Functional Identification And Alteration And Crystal Structure Elucidation Of A Novel Isocitrate Dehydrogenase From The Smallest Unicellular Marine Green Alga

Isocitrate dehydrogenase(IDH)catalyzes the second limiting step in tricarboxylic acid cycle,which catalyzes the dehydrogenation and decarboxylation of isocitrate(ICT)to yield NAD(P)H,CO₂ and?-ketoglutarate(?-KG).As a key metabolic enzyme,IDH is distributed in all living organisms.According to the coenzyme specificity,IDH falls into NAD⁺-dependent IDH(NAD-IDH)and NADP⁺-dependent IDH(NADP-IDH).In eukaryotes,NAD-IDH and NADP-IDH play different biological roles.The former is mainly involved in energy production,while the latter mainly participates in anabolism as well as defense against oxidative damage.Based on the amino acid sequence,IDH can be divided into Type I IDH and Type II IDH.The former consists of both NAD-IDH and NADP-IDH while the latter only contains NADP-IDH.Phylogenetic analysis and competition studies have revealed that in Type I subfamily,NADP-IDH was evolved from NAD-IDH,which was the adaptive result for bacteria to produce more NADPH to overcome the environment with poor carbon source.In Type II subfamily,NAD-IDH could exist if NADP-IDH underwent the similar functional evolution process.The phylogenetic analysis have shown that there are three novel clades of NAD-IDH in Type II subfamily.One of them is putative eukaryotic homodimeric NAD-IDH,and its representative member is IDH from a unicellular green alga Ostreococcus tauri(Ot IDH),the smallest eukaryote in the world.In this study,the structure and function of Ot IDH are determined,including the detailed enzymatic characterization,the alteration of coenzyme specificity and the elucidation of crystal structure.1.Detailed enzymatic characterization of Ot IDHThe studies on the enzymology revealed that Ot IDH is a homodimeric enzyme which is a eukaryotic NAD-IDH in Type II subfamily.Ot IDH is an approximately 93 k Da enzyme that exhibits a 42-fold(with Mn²⁺)or 51-fold(with Mg²⁺)specificity for NAD⁺over NADP⁺,respectively.At present,all reported eukaryotic NAD-IDHs are found to be hetero-oligomers,whereas Ot IDH is a unique eukaryotic homodimeric enzyme.Furthermore,all known members in Type II subfamily are NADP-IDHs,whereas Ot IDH is a NAD-IDH in Type II subfamily with ancient trait.Interestingly,Ot IDH shows an allosteric regulation by ICT(n _H?2.0)unlike bacterial NAD-IDH,and a significantly high affinity for ICT(S_0.5<10?M)unlike its hetero-oligomeric homologs.Like other plant NAD-IDHs,NADH is a competitive inhibitor for the NAD⁺-linked activity of Ot IDH(K_i=0.14±0.01 m M),but NADPH is not its non-competitive inhibitor.The optimum temperature of Ot IDH was approximately 45°C with either Mg²⁺or Mn²⁺as an activator,which was slightly higher than that of NAD-IDH from Chlamydomonas reinhardtii(40°C),but lower than that of NAD-IDH from Zymomonas mobilis(55°C).The optimum p H for Ot IDH catalysis was around 8.0 with Mn²⁺and 8.5 with Mg²⁺,respectively,which is consistent with that of Z.mobilis NAD-IDH.The thermostability testing showed that Ot IDH retained at least 88%activity after incubation at 40°C for 20 min,but rapidly lost the activity above this temperature.Ot IDH is a metal ion-dependent enzyme,and Mn²⁺was its best activator,followed by Mg²⁺.Other divalent cations(Co²⁺,Cu²⁺,Ni²⁺and Zn²⁺)and monovalent cations(K⁺and Na⁺)displayed either a very weak activating effect or no effect.Zn²⁺was a strong inhibitor on Ot IDH activity,while other divalent cations had different degree of inhibitory effect.The monovalent cations had no effect on Ot IDH activity.AMP or ADP or ATP at low concentrations(0.01 m M or 0.1 m M)and 1 m M ADP had no effect on Ot IDH activity,but 1m M AMP or ATP had slightly inhibitory effect on Ot IDH activity.?