Abstract
The eukaryotic serine racemase from Dictyostelium discoideum is a fold-type II pyridoxal 5′-phosphate (PLP)-dependent enzyme that catalyzes racemization and dehydration of both isomers of serine. In the present study, the catalytic mechanism and role of the active site residues of the enzyme were examined by site-directed mutagenesis. Mutation of the PLP-binding lysine (K56) to alanine abolished both serine racemase and dehydrase activities. Incubation of d- and l-serine with the resultant mutant enzyme, K56A, resulted in the accumulation of PLP-serine external aldimine, while less amounts of pyruvate, α-aminoacrylate, antipodal serine and quinonoid intermediate were formed. An alanine mutation of Ser81 (S81) located on the opposite side of K56 against the PLP plane converted the enzyme from serine racemase to l-serine dehydrase; S81A showed no racemase activity and had significantly reduced d-serine dehydrase activity, but it completely retained its l-serine dehydrase activity. Water molecule(s) at the active site of the S81A mutant enzyme probably drove d-serine dehydration by abstracting the α-hydrogen in d-serine. Our data suggest that the abstraction and addition of α-hydrogen to l- and d-serine are conducted by K56 and S81 at the si- and re-sides, respectively, of PLP.
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Abbreviations
- SR:
-
Serine racemase
- DdSR:
-
Serine racemase of Dictyostelium discoideum
- SpSR:
-
Serine racemase of Schizosaccharomyces pombe
- PLP:
-
Pyridoxal 5′-phosphate
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Acknowledgments
This work was supported in part by the Program for Promotion of Basic and Applied Researches for Innovation in Bio-oriented Industry (BRAIN) (to T. Y.), Grant-in-Aid for Research 22380058 (to T. Y.) from the Ministry of Education, Culture, Sports, Science, and Technology, Grant-in-Aid for Research Activity Start-up 22880014 (to T. I) and Grant-in-Aid for Young Scientists (B) 24780098 (to T. I.).
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T. Ito amd M. Maekawa contributed equally to this work.
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Ito, T., Maekawa, M., Hayashi, S. et al. Catalytic mechanism of serine racemase from Dictyostelium discoideum . Amino Acids 44, 1073–1084 (2013). https://doi.org/10.1007/s00726-012-1442-4
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DOI: https://doi.org/10.1007/s00726-012-1442-4