biostudies-literatureIkushiro HJapan Foundation for Applied EnzymologyToyo Institute of Food TechnologyJapan Society for the Promotion of Science105728https://www.ebi.ac.uk/biostudies/studies/S-EPMC10912632biostudies-literatureUnknown300(3)Serine palmitoyltransferase (SPT) catalyzes the pyridoxal-5'-phosphate (PLP)-dependent decarboxylative condensation of l-serine and palmitoyl-CoA to form 3-ketodihydrosphingosine (KDS). Although SPT was shown to synthesize corresponding products from amino acids other than l-serine, it is still arguable whether SPT catalyzes the reaction with d-serine, which is a question of biological importance. Using high substrate and enzyme concentrations, KDS was detected after the incubation of SPT from Sphingobacterium multivorum with d-serine and palmitoyl-CoA. Furthermore, the KDS comprised equal amounts of 2S and 2R isomers. ¹H-NMR study showed a slow hydrogen-deuterium exchange at Cα of serine mediated by SPT. We further confirmed that SPT catalyzed the racemization of serine. The rate of the KDS formation from d-serine was comparable to those for the α-hydrogen exchange and the racemization reaction. The structure of the d-serine-soaked crystal (1.65 Å resolution) showed a distinct electron density of the PLP-l-serine aldimine, interpreted as the racemized product trapped in the active site. The structure of the α-methyl-d-serine-soaked crystal (1.70 Å resolution) showed the PLP-α-methyl-d-serine aldimine, mimicking the d-serine-SPT complex prior to racemization. Based on these enzymological and structural analyses, the synthesis of KDS from d-serine was explained as the result of the slow racemization to l-serine, followed by the reaction with palmitoyl-CoA, and SPT would not catalyze the direct condensation between d-serine and palmitoyl-CoA. It was also shown that the S. multivorum SPT catalyzed the racemization of the product KDS, which would explain the presence of (2R)-KDS in the reaction products.The Journal of biological chemistryRacemization of the substrate and product by serine palmitoyltransferase from Sphingobacterium multivorum yields two enantiomers of the product from d-serine.PMC109126322544003622K06153Sawai TMiyahara ITakahashi AIkushiro HMurai YGoto HHirabayashi YYano TKamiya NMurakami TIkushiro SIMonde KHonda TfalseRacemization of the substrate and product by serine palmitoyltransferase from Sphingobacterium multivorum yields two enantiomers of the product from d-serine.Serine palmitoyltransferase (SPT) catalyzes the pyridoxal-5'-phosphate (PLP)-dependent decarboxylative condensation of l-serine and palmitoyl-CoA to form 3-ketodihydrosphingosine (KDS). Although SPT was shown to synthesize corresponding products from amino acids other than l-serine, it is still arguable whether SPT catalyzes the reaction with d-serine, which is a question of biological importance. Using high substrate and enzyme concentrations, KDS was detected after the incubation of SPT from Sphingobacterium multivorum with d-serine and palmitoyl-CoA. Furthermore, the KDS comprised equal amounts of 2S and 2R isomers. ¹H-NMR study showed a slow hydrogen-deuterium exchange at Cα of serine mediated by SPT. We further confirmed that SPT catalyzed the racemization of serine. The rate of the KDS formation from d-serine was comparable to those for the α-hydrogen exchange and the racemization reaction. The structure of the d-serine-soaked crystal (1.65 Å resolution) showed a distinct electron density of the PLP-l-serine aldimine, interpreted as the racemized product trapped in the active site. The structure of the α-methyl-d-serine-soaked crystal (1.70 Å resolution) showed the PLP-α-methyl-d-serine aldimine, mimicking the d-serine-SPT complex prior to racemization. Based on these enzymological and structural analyses, the synthesis of KDS from d-serine was explained as the result of the slow racemization to l-serine, followed by the reaction with palmitoyl-CoA, and SPT would not catalyze the direct condensation between d-serine and palmitoyl-CoA. It was also shown that the S. multivorum SPT catalyzed the racemization of the product KDS, which would explain the presence of (2R)-KDS in the reaction products.2024-01-01T00:00:00Z2024 Mar2026-06-17T06:36:05.059Z2026-06-17T03:07:03.202ZS-EPMC109126323832574010.1016/j.jbc.2024.105728