Structural biology of sphingolipid synthesis

Abstract

As most eukaryotic enzymes involved in sphingolipid synthesis are membrane-bound proteins analyses of their biochemical and structural are difficult, but sphingolipid-containing bacteria are useful alternatives for enzyme sources. We studied serine palmitoyltransferase (SPT), the first enzyme of the biosynthetic pathway, using this method. To study recombinant SPT's enzymatic properties, we cloned bacterial SPT genes and then over-expressed them in Escherichia coli and purified the water-soluble enzymes. One yielded crystals good enough for X-ray crystallographic analysis, and the structure of SPT in a complex with the amino acid substrate, L-serine, was successfully determined at 2.3 Å resolution. SPT is a homodimer with two pyridoxal 5′-phosphate (PLP) molecules located at the dimer interface. Both subunits contribute side chains to the active sites. The electron density map indicates that a Schiff base is formed between L-serine and PLP in the crystal, so any reaction would stop at the external aldimine intermediate if the co-substrate, palmitoyl-CoA, were absent. Highly conserved amino acids among bacterial and eukaryotic SPTs are also located in the three dimensional structure of this enzyme, and their possible roles in the function of SPT are discussed. © Springer-Verlag Tokyo 2006. All rights reserved.