Abstract
Serine palmitoyltransferase (SPT) is a key enzyme of sphingolipid biosynthesis and catalyzes the decarboxylative condensation of L-serine with palmitoyl-CoA to form 3-ketodihydrosphingosine. Eukaryote SPTs are known as heterodimers composed of tightly membrane-bound subunits, which makes their detailed analysis difficult. Therefore, as the alternative enzyme source, we focused into the bacteria that contain a large amount of sphingolipid as their cellular component. We have purified a water-soluble homodimeric SPT from Sphingomonas paucimobilis 2395^T first, then cloned SPT genes from several bacterial species and have overproduced them in E. coli. Using these bacterial enzymes, the reaction mechanism of SPT was analyzed in detail and X-ray crystallographic analysis of its structure in a complex with the amino acid substrate, L-serine were performed. These results allow us to understand the structure-function relationship of eukaryote SPT.
