title: Role of a conserved arginine residue during catalysis in serine palmitoyltransferase.
abstract: All sphingolipid-producing organisms require the pyridoxal 5'-phosphate (PLP)-dependent serine palmitoyltransferase (SPT) to catalyse the first reaction on the de novo sphingolipid biosynthetic pathway. SPT is a member of the alpha oxoamine synthase (AOS) family that catalyses a Claisen-like condensation of palmitoyl-CoA and L-serine to form 3-ketodihydrosphingosine (KDS). Protein sequence alignment across various species reveals an arginine residue, not involved in PLP binding, to be strictly conserved in all prokaryotic SPTs, the lcb2 subunits of eukaryotic SPTs and all members of the AOS family. Here we use UV-vis spectroscopy and site-directed mutagenesis, in combination with a substrate analogue, to show that the equivalent residue (R370) in the SPT from Sphingomonas wittichii is required to form the key PLP:L-serine quinonoid intermediate that condenses with palmitoyl-CoA and thus plays an essential role enzyme catalysis.
authors.affiliation: School of Chemistry, EaStCHEM, University of Edinburgh, Edinburgh, Scotland, UK.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Lowther
authors.forename: Guillaume
authors.initials: G
authors.lastname: Charmier
authors.forename: Marine C
authors.initials: MC
authors.lastname: Raman
authors.forename: Hiroko
authors.initials: H
authors.lastname: Ikushiro
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Hayashi
authors.forename: Dominic J
authors.initials: DJ
authors.lastname: Campopiano
mesh_terms: D001120:Arginine / Q000502:physiology*; D002384:Catalysis; D016297:Mutagenesis, Site-Directed; D010171:Palmitoyl Coenzyme A; D051102:Serine C-Palmitoyltransferase / Q000737:chemistry / Q000378:metabolism*; D013057:Spectrum Analysis; D020578:Sphingomonas / Q000201:enzymology*; D013379:Substrate Specificity
chemical_list: D010171:Palmitoyl Coenzyme A; D001120:Arginine; D051102:Serine C-Palmitoyltransferase