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J. Biol. Chem., Vol. 278, Issue 35, 32733-32743, August 29, 2003

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Purification, Characterization, and Identification of a Sphingomyelin Synthase from Pseudomonas aeruginosa

PlcH IS A MULTIFUNCTIONAL ENZYME^*

Chiara Luberto  , Martin J. Stonehouse ¶, Elizabeth A. Collins , Norma Marchesini , Samer El-Bawab  ||, Adriana I. Vasil ¶, Michael L. Vasil ¶ and Yusuf A. Hannun 

From the Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425 and the ^¶Department of Microbiology, University of Colorado Health Sciences Center, Denver, Colorado 80262

Sphingomyelin synthase is the enzyme that synthesizes sphingomyelin (SM) in mammalian cells by transferring a phosphorylcholine moiety from phosphatidylcholine to ceramide. Despite its importance, the gene and/or the protein responsible for this activity has not yet been identified. Here we report the purification, identification, and biochemical characterization of an enzymatic activity that synthesizes SM in Pseudomonas aeruginosa. SM synthase-like activity was found secreted in the culture medium of P. aeruginosa, strains PA01 and PAK, whereas it could not be detected in cultures of Escherichia coli. From the medium of PAK cultures, SM synthase was purified through sequential chromatographic columns. After separation on polyacrylamide-SDS gels and visualization by silver staining, the purified enzyme showed two bands, one of 75 kDa and one of 30–35 kDa. Interestingly, the highly purified SM synthase preparation also showed neutral sphingomyelinase activity. We therefore investigated whether the protein we purified as SM synthase could actually be the previously identified PlcH, a 78-kDa phospholipase C known to hydrolyze phosphatidylcholine and SM in P. aeruginosa. First, the purified SM synthase preparation contained a 78-kDa protein that reacted with monoclonal antibodies raised against purified PlcH. Second, purified PlcH showed SM synthase activity. Third, using different knockout mutant strains for the PlcH operon, PlcH was found to be necessary for SM synthase activity in P. aeruginosa. Interestingly, SM synthase activity was specific to the Pseudomonas PlcH as other bacterial phospholipases did not display SM synthase activity. Biochemical studies on the Pseudomonas SM synthase confirmed that it is a transferase, similar to the mammalian enzyme, that specifically recognizes the choline head-group and the primary hydroxyl on ceramide. This SM synthase did not have reverse transferase activity. In conclusion, the Pseudomonas PlcH also exerts SM synthase activity; therefore, for the first time, we have identified a structural gene for a SM synthase.

Received for publication, January 28, 2003 , and in revised form, June 2, 2003.

This paper is dedicated to the memory of Prof. Gianna Maria Bartoli.

^* This work was supported in part by National Institutes of Health Grants HL43707 (to Y. A. H.) and HL62608 (to M. L. V.). 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.

^|| Present address: Diabetic Microangiopathy Research Unit, Merck-Santé/INSERM U352, INSA-Lyon, 69621 Villeurbanne, France.

To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Medical University of South Carolina, STB Rm. 652, Charleston, SC 29425. Tel.: 843-876-5214; Fax: 843-876-5172; E-mail: luberto{at}musc.edu.

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