Abstract
Recently, we cloned and characterized eleven serine and aspartate racemases (SerR and AspR, respectively) from animals. These SerRs and AspRs are not separated by their racemase functions and form a serine/aspartate racemase family cluster based on phylogenetic analysis. Moreover, we have proposed that the AspR-specific triple serine loop region at amino acid positions 150–152 may be responsible for the large AspR activity. In the present study, to test this hypothesis, we prepared and characterized fourteen mutants in this region of animal SerRs and AspRs. The large AspR activity in Acropora and Crassostrea AspR was reduced to <0.04% of wild-type after substitution of the triple serine loop region. Conversely, introducing the triple serine loop region into Acropora, Crassostrea, and Penaeus SerR drastically increased the AspR activity. Those mutants showed similar or higher substrate affinity for aspartate than serine and showed 11–683-fold higher kcat and 28–351-fold higher kcat/Km values for aspartate than serine racemization. Furthermore, we introduced serine residues in all combinations at position 150–152 in mouse SerR. These mutants revealed that a change in the enzyme function from SerR to AspR can be caused by introduction of Ser151 and Ser152, and addition of the third serine residue at position 150 further enhances the enzyme specificity for aspartate due to a decrease in the serine racemase and serine dehydratase activity. Here, we provide convincing evidence that the AspR gene has evolved from the SerR gene by acquisition of the triple serine loop region.
Original language | English |
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Pages (from-to) | 1743-1754 |
Number of pages | 12 |
Journal | Amino Acids |
Volume | 49 |
Issue number | 10 |
DOIs | |
State | Published - Oct 1 2017 |
Bibliographical note
Publisher Copyright:© 2017, Springer-Verlag GmbH Austria.
Funding
This work was supported by a Grant-in-Aid for Scientific Research in Japan to KU (24770068 and 15K07152).
Funders | Funder number |
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Japan Society for the Promotion of Science | 15K07152 |
Japan Society for the Promotion of Science |
Keywords
- Aspartate racemase
- Serine racemase
- d-Amino acid
- d-Asp
- d-Ser
ASJC Scopus subject areas
- Biochemistry
- Clinical Biochemistry
- Organic Chemistry