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

Abstract

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.

Biochemical and Phylogenetic Characterization of a Novel NADP+-Specific Isocitrate Dehydrogenase From the Marine Microalga Phaeodactylum tricornutum

“…Cell debris was removed by centrifugation at 12,000 × g for 20 min at 4°C. Finally, recombinant PtIDH2 and its mutated forms fused with a 6×His tag were purified with TALON Metal Affinity Resin using a previously described method ( Huang et al, 2020 ). The purity and subunit molecular mass of the recombinant enzyme were analyzed by 12% SDS–PAGE.…”

“…The molecular mass of the recombinant PtIDH2 was estimated by size-exclusion chromatography (SEC) on the ÄKTA pure protein purification system equipped with a Superdex 200 (10/300) Increase column (GE Healthcare Life Sciences, Pittsburgh, PA, United States ). The columns were calibrated using the Gel Filtration HMW Calibration Kit (GE Healthcare) as described elsewhere ( Huang et al, 2020 ). Five protein standards were used to calibrate the gels: ovalbumin (44 kDa), conalbumin (75 kDa), aldolase (158 kDa), ferritin (440 kDa), and thyroglobulin (669 kDa).…”

“…Data are expressed as means ± SD of at least three independent measurements. optimal activator for most monomeric IDHs, including Streptomyces lividans IDH, as well as for type II homodimeric IDHs such as P. tricornutum IDH1 and Bifidobacterium longum IDH (Zhang et al, 2009;Huang et al, 2016;Huang et al, 2020).…”

“…Additionally, the increased characterization of IDH family members has allowed for the expansion and refinement of the classification of the IDH family of proteins. Using bioinformatic analysis, we have found that, in addition to typical type II homodimer NAD + -specific IDHs ( Tang et al, 2015 ; Huang et al, 2020 ), eukaryotes further contain a class of putative type III NADP + -specific IDHs, represented by the potential monomeric NADP-IDH from Phaeodactylum tricornutum , a unicellular marine eukaryotic microalga.…”

“…Analysis of the P. tricornutum genome has revealed several unique characteristics, including the presence of hundreds of genes transferred from bacteria, suggesting that P. tricornutum has a complicated evolutionary history. We have previously found that P. tricornutum contains two putative IDHs, and characterized in detail the biochemical properties and crystal structure of PtIDH1, a type II homodimeric NAD-IDH ( Huang et al, 2020 ). In the current study, the enzymology of a putative type III NADP-IDH from P. tricornutum (PtIDH2) was characterized, including its oligomeric form in solution, kinetics, and thermostability, as well as how its activity is affected by pH, temperature, and metal ions.…”