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.
Highlights
Isocitrate dehydrogenase (IDH) plays a critical role in the tricarboxylic acid (TCA) cycle and catalyzes the oxidative decarboxylation of isocitrate to generate CO2 and α-ketoglutarate (α-KG) with divalent metal ions (Mg2+ or Mn2+) while reducing NAD(P)+ to NAD(P)H [1]
We investigated the overexpression, purification, and detailed biochemical characteristics of a type II homodimeric NAD-IDH from P. tricornutum (PtIDH1)
NADH was a competitive inhibitor of PtIDH1 (Ki = 0.45 ± 0.08 mM) (Figure 5D), which is consistent with O. tauri NAD-IDH (Ki = 0.14 mM) and C. reinhardtii NAD-IDH [12,32]
Summary
Isocitrate dehydrogenase (IDH) plays a critical role in the tricarboxylic acid (TCA) cycle and catalyzes the oxidative decarboxylation of isocitrate to generate CO2 and α-ketoglutarate (α-KG) with divalent metal ions (Mg2+ or Mn2+) while reducing NAD(P)+ to NAD(P)H [1]. Eukaryotic mitochondrial or cytoplasmic homodimeric NADP-IDHs and a few bacterial NAD(P)-IDHs are categorized into the type II subfamily. Due to the lack of detailed structural information, the differences in the catalytic mechanism between eukaryotic and prokaryotic homodimeric NAD-IDH are currently still unclear. We investigated the overexpression, purification, and detailed biochemical characteristics of a type II homodimeric NAD-IDH from P. tricornutum (PtIDH1). PtIDH1 is the first eukaryotic homodimeric NAD-IDH structure proposed in the type II subfamily, and it exhibits a unique EF-hand domain at the C-terminal. The structure of PtIDH1 was compared with other IDH structures from H. sapiens (PDB entry: 1T0L) and A. thiooxidans (PDB entry: 2D4V) These results provide basic knowledge of eukaryotic homodimeric NAD-IDH and refine the phylogeny and structural information of the IDH family
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