶ functions as a gatekeeper that controls the order of substrate addition. These data assign specific roles for each active site residue and lead to a revised general mechanism for this important class of enzymes.

Received for publication, December 12, 2005 , and in revised form, April 13, 2006.

The atomic coordinates and structure factors (code 2ALM) 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 by National Institutes of Health Grant GM34496, Cancer Center (CORE) Support Grant CA 21765, and the American Lebanese Syrian Associated Charities. SER-CAT-supporting institutions may be found on the World Wide Web at www.ser.anl.gov/new/index.html. Use of the Advanced Photon Source was supported by the U.S. Dept. of Energy, Basic Energy Sciences, Office of Science, under Contract W-31-109-Eng-38. 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 To whom correspondence should be addressed: St. Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, TN 38105-2794. Tel.: 901-495-3491; Fax: 901-495-3099; E-mail: charles.rock{at}stjude.org.

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