2. Academic Validation
  3. Racemization of the substrate and product by serine palmitoyltransferase from Sphingobacterium multivorum yields two enantiomers of the product from d-serine

Racemization of the substrate and product by serine palmitoyltransferase from Sphingobacterium multivorum yields two enantiomers of the product from d-serine

  • J Biol Chem. 2024 Mar;300(3):105728. doi: 10.1016/j.jbc.2024.105728.
Hiroko Ikushiro 1,  Takumi Honda 2,  Yuta Murai 3,  Taiki Murakami 4,  Aya Takahashi 4,  Taiki Sawai 5,  Haruna Goto 5,  Shin-Ichi Ikushiro 6,  Ikuko Miyahara 4,  Yoshio Hirabayashi 7,  Nobuo Kamiya 8,  Kenji Monde 9,  Takato Yano 10
Affiliations

Affiliations

  • 1 Department of Biochemistry, Faculty of Medicine, Osaka Medical and Pharmaceutical University, Osaka, Japan. Electronic address: [email protected].
  • 2 Graduate School of Life Science, Hokkaido University, Sapporo, Japan.
  • 3 Graduate School of Life Science, Hokkaido University, Sapporo, Japan; Frontier Research Center for Advanced Material and Life Science, Faculty of Advanced Life Science, Hokkaido University, Sapporo, Hokkaido, Japan; Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Sapporo, Hokkaido, Japan. Electronic address: [email protected].
  • 4 Department of Chemistry, Graduate School of Science, Osaka Metropolitan University, Osaka, Japan.
  • 5 Department of Biochemistry, Faculty of Medicine, Osaka Medical and Pharmaceutical University, Osaka, Japan.
  • 6 Department of Biotechnology, Faculty of Engineering, Toyama Prefectural University, Imizu, Toyama, Japan.
  • 7 RIKEN Cluster for Pioneering Research, RIKEN, Wako, Saitama, Japan; Institute for Environmental and Gender-Specific Medicine, Juntendo University Graduate School of Medicine, Chiba, Japan.
  • 8 Research Center for Artificial Photosynthesis, Osaka Metropolitan University, Osaka, Japan.
  • 9 Graduate School of Life Science, Hokkaido University, Sapporo, Japan; Frontier Research Center for Advanced Material and Life Science, Faculty of Advanced Life Science, Hokkaido University, Sapporo, Hokkaido, Japan.
  • 10 Department of Biochemistry, Faculty of Medicine, Osaka Medical and Pharmaceutical University, Osaka, Japan. Electronic address: [email protected].
PMID: 38325740 DOI: 10.1016/j.jbc.2024.105728
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. 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.

Keywords

PLP-dependent enzyme; X-ray crystallography; crystal structure; racemization; serine palmitoyltransferase; sphingolipid.

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