title: Purification, characterization, and identification of a sphingomyelin synthase from Pseudomonas aeruginosa. PlcH is a multifunctional enzyme.
abstract: 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 approximately 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425, USA. luberto@musc.edu
authors.forename: Chiara
authors.initials: C
authors.lastname: Luberto
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Stonehouse
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Collins
authors.forename: Norma
authors.initials: N
authors.lastname: Marchesini
authors.forename: Samer
authors.initials: S
authors.lastname: El-Bawab
authors.forename: Adriana I
authors.initials: AI
authors.lastname: Vasil
authors.forename: Michael L
authors.initials: ML
authors.lastname: Vasil
authors.forename: Yusuf A
authors.initials: YA
authors.lastname: Hannun
mesh_terms: D002845:Chromatography; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D009154:Mutation; D010713:Phosphatidylcholines / Q000737:chemistry; D010767:Phosphorylcholine / Q000737:chemistry; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D012685:Sepharose / Q000494:pharmacology; D016622:Silver Staining; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism; D013379:Substrate Specificity; D013997:Time Factors; D017855:Transferases (Other Substituted Phosphate Groups) / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D019076:Transgenes
chemical_list: D010713:Phosphatidylcholines; D010767:Phosphorylcholine; D012685:Sepharose; D017855:Transferases (Other Substituted Phosphate Groups); C070186:phosphatidylcholine-ceramide phosphocholine transferase; C477519:sphingomyelin synthase, Pseudomonas aeruginosa; D013108:Sphingomyelin Phosphodiesterase