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Originally published In Press as doi:10.1074/jbc.M304091200 on July 10, 2003

J. Biol. Chem., Vol. 278, Issue 38, 36897-36904, September 19, 2003
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Crystal Structure of the Monomeric Isocitrate Dehydrogenase in the Presence of NADP+

INSIGHT INTO THE COFACTOR RECOGNITION, CATALYSIS, AND EVOLUTION*

Yoshiaki Yasutake, Seiya Watanabe {ddagger}, Min Yao, Yasuhiro Takada, Noriyuki Fukunaga and Isao Tanaka §

From the Division of Biological Sciences, Graduate School of Science, Hokkaido University, Kita-10, Nishi-8, Kita-ku, Sapporo, Hokkaido 060-0810, Japan

Received for publication, April 18, 2003 , and in revised form, July 9, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
NADP+-dependent monomeric isocitrate dehydrogenase (IDH) from the nitrogen-fixing bacterium Azotobacter vinelandii (AvIDH) is one of members of the {beta}-decarboxylating dehydrogenase family and catalyzes the dehydration and decarboxylation of isocitrate to yield 2-oxoglutrate and CO2 in the Krebs cycle. We solved the crystal structure of the AvIDH in complex with cofactor NADP+ (AvIDH-NADP+ complex). The final refined model shows the closed form that has never been detected in any previously solved structures of {beta}-decarboxylating dehydrogenases. The structure also reveals all of the residues that interact with NADP+. The structure-based sequence alignment reveals that these residues were not conserved in any other dimeric NADP+-dependent IDHs. Therefore the NADP+ specificity of the monomeric and dimeric IDHs was independently acquired through the evolutional process. The AvIDH was known to show an exceptionally high turnover rate. The structure of the AvIDH-NADP+ complex indicates that one loop, which is not present in the Escherichia coli IDHs, reliably stabilizes the conformation of the nicotinamide mononucleotide of the bound NADP+ by forming a few hydrogen bonds, and such interactions are considered to be important for the monomeric enzyme to initiate the hydride transfer reaction immediately. Finally, the structure of the AvIDH is compared with that of other dimeric NADP-IDHs. Several structural features demonstrate that the monomeric IDHs are structurally more related to the eukaryotic dimeric IDHs than to the bacterial dimeric IDHs.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Isocitrate dehydrogenase (IDH)1 is one of the enzymes of the Krebs cycle and catalyzes the metal ion- and NAD(P)+-dependent hydride transfer at C2 and then the metal ion-dependent decarboxylation at C3 of 2R,3S-isocitrate to generate 2-oxoglutarate and CO2 with a concomitant reduction of NAD(P)+ to NAD(P)H (1). The product 2-oxoglutarate is a key material for the biosynthesis of glutamate by the action of glutamate dehydrogenase. IDHs are ubiquitously distributed in a wide variety of both prokaryotes and eukaryotes. Most prokaryotes possess only homodimeric NADP+-dependent IDHs (EC 1.1.1.42 [EC] , NADP-IDHs), whereas eukaryotes possess both homodimeric NADP-IDHs and hetero-oligomeric NAD+-dependent IDHs (EC 1.1.1.41 [EC] , NAD-IDHs). The hetero-oligomeric NAD-IDHs are known to be one of the allosterically regulated enzymes and participate in the supply of NADH in order to produce ATP by the respiratory chain reaction in the mitochondria (2, 3). In addition to IDHs, homodimeric NAD+-dependent isopropylmalate dehydrogenases (EC 1.1.1.85 [EC] , NAD-IPMDHs) also catalyze the metal ion- and NAD+-dependent dehydration and decarboxylation of 2-isopropylmalate, a structurally similar compound to 2R,3S-isocitrate (4, 5). The structures of NADIPMDH from a few species of bacteria have already been determined (6-9). The structure analyses revealed that IDHs and IPMDHs were structurally homologous, and the similar structure at the catalytic site suggests that the catalytic mechanism must be identical. Therefore the IDHs and IPMDHs form the divergent protein family of the metal-dependent {beta}-decarboxylating dehydrogenases (9). Recently, tartrate dehydrogenase and homoisocitrate dehydrogenase have also been suggested to be members of this family (10-12).

