Studies on the molecular mechanism of sphingolipid metabolism based on the crystal structure of the enzymes

测定了丝氨酸棕榈酰转移酶与L-丝氨酸的络合物的晶体结构。通过对突变体SPTS的分析，实验提出了催化残基组氨酸在反应机理中的多功能作用：组氨酸残基固定了L-丝氨酸和反应产物的构象，防止了不利的副反应，并通过酸催化剂的作用促进了克莱森式缩合和脱羧化。成功地在大肠杆菌中大量生产了1-磷酸鞘氨醇。利用纯化的重组酶和荧光标记底物建立了检测体系，并对其酶学性质进行了详细的分析。1-磷酸鞘氨醇也得到结晶，重复性较好。

The crystal structural of serine palmitoyltransferase complexed with L-serine was determined. The multifunctional role of the catalytic residue, histidine, in the reaction mechanism was experimentally proposed by the analyses of the mutant SPTs, as follows ; The histidine residue fixes the conformation of both of L-serine and reaction product preventing the unfavorable side reactions, and enhances the Claisen-type condensation and decarboxylation by its function as an acid catalyst. Sphingosine 1-phosphate was overproduced successfully in E. coli. Assay system using the purified recombinant enzyme and fluorescent labeled-substrate was developed and enzymatic property was analyzed in detail. Sphingosine 1-phosphate was also crystallized with good reproducibility.