A unique homodimeric NAD⁺-linked isocitrate dehydrogenase from the smallest autotrophic eukaryote Ostreococcus tauri.
In eukaryotes, NAD(+)-dependent isocitrate dehydrogenase (IDH) is strictly mitochondrial and is a key enzyme in the Krebs cycle. To date, all known NAD(+)-specific IDHs (NAD-IDHs) in the mitochondria are believed to be heteromeric in solution. Here, a unique homodimeric NAD-IDH from Ostreococcus tauri (OtIDH), the smallest autotrophic picoeukaryote, was unveiled. Active OtIDH has a molecular weight of ∼93 kDa with each subunit of 46.7 kDa. In the presence of Mn(2+) and Mg(2+), OtIDH displayed 42-fold and 51-fold preference for NAD(+) over NADP(+), respectively. Interestingly, OtIDH exhibited a sigmoidal kinetic behavior in response to isocitrate unlike other homodimeric homologs, and a remarkably high affinity for isocitrate (S0.5 < 10 μM) unlike other hetero-oligomeric homologs. Furthermore, its coenzyme specificity can be completely converted from NAD(+) (ancient trait) to NADP(+) (adaptive trait) by rational mutagenesis based on the evolutionary trace. Mutants D344R and D344R/M345H displayed a 15-fold and 72-fold preference for NADP(+) over NAD(+), respectively, indicating that D344 and M345 are the determinants of NAD(+) specificity. These findings also suggest that OtIDH may be an ancestral form of type II IDHs (all reported members are NADP(+)-linked enzymes) and may have evolved into NADP(+)-dependent IDH for adaptation to the increased demand of NADPH under carbon starvation.
Institute of Molecular Biology and Biotechnology, College of Life Sciences, Anhui Normal University, Wuhu, China.
Wang-Gang
WG
Tang
Institute of Molecular Biology and Biotechnology, College of Life Sciences, Anhui Normal University, Wuhu, China.
Ping
P
Song
Institute of Molecular Biology and Biotechnology, College of Life Sciences, Anhui Normal University, Wuhu, China.
Zheng-Yu
ZY
Cao
Institute of Molecular Biology and Biotechnology, College of Life Sciences, Anhui Normal University, Wuhu, China.
Peng
P
Wang
Institute of Molecular Biology and Biotechnology, College of Life Sciences, Anhui Normal University, Wuhu, China gpz1996@yahoo.com.
Guo-Ping
GP
Zhu
D020418:Algal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D000460:Chlorophyta / Q000201:enzymology* / Q000235:genetics; D002942:Circular Dichroism; D007521:Isocitrate Dehydrogenase / Q000737:chemistry* / Q000145:classification / Q000378:metabolism; D007523:Isocitrates / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D008970:Molecular Weight; D009154:Mutation; D009243:NAD / Q000737:chemistry* / Q000378:metabolism; D009249:NADP / Q000737:chemistry / Q000378:metabolism; D010802:Phylogeny; D011485:Protein Binding; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D020418:Algal Proteins; D007523:Isocitrates; D009243:NAD; D008345:Manganese; D009249:NADP; C034219:isocitric acid; D007521:Isocitrate Dehydrogenase; D008274:Magnesium
adaptation; coenzyme specificity determinants; kinetics; phylogenetic analysis