The Escherichia coli IDH (EcIDH) has been studied most extensively (13-24). The EcIDH is an Mg2+- and NADP+-dependent dimeric enzyme consisting of two identical subunits with a molecular mass of 45 kDa. At present, >20 structures of the EcIDH have been solved including those for free enzymes, various complexes, and mutants. The catalytic site of the EcIDH was located at the intersubunit interface, and the isocitrate-Mg2+ was recognized with several residues derived from both subunits. Very recently, the structure of the porcine heart mitochondrial NADP-IDH (PcIDH) in complex with isocitrate-Mn2+ was reported previously (25). Although the sequence identity between EcIDH and PcIDH is not high (16%), both structures are homologous and the structure-based sequence alignment showed that all of the residues for substrate and metal ion binding in both enzymes were completely conserved.

In contrast to the homodimeric IDHs, the monomeric NADPIDHs with a molecular mass of 80-100 kDa were found in several taxonomically unrelated eubacteria. The nitrogen-fixing bacterium Azotobacter vinelandii is one of bacteria that possess the monomeric IDH. Interestingly, the cytoplasm of this organism contains the abundant monomeric IDH with a concentration of ~1-2% of the total soluble protein (26). Furthermore, the enzyme showed unusually high activity as Barrera et al. (27) reported that the calculated turnover number of the enzyme was 56,000. These findings suggested that a large amount of NADPH was needed for the energy production and for providing the reductive environment to protect the abundant nitrogenase from oxidative damage. The Corynebacterium glutamicum is well known as a bacterium used for the production of approximately a million tons of L-glutamate every year (28). This organism also contains the highly active and highly specific monomeric IDH (29). The psychrophilic marine bacterium Colwellia maris is a unique organism that possesses both homodimeric and monomeric IDHs. The gene encoding the monomeric enzyme was found to be a low temperature-inducible gene, and the enzyme exhibited the cold adaptation. Therefore the monomeric IDH was essential for the survival of this bacterium in low temperature environments (30-32). Additionally, recent genome projects have revealed several putative genes encoding the monomeric IDHs in extra bacteria, such as Vibrio cholerae, Pseudomonas aeruginosa and Xylella fastidiosa.

For the dimeric NADP- and hetero-oligomeric NAD-IDHs, the subunit assembly is an indispensable process because the catalytic sites are located at the intersubunit cleft and are formed with residues from both subunits. In contrast, the monomeric IDHs apparently catalyze the reaction identical to that of the dimeric IDH with a single polypeptide chain. We recently solved the crystal structure of the monomeric A. vinelandii IDH (AvIDH) in complex with isocitrate-Mn2+ (33, 34). Despite the absence of significant sequence similarities, the folding topology of the AvIDH was surprisingly related to that of the dimeric IDH. The pseudo 2-fold symmetry in the large domain (domain II) indicated that the AvIDH must have arisen through partial gene duplication. The duplicated structure comprised a portion corresponding to another subunit of the dimeric IDH, and such unique folding enabled a single polypeptide chain to form a catalytic site that is homologous to that of the dimeric IDH (34).

The structure of the AvIDH-isocitrate-Mn2+ complex also revealed the evolutional relationship between the monomeric and dimeric IDHs. However, it is still not clear how the monomeric IDH recognizes the NADP+ and why the enzyme shows a particularly high turnover rate. The structure-based sequence alignment showed that the NADP+ binding residues of the dimeric EcIDH were not conserved in the AvIDH. Furthermore, the structure of the putative NADP+ binding site of the AvIDH was somehow different from that of the EcIDH structure (34). These findings suggested that the manner of NADP+ recognition is different between monomeric and dimeric IDHs. Here we describe the crystal structure of the AvIDH in complex with NADP+. Together with the previous structure of the AvIDH-isocitrate-Mn2+ complex, the mechanism of NADP+ recognition, the catalytic scheme with high activity, and the molecular evolution of the monomeric IDHs are discussed.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
 REFERENCES
 
Purification and Crystallization of the AvIDH—The nitrogen-fixing bacterium A. vinelandii (IAM1078) was cultured, and the monomeric IDH with high activity was directly isolated from the cell extract. The detailed procedures for the enzyme purification and the initial screenings of the crystallization condition were described previously (33).

