Purification, characterization, and identification of a sphingomyelin synthase from Pseudomonas aeruginosa -: PlcH is a multifunctional enzyme
Purification, characterization, and identification of a sphingomyelin synthase from Pseudomonas aeruginosa -: PlcH is a multifunctional enzyme
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DOI：
10.1074/jbc.m300932200
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发表时间：
2003-08-29
影响因子：
4.8
通讯作者：
Hannun, YA
中科院分区：
文献类型：
--
作者：
Luberto, C;Stonehouse, MJ;Hannun, YA
鞘磷脂合成酶是在哺乳动物细胞中通过将磷酸胆碱部分从磷脂酰胆碱转移到神经酰胺来合成鞘磷脂（SM）的酶。尽管它的重要性，基因和/或蛋白质负责这一活动尚未确定。在这里，我们报告的纯化，鉴定和生化特性的酶活性，合成SM在铜绿假单胞菌。铜绿假单胞菌、菌株PA 01和PAK的培养基中发现分泌SM的类似酶活性，而在大肠杆菌的培养物中不能检测到SM的类似酶活性。从PAK培养物的培养基中，通过连续色谱柱纯化SM合酶。经聚丙烯酰胺-SDS凝胶分离和银染显色后，纯化的酶显示两条带，一条类似于75 kDa，一条30- 35 kDa。有趣的是，高度纯化的SM合酶制剂也显示中性鞘磷脂酶活性。因此，我们研究了我们作为SM合酶纯化的蛋白质是否实际上可能是先前鉴定的PlcH，一种已知在铜绿假单胞菌中水解磷脂酰胆碱和SM的78 kDa磷脂酶C。首先，纯化的SM合酶制剂含有78 kDa的蛋白质，该蛋白质与针对纯化的PlcH产生的单克隆抗体反应。第二，纯化的PlcH显示SM合酶活性。第三，使用PlcH操纵子的不同敲除突变株，发现PlcH对于铜绿假单胞菌中的SM合酶活性是必需的。有趣的是，SM合酶活性是假单胞菌PlcH所特有的，因为其他细菌磷脂酶不显示SM合酶活性。对假单胞菌SM合酶的生化研究证实，它是一种转移酶，类似于哺乳动物的酶，特异性识别胆碱头基和神经酰胺上的伯羟基。该SM合酶不具有逆转录酶活性。总之，假单胞菌PlcH也发挥SM合酶活性，因此，我们第一次确定了SM合酶的结构基因。
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 similar to 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.