1. Racemization of the substrate and product by serine palmitoyltransferase from Sphingobacterium multivorum yields two enantiomers of the product from d-serine.
- Author
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Ikushiro H, Honda T, Murai Y, Murakami T, Takahashi A, Sawai T, Goto H, Ikushiro SI, Miyahara I, Hirabayashi Y, Kamiya N, Monde K, and Yano T
- Subjects
- Catalytic Domain, Crystallization, Deuterium Exchange Measurement, Electrons, Hydrogen metabolism, Palmitoyl Coenzyme A metabolism, Sphingosine analogs & derivatives, Sphingosine biosynthesis, Sphingosine metabolism, Stereoisomerism, Substrate Specificity, Serine analogs & derivatives, Serine metabolism, Serine C-Palmitoyltransferase chemistry, Serine C-Palmitoyltransferase metabolism, Sphingobacterium enzymology, Sphingobacterium metabolism
- Abstract
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.
1 H-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., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)- Published
- 2024
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