To obtain the crystals of AvIDH in complex with isocitrate-Ca2+ and NADP+, the pseudo-Michaelis quaternary complex, we further performed an optimization of the crystallization condition by the hanging-drop vapor diffusion technique at room temperature (291 K) using 24-well tissue culture plates (Linbro plates, ICN Biomedicals). The protein solution was prepared by adjusting 8 mg of protein/ml in 100 mM HEPES-NaOH, pH 7.0, and 10% (v/v) glycerol. The hanging drops were made by mixing 2.5 µl of protein solution with 1.5-4.5 µl of precipitating solution containing 100 mM HEPES-NaOH, pH 7.0, 24-28% (w/v) polyethylene glycol 6000, 20% (v/v) glycerol, 1-10 mM 2R,3S-isocitrate, 1-10 mM CaCl2, and 1-10 mM {beta}-NADP+ and were equilibrated to a 500-µl precipitating solution. Although most crystals were needle-shaped and clustered, a few single crystals also grew within one month. As described later, the clear electron density showed that only the NADP+ bound to the enzyme.

X-ray Diffraction Study—The x-ray diffraction experiment was performed on the beamline BL41XU (35) at the third generation synchrotron radiation facility, SPring-8 (Harima, Japan). The flash-cooled crystals of the AvIDH complexed with NADP+ using the CryoLoop (Hampton Research) diffracted with extremely high mosaicity. Therefore the best single crystal was mounted in a glass capillary (Hampton Research) in advance and then the crystal was flash-cooled as the cryogenic nitrogen stream passed over the capillary.2 The diffraction data were collected on a charge-coupled device (CCD) detector (MAR Research) using a wavelength of 0.9 Å up to a resolution of 3.2 Å. The crystal was found to belong to the monoclinic system, the space group P21 with unit cell parameters a = 113.5, b = 110.4, c = 133.7 Å and {beta} = 90.2°. The diffraction data were integrated, scaled, and merged using the program MOSFLM (36) and SCALA (37). The crystallographic data and the processing statistics are summarized in Table I.


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  		TABLE I
Data collection and refinement statistics

 

Phasing—The initial phase was obtained by the molecular replacement method using the programs AMoRe (38) and MOLREP (37), employing the previous structure of the AvIDH-isocitrate-Mn2+ complex (Protein Data Bank (PDB) accession code 1ITW [PDB] ) as a search model. The first run of the molecular replacement using a whole-length search model was a failure. To provide a more suitable search model, three fragments that protrude from the globular domain with high temperature factors were deleted (95 of 740 residues) from the model. We again performed the cross-rotation and translation search using this modified model, and the four correct locations of the molecule in the asymmetric unit were successfully determined.

Model Refinement—The model refinement of the AvIDH·NAPD+ complex was performed with the program CNS (39) using reflections ranging from 10- to 3.2-Å resolution with the F/{sigma} amplitude cutoff criteria of 2.4. To monitor the refinement, we set aside 10% of all reflections for the calculation of the free R-factor (Rfree) and the noncrystallographic symmetry restraints with weights of 500 kcal/Å2 were introduced for all of the atoms of protein molecules during all stages of model refinement. After the first cycle of rigid-body, positional, simulated annealing, and the individual B-factor refinement, the crystallographic R-factor (Rcryst) and the Rfree were reduced from 36.5 and 39.2% to 33.3 and 37.5%, respectively. At this step, a SIGMAA-weighted mFo - DFc map calculated using this initial refined model showed that the large domain (domain II) was rotated to a certain degree and the location of the domain was corrected manually on the graphic program O (40). The SIGMAA-weighted mFo - DFc map also allowed us to recognize that the NADP+ is bound to the enzyme. The model still included the incorrect parts, and we therefore corrected the model very carefully based on the SIGMAA-weighted mFo - DFc omit map calculated at the end of each refinement cycle. After the manual model corrections were mostly completed, four NADP+ molecules (one for each monomer) were added to the model. The topology and parameter files of the NADP+ molecule were obtained from the Hetero-compound Information Center of Uppsala on the Uppsala web site (41). After the iterative cycles of positional and individual B-factor refinement and manual model fitting, the Rcryst and the Rfree were reduced to 27.5 and 30.3%, respectively. The water molecules were automatically located by peak-searching on the SIGMAA-weighted mFo - DFc map, and some of the molecules that occupied the irrelevant positions were deleted on the basis of real space correlation coefficient and/or maximum density level using the procedure in the program CNS (39). Finally, the Rcryst and Rfree were converged to 26.0 and 29.7%, respectively. The stereochemical quality of the final refined model was analyzed with the program PROCHECK (42). The refinement statistics are summarized in Table I.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
REFERENCES
 
