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J. Biol. Chem., Vol. 281, Issue 26, 18025-18032, June 30, 2006

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Structure of RhlG, an Essential -Ketoacyl Reductase in the Rhamnolipid Biosynthetic Pathway of Pseudomonas aeruginosa^*

Darcie J. Miller, Yong-Mei Zhang, Charles O. Rock^¶, and Stephen W. White^¶1

From the Departments of Structural Biology, and Infectious Diseases, St. Jude Children's Research Hospital, Memphis, Tennessee 38105 and the ^¶Department of Molecular Sciences, University of Tennessee Health Science Center, Memphis, Tennessee 38163

Rhamnolipids are extracellular biosurfactants and virulence factors secreted by the opportunistic human pathogen Pseudomonas aeruginosa that are required for swarming motility. The rhlG gene is essential for rhamnolipid formation, and the RhlG enzyme is thought to divert fatty acid synthesis intermediates into the rhamnolipid biosynthetic pathway based on its similarity to FabG, the -ketoacyl-acyl carrier protein (ACP) reductase of type II fatty acid synthesis. Crystallographic analysis reveals that the overall structures of the RhlG·NADP⁺ and FabG·NADP⁺ complexes are indeed similar, but there are key differences related to function. RhlG does not undergo the conformational changes upon NADP(H) binding at the active site that in FabG are the structural basis of negative allostery. Also, the acyl chain-binding pocket of RhlG is narrow and rigid compared with the larger, flexible substrate-binding subdomain in FabG. Finally, RhlG lacks a positively charged/hydrophobic surface feature adjacent to the active site that is found on enzymes like FabG that recognize the ACP of fatty acid synthesis. RhlG catalyzed the NADPH-dependent reduction of -ketodecanoyl-ACP to -D-hydroxydecanoyl-ACP. However, the enzyme was 2000-fold less active than FabG in carrying out the same reaction. These structural and biochemical studies establish RhlG as a NADPH-dependent -ketoacyl reductase of the SDR protein superfamily and further suggest that the ACP of fatty acid synthesis does not carry the substrates for RhlG.

Received for publication, February 22, 2006 , and in revised form, April 17, 2006.

^* This work was supported by National Institutes of Health Grants GM 34496 (to C. O. R.), Cancer Center (CORE) Support Grant CA 21765, and the American Lebanese Syrian Associated Charities. 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 atomic coordinates and structure factors (code 2B4Q) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

¹ To whom correspondence should be addressed: St. Jude Children's Research Hospital, Dept. of Structural Biology, 332 N. Lauderdale St., MS# 311, Memphis, TN 38105. Tel.: 901-495-3040; Fax: 901-495-3032; E-mail: stephen.white{at}stjude.org.

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