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）催化了鞘脂生物合成的第一个和限制的步骤反应。 SPT活性的变化直接影响了地窖鞘脂的总量和种类。因此，阐明SPT的调节机制非常重要。（1）我们克隆了各种鞘脂含量细菌的SPT基因，构建了大肠杆菌中SPT的过表达系统，并建立了每种（2）的纯化方法，我们成功地合成了棕榈酸-COA的非反应类似物的类似物，palmitoyl-CoA，s-（2- oxoxoheptadecylecyl）-CoA。使用纯化的重组SPT，详细检查了SPT与L链氨酸和S-（2-氧HopTadecyl）-COA的反应。通过向SPT添加L塞林，外部醛氨酸中间体是通过Michaelis Complex形成的。当将S-（2-氧二甲苯基）-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-酮二氢磷酸酯，但是……检测到奎诺酮中间体的更多新吸收峰。该三元复合物的时间解析光谱的动力学分析表明，s-（2-氧二甲苯基）-COA与SPT的结合会影响形成。奎诺酮中间体和SPT反应的积累在奎诺酮中间体和以前的外部醛氨酸中间体之间。（3）通过NMR检查了与奎诺尼形成速率相对应的SPT的L-塞林的α-二苯甲酸的速率。 NMR的研究表明，在SPT-L-丝氨酸外部醛氨酸复合物中，L链CA的氢 - 居民交换非常慢，但是通过添加S-（2-氧霍普二烷基）-COA，速率提高了100倍，显示了SPT中的显着底物协同作用。（4）我们成功地结晶了SPT。已经确定了SPT ・ l-serine外部醛酸复合物。基于晶体学数据，有关配置氨基酸在SPT催化中保留的作用的讨论变得可能是可能的。较少的