Structure Description—The structure of the AvIDH in complex with NADP+ was solved by the molecular replacement method, and the model refinement was performed against 3.2-Å resolution. The AvIDH is an 80-kDa monomeric enzyme consisting of 741 amino acid residues, and the asymmetric unit contains four monomers with a Matthews' coefficient VM value of 2.61 Å3 Da-1 (43). The SIGMAA-weighted mFo - DFc omit map showed unambiguously that the NADP+ was bound to the enzyme, whereas the isocitrate and Ca2+, which we added to the mother liquor in expectation of their binding to the enzyme, were not found at the catalytic site. The final refined model was composed of four polypeptide chains (each contained residues 3-740), four NADP+ molecules at the active sites, and 324 oxygen atoms of water molecules with the crystallographic R-factor and the Rfree factor of 26.0 and 29.7%, respectively.

The structure of the AvIDH-NADP+ complex was comprised of 27 {alpha}-helices and 18 {beta}-strands with a folding topology similar to the structure of the AvIDH-isocitrate-Mn2+ complex that we reported recently (Fig. 1) (34). The structure of the AvIDH was divided into two distinct domains: a small domain (domain I) and a large domain (domain II). Domain I was comprised of five {beta}-strands and 14 {alpha}-helices and includes both N- and C-terminal segments. Domain II was comprised of 16 {beta}-strands and 13 {alpha}-helices and was formed by two topologically identical motifs, which were tightly intertwined and related by a pseudo 2-fold symmetry. This interaction between two motifs structurally corresponds to the intensive and expansive intersubunit interface in the structure of the dimeric IDH (34).



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  		FIG. 1.
Ribbon diagram of the AvIDH in complex with cofactor NADP+ The structure of the AvIDH consists of two domains, a small domain (Domain I) and a large domain (Domain II). The NADP+ was located at the interdomain cleft and interacted with domain I. The model is colored according to the sequence by a rainbow color ramp from blue at the N-terminal segment to red at the C-terminal segment. The bound NADP+ is represented as a ball-and-stick model. The figure is generated using the program MOLSCRIPT (60) and Raster3D (61).

 

Domain Movement—In the structure of the AvIDH-NADP+ complex, a large conformational change was found to occur. By comparison with the previous structure of the AvIDH-isocitrate-Mn2+ complex, the two domains of the AvIDH were relatively rotated by an angle of 9° and, accordingly, the present structure showed a more closed conformation than the previous structure of the isocitrate-Mn2+ complex (Fig. 2). This closed structure seemed to be an inactive form, because the structure did not provide enough space for substrate-metal ion binding (see "Inhibition by the Ca2+"). Within each domain, the main chains can be very well superimposed. This rigid-body movement is an example of the typical hinge motions that occur in several proteins with multidomain structures. Since both the N- and C-terminal segments were in domain I, the hinges must be located at the incoming and outgoing chain in the vicinity of the connecting region between the two domains. The Ramachandran analysis indicated that the greater than 30° rotations of the {phi}- and/or {psi}-angles between the two structures occurred in the residues 137-138, 142-144, and 560-565. The former two regions lie at the loop between {alpha}6 and {beta}6, and the latter lies at the loop between {alpha}19 and {beta}5. Both of these loops are located exactly at the connecting region between the two domains.



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  		FIG. 2.
Structural comparison between the open and closed form of the AvIDH. A, the previous structure of the AvIDH-isocitrate-Mn2+ complex. B, the present structure of the AvIDH-NADP+ complex. The AvIDH-NADP+ complex showed a more closed form than the previous AvIDH-isocitrate-Mn2+ complex by the rigid-body domain movement. Although the crystal of the AvIDH was obtained in a solution containing isocitrate and Ca2+, the isocitrate and Ca2+ were not found in the present closed structure. The molecular surfaces were generated using the program GRASP (62).

 

One question then arises as to why the enzyme formed the closed structure. The rigid-body movement does not spawn additional hydrogen bonds, ion pairs, or hydrophobic interactions between the two domains. This finding suggests that such conformational changes must occur easily if the small free energy barrier is surmounted. Therefore the closed structure might have been formed in the process of the crystallization. It is likely that the crystal lattice formation has forced the flexible molecule into the closed structure. Actually, the condition of the molecular contacts in the crystal lattice was quite different between the previous open form and the present closed form.

