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

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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.