Biochemical Characterization and Crystal Structure of a Novel NAD<sup>+</sup>-Dependent Isocitrate Dehydrogenase from <i>Phaeodactylum tricornutum</i>
The marine diatom <i>Phaeodactylum tricornutum</i> originated from a series of secondary symbiotic events and has been used as a model organism for studying diatom biology. A novel type II homodimeric isocitrate dehydrogenase from <i>P. tricornutum</i> (PtIDH1) was expressed,...
| Main Authors: | , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2020-08-01
|
| Series: | International Journal of Molecular Sciences |
| Subjects: | |
| Online Access: | https://www.mdpi.com/1422-0067/21/16/5915 |
| Summary: | The marine diatom <i>Phaeodactylum tricornutum</i> originated from a series of secondary symbiotic events and has been used as a model organism for studying diatom biology. A novel type II homodimeric isocitrate dehydrogenase from <i>P. tricornutum</i> (PtIDH1) was expressed, purified, and identified in detail through enzymatic characterization. Kinetic analysis showed that PtIDH1 is NAD<sup>+</sup>-dependent and has no detectable activity with NADP<sup>+</sup>. The catalytic efficiency of PtIDH1 for NAD<sup>+</sup> is 0.16 μM<sup>−1</sup>·s<sup>−1</sup> and 0.09 μM<sup>−1</sup>·s<sup>−1</sup> in the presence of Mn<sup>2+</sup> and Mg<sup>2+</sup>, respectively. Unlike other bacterial homodimeric NAD-IDHs, PtIDH1 activity was allosterically regulated by the isocitrate. Furthermore, the dimeric structure of PtIDH1 was determined at 2.8 Å resolution, and each subunit was resolved into four domains, similar to the eukaryotic homodimeric NADP-IDH in the type II subfamily. Interestingly, a unique and novel C-terminal EF-hand domain was first defined in PtIDH1. Deletion of this domain disrupted the intact dimeric structure and activity. Mutation of the four Ca<sup>2+</sup>-binding sites in the EF-hand significantly reduced the calcium tolerance of PtIDH1. Thus, we suggest that the EF-hand domain could be involved in the dimerization and Ca<sup>2+</sup>-coordination of PtIDH1. The current report, on the first structure of type II eukaryotic NAD-IDH, provides new information for further investigation of the evolution of the IDH family. |
|---|---|
| ISSN: | 1661-6596 1422-0067 |