Biochemical Characterization and Crystal Structure of a Novel NAD⁺-Dependent Isocitrate Dehydrogenase from Phaeodactylum tricornutum

The marine diatom Phaeodactylum tricornutum 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 P. tricornutum (PtIDH1) was expressed, purified, and identified in detail through enzymatic characterization. Kinetic analysis showed that PtIDH1 is NAD⁺-dependent and has no detectable activity with NADP⁺. The catalytic efficiency of PtIDH1 for NAD⁺ is 0.16 μM^-1·s^-1 and 0.09 μM^-1·s^-1 in the presence of Mn²⁺ and Mg²⁺, 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 Ca²⁺-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 Ca²⁺-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.