Similar domain movement was also reported previously in the structure of the EcIDH (19) and IPMDH from Thermus thermophilus (TtIPMDH) (6, 9, 44). However, these conformational changes were distinct in that the structures were transformed into a more open conformation. In the case of the EcIDH, the domain rotation opened up the active site cleft and further exposed the residue Ser113 to the solvent (20). This open conformation thus appeared to be a key structure for the regulatory phosphorylation of Ser113 catalyzed by the bifunctional IDH kinase/phosphatase (20, 45). The TtIPMDH is also known to convert its structure into a more open form in the absence of substrate, although the biochemical implications of the open form have not been elucidated. Considering these conformational changes, the structurally homologous {beta}-decarboxylating dehydrogenases seem to easily cause the domain movement under the substrate-free condition. The substrate binding to the enzyme apparently plays a role in fixing the mutual movement of the two domains, because the substrate interacts with residues belonging to both domains.

Inhibition by the Ca2+—The true substrate of IDH is not free isocitrate but metal-chelating isocitrate (46, 47). The isocitrate-Ca2+ was known to bind to the EcIDH, to act as a competitive inhibitor of isocitrate-Mg2+, and to cause the complete inhibition of the enzyme activation (16). The crystal structure of the EcIDH-isocitrate-Ca2+-NADP+, a pseudo Michaelis quaternary complex, provided evidence that very small structural changes contributed large catalytic consequences (16, 21). Similarly, Barrera and Jurtshuk (27) reported that no activation of the AvIDH was obtained in the presence of Ca2+ alone. Although crystals of the AvIDH were successfully obtained in the solution containing isocitrate, Ca2+, and NADP+, the structure analysis disclosed that the isocitrate-Ca2+ did not bind to the enzyme and alternatively that the molecule has adopted a more closed conformation in complex with NADP+. In this closed structure, two aspartate residues (Asp350 and Asp548), which should have coordinated with Mn2+, were considerably close to each other due to the closeness of the two domains and not enough space was observed for the binding of isocitrate-Ca2+.It is likely that the closed structure was consequently adopted because of the low affinity of isocitrate-Ca2+ to the monomeric enzyme. In other words, the substrate-binding pocket of the AvIDH has an ability to discriminate between two metal-chelating substrates, isocitrate-Mn2+ and isocitrate-Ca2+. Thus the inhibition mechanism by the Ca2+ was absolutely different between the dimeric EcIDH and monomeric AvIDH.

NADP+ Recognition—In the present analysis, the electron density corresponding to the overall NADP+ molecule was obviously recognized (Fig. 3). The NADP+ was located in the active site cleft between the two domains and was found to interact with several residues belonging to domain I (Figs. 4 and 6). The clear electron density also showed that both the adenine ring and the nicotinamide ring of the bound NADP+ molecule were in the anti-conformation with respect to each ribose.



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  		FIG. 3.
The SIGMAA-weighted mFo - DFc electron density maps. A, a portion of the regions that were excluded in the phasing model (residues Phe274-Leu286). B, cofactor NADP+. The electron density map was calculated by the SIGMAA-weighted mFo - DFc difference Fourier synthesis with a resolution range of 10-3.2 Å at an early stage of refinement. The maps are less biased by the model because these regions are not included in the model refinement. The final refined model is also displayed. The figures were prepared using the program O (40).

 


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  		FIG. 4.
The NADP+ recognition site of the AvIDH and EcIDH. A, the stereoview of the NADP+ recognition site of the AvIDH. The specificity for NADP+ is primarily conferred by the interactions between His589, Arg600, and Arg649 and 2'-phosphomonoester. The residues Asn85, Ser87, and Ser585 also interact with the nicotinamide mononucleotide, and these interactions play a role in stabilizing the conformation of the nicotinamide ring moiety of bound NADP+. B, the stereoview of the NADP+ recognition site of the EcIDH (PDB code 1AI2 [PDB] ). Three residues, Tyr345, Tyr391, and Arg395, which are not conserved in the AvIDH, recognize the 2'-phosphomonoester. The residues structurally corresponding to Asn85 and Ser87 of the AvIDH are not present in the EcIDH. The figures were generated using the program MOLSCRIPT (60) and Raster3D (61).

 


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  		FIG. 6.
Schematic diagram showing the interactions of the cofactor NADP+, isocitrate-Mn2+, and nearby residues. The residues shown in the thick square (His589, Arg600, and Arg649) denote those essential for the cofactor recognition (see "Results and Discussion"). In this diagram, isocitrate-Mn2+, Asp350, and three water molecules (w843, w190, and w1378) are also included from the structure of 1ITW [PDB] . The thick dotted line represents the pathway of hydride transfer.

