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J. Biol. Chem., Vol. 282, Issue 15, 11339-11346, April 13, 2007

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Topology and Active Site of PlsY

THE BACTERIAL ACYLPHOSPHATE:GLYCEROL-3-PHOSPHATE ACYLTRANSFERASE^*

Ying-Jie Lu¹, Fan Zhang¹, Kimberly D. Grimes, Richard E. Lee, and Charles O. Rock²

From the Department of Infectious Diseases, St. Jude Children's Research Hospital, Memphis, Tennessee 38105-2794 and the Department of Pharmaceutical Sciences, University of Tennessee Health Science Center, Memphis, Tennessee 8163

The most widely distributed biosynthetic pathway to initiate phosphatidic acid formation in bacterial membrane phospholipid biosynthesis involves the conversion of acyl-acyl carrier protein to acylphosphate by PlsX and the transfer of the acyl group from acylphosphate to glycerol 3-phosphate by an integral membrane protein, PlsY. The membrane topology of Streptococcus pneumoniae PlsY was determined using the substituted cysteine accessibility method. PlsY has five membrane-spanning segments with the amino terminus and two short loops located on the external face of the membrane. Each of the three larger cytoplasmic domains contains a highly conserved sequence motif. Site-directed mutagenesis revealed that each conserved domain was critical for PlsY catalysis. Motif 1 had an essential serine and arginine residue. Motif 2 had the characteristics of a phosphate-binding loop. Mutations of the conserved glycines in motif 2 to alanines resulted in a K_m defect for glycerol 3-phosphate binding leading to the conclusion that this motif corresponded to the glycerol 3-phosphate binding site. Motif 3 contained a conserved histidine and asparagine that were important for activity and a glutamate that was critical to the structural integrity of PlsY. PlsY was noncompetitively inhibited by palmitoyl-CoA. These data define the membrane architecture and the critical active site residues in the PlsY family of bacterial acyltransferases.

Received for publication, January 12, 2007 , and in revised form, February 16, 2007.

^* This work was supported by National Institutes of Health Grant GM 34496, Cancer Center (CORE) Support Grant CA 21765, and the American Lebanese and 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.

¹ These authors contributed equally to this work.

² 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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