-KG at low concentrations(?1 m M)had no effect on Ot IDH activity,but inhibited the activity at high concentrations(?5 m M).L-glutamate,an inbititor of NAD-IDHs from Neurospora crassa and Rhodosporidium toruloides,showed no effect on Ot IDH activity.2.Conversion of coenzyme specificity of Ot IDH and its evolutionary mechanismTwo mutant enzymes of Ot IDH,single point mutant D344R and double point mutant D344R/M345H,were designed and constructed based on the amino acid sequence alignment.Gel filtration chromatography revealed that these two mutants retained dimeric structures,and the circular dichroism(CD)spectra analysis showed that their CD spectra were similar to that of wild-type enzyme.Thus,these mutations had no significant effect on the oligomeric state and secondary structure of Ot IDH.Kinetic analysis revealed that D344R and D344R/M345H displayd 15-fold and 72-fold preference for NADP⁺over NAD⁺,respectively.Therefore,the coenzyme specificity of Ot IDH can be completely converted from NAD⁺(ancient phenotype)to NADP⁺(adaptive phenotype)by a factor of 630 for D344R and a factor of 3024 for D344R/M345H through rational site-directed mutagenesis.It has been proved that the alteration of the coenzyme specificity of Type I NAD-IDH was an adaptive result.Ot IDH,a NAD-IDH in Type II subfamily,can be completely changed into a NADP⁺-specific enzyme,suggesting that Type II NADP-IDH may have a similar evolutionary mechanism to Type I NADP-IDH.During the evolutionary history,the coenzyme specificity of Type II NADP-IDH was evolved from NAD⁺to meet the increased demand of NADPH during growth on poor carbon sources.Evidently,the evolutionary mechanism may be common for all members of Type I and II IDHs.3.Determination of crystal structure of Ot IDHThe crystal structures of Ot IDH without ligand or with ligand were determined by single-wavelength anomalous dispersion(SAD)and molecular replacement(MR)method.Four structures were obtained,including unliganded Ot IDH(Ot IDH-Apo),Ot IDH in complex with NAD⁺and Mg²⁺(Ot IDH-NAD⁺-Mg²⁺),Ot IDH in complex with NAD⁺and citrate(CIT)(Ot IDH-NAD⁺-CIT),and Ot IDH in complex with ICT and Mg²⁺(Ot IDH-ICT-Mg²⁺).The resolution of these four structures were 1.75(?),1.78(?),1.87(?),and 1.80(?),respectively.Structural similarity search revealed that Ot IDH shares the highest structural similarity to NADP-IDH in Type II subfamily,due to their similar distance between N and C termini,the insertion of two helices(?4 and?11)as well as similar clasp domains.Based on the structural comparison,the crystal structure of Ot IDH was evidently different from reported bacterial NAD-IDH and yeast NAD-IDH in the three aspects mentioned above.Based on the complex structures of Ot IDH with ligand,all residues involved in the interaction with both coenzyme and substrate can be identified.As compared with the bacterial homodimeric NAD-IDH,all residues involved in the substrate-binding pocket of Ot IDH are highly conserved,but the amino acids in the NAD⁺-binding pocket are different.It's noted that Lys283*(the“*”denotes the residue in another subunit)forms a unique hydrogen bond with the 3'-OH of adenine ribose moiety of NAD⁺.Furthermore,the pairwise comparisons among four structures illustrated lots of ligand-induced conformation changes in both active site and overall structure.Finally,it was proposed that Ot IDH employed a catalytic mechanism using Tyr159-Asp302-Lys234*as a catalytic triad.Based on above results,it is proposed that eukaryotic homodimeric NAD-IDH,such as Ot IDH,could be the ancient form of eukaryotic homodimeric NADP-IDH.Under carbon starvation,Ot IDH and Ot IDH-like NAD-IDHs may evolve into NADP-IDHs for adaptation to the elevated demand of NADPH for cellular biosynthesis.