 

The adenine moiety was close to the side chains of His589 and Trp601 and the main chain of Asp602, and primarily the hydrophobic interactions are formed. Among these residues, the main chain carbonyl oxygen and amide nitrogen of the Asp602 formed the hydrogen bonds to the N6 and N1 of the adenine, respectively. The side chains of His589, Arg600, and Arg649 interacted with the 2'-phosphomonoester of the NADP+ by the hydrogen bonds or ion pairs. Although the adenine ribose and the diphosphate moiety of the NADP+ lay in the vicinity of the main chain of Gly584-Ser585-Ala586, apparent hydrogen bonds were not formed. Therefore these interactions were considered to be built up by the van der Waals forces. For the nicotinamide ribose moiety, several hydrogen bonds were detected. The endocyclic oxygen atom of the nicotinamide ribose formed two hydrogen bonds with the side chain and the main chain amide nitrogen of Ser585, and the 2'-hydroxyl of ribose interacted with the main chain amide nitrogen of Ser87. Furthermore the side chains of Asn85 seemed to interact with the amide group of the nicotinamide moiety, although the electron density corresponding to the amide group was not clear.

Based on the present structure, the NADP+ specificity of the AvIDH was predominantly conferred by the interactions between the side chains of His589, Arg600, and Arg649 and the 2'-phosphomonoester. Although these three residues are completely conserved within monomeric IDHs, they are not conserved or not found in sequences of dimeric NADP+-dependent IDHs. For example, the structure-based sequence alignment showed that the His589 and Arg649 of the AvIDH are substituted with the Tyr345 and Tyr391 in the EcIDH, respectively, as shown in Figs. 4 and 5. Indeed the structures of the EcIDHNADP+ complex revealed that these two tyrosine residues interacted with 2'-phosphomonoester (14, 15, 20). The Arg600 of the AvIDH is located at the connecting loop between helices {alpha}20 and {alpha}21. Interestingly, in the EcIDH, the structure corresponding to helix {alpha}20 and the sequential loop are never present and thus this arginine is considered to be a specific residue for only the monomeric IDHs (Fig. 4). These findings suggest that, in contrast to the conservation of most of the substrate and metal ion binding residues in all of the IDHs, the monomeric and dimeric IDHs might have independently acquired NADP+ specificity.



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  		FIG. 5.
Structure-based sequence alignment among {beta}-decarboxylating dehydrogenases. Only regions including substrate, metal ion, and NAD(P)+ binding residues (boldface letters) are shown. The letters boxed in gray are residues for NAD(P)+ recognition. The active residues in parentheses actually participate in the other active site. AvIDH, NADP+-dependent monomer; CmIDH, NADP+-dependent monomer; PcIDH, eukaryotic NADP+-dependent homodimer; CsIDH, Saccharomyces cerevisiae IDH (eukaryotic NADP+-dependent homodimer); EcIDH, prokaryotic NADP+-dependent homodimer; BsIDH, prokaryotic NADP+-dependent homodimer; CsIDH2, S. cerevisiae IDH subunit 2 (eukaryotic NAD+-dependent hetero-oligomer); HmIDH{alpha}, human IDH {alpha} subunit (eukaryotic NAD+-dependent hetero-oligomer); TtIPMDH, NAD+-dependent homodimer; and EcIPMDH, E. coli IPMDH (NAD+-dependent homodimer). Of these enzymes, the structures of the AvIDH, EcIDH, BsIDH, PcIDH, TtIPMDH, and EcIPMDH are already determined. The alignment was partly performed using the program ClustalW (63).

 

Catalysis—The general kinetic mechanism of the IDH can be divided into five steps as follows: (i) the formation of enzyme-substrate-cofactors, ES Michaelis complex, and the abstraction of hydroxyl proton at C2 of isocitrate; (ii) hydride transfer from isocitrate to NADP+ to generate oxalosuccinate and NADPH; (iii) decarboxylation of oxalosuccinate; (iv) dissociation of CO2; and (v) dissociation of enzyme, product, and cofactors. Steps ii-iv proceed very rapidly (22). Now the two structures of the AvIDH-isocitrate-Mn2+ and -NADP+ complex allow us to discuss the catalytic mechanism of the monomeric IDH. The bound NADP+ interacted only with residues of domain I, and the domain structure of two complexes could be superimposed very well with low r.m.s.d. These findings reliably provided us with the model of AvIDH-isocitrate-Mn2