Elucidation of regulation mechanism of sphingolipid biosynthesis and degradation based on protein structure.

基于蛋白质结构阐明鞘脂生物合成和降解的调控机制。

基本信息

批准号：
18570114
负责人：
IKUSHIRO Hiroko
金额：
$ 2.63万
依托单位：
Osaka Medical College
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

项目摘要

Serine palmitoyltransferase (SPT) catalyzes the first and rate-limiting step reaction of the sphingolipid biosynthesis. The change of the SPT activity influences directly the total amount and the kind of the cellar sphingolipids. Therefore, the elucidation of the regulation mechanism of SPT is very important. (1) We cloned the SPT genes of the various kinds of the sphingolipid-content bacteria, constructed the overexpression system of SPT in E. coli, and established the purification method of each recombinant SPT. (2) We succeeded in synthesis of a non-reactive analogue of palmitoyl-CoA, S-(2-oxoheptadecyl)-CoA. Using the purified recombinant SPT, the reaction of SPT with L-serine and S-(2-oxoheptadecyl)-CoA was examined in detail. By the addition of L-serin to SPT, the external aldimine intermediate is formed via the Michaelis complex. When S-(2-oxoheptadecyl)-CoA was added to this SPT-L-serine binary complex, it was not converted to the final product 3-ketodihydrosphingosine, but the … More new absorption peak of the quinonoid intermediate was detected. The kinetic analysis of time resolved spectra of this ternary complex showed that the binding of the S-(2-oxoheptadecyl)-CoA to the SPT induces the formation. And accumulation of the quinonoid intermediate and the SPT reaction is in equilibrium between the quinonid intermediate and the former external aldimine intermediate. (3) The rate of the α-deprotonation of L-serin by SPT, that corresponds to the rate of the quinonoid formation, was examined by NMR. NMR studies showed that the hydrogen-deuterium exchange at Ca of L-serine is very slow in the SPT-L-serine external aldimine complex, but the rate is 100-fold increased by the addition of S-(2-oxoheptadecyl)-CoA, showing a remarkable substrate synergism in SPT. (4) We succeeded in crystallization of SPT. The structure of SPT・L-serine external aldimine complex had been determined. Based on the crystallographic data, the discussion about the role of the conserved amino acid residues in the SPT catalysis became possible. Less

丝氨酸棕榈酰转移酶(SPT)催化鞘脂生物合成的第一步反应和限速反应，其活性的变化直接影响细胞内鞘脂的总量和种类，因此对其调控机制的阐明非常重要。 (1)克隆了各类含鞘脂细菌的SPT基因，构建了SPT基因的过表达系统。 (2)利用纯化的重组SPT，成功合成了棕榈酰辅酶A的非反应性类似物S-(2-氧代十七烷基)-CoA。通过向SPT中添加L-丝氨酸，详细研究了SPT与L-丝氨酸和S-(2-氧代十七烷基)-CoA的反应。醛亚胺中间体是通过 Michaelis 复合物形成的。当 S-(2-氧代十七烷基)-CoA 添加到该 SPT-L-丝氨酸二元复合物中时，它没有转化为最终产物 3-酮二氢鞘氨醇，但出现了新的吸收峰。该三元复合物的时间分辨光谱的动力学分析表明，S-(2-氧代十七烷基)-CoA 与醌类中间体的结合。 SPT 诱导醌类中间体的形成，并且 SPT 反应在醌类中间体和前外部醛亚胺中间体之间处于平衡状态。 (3) SPT 对 L-丝氨酸的 α-去质子化反应的速率。通过NMR研究表明，在SPT-L-丝氨酸外部醛亚胺中，L-丝氨酸Ca处的氢-氘交换非常慢。复合物，但通过添加S-(2-氧代十七烷基)-CoA，其速率提高了100倍，在SPT中显示出显着的底物协同作用。 (4)我们成功地实现了SPT・L-丝氨酸的结构。基于晶体学数据，关于保守氨基酸残基在SPT催化中的作用的讨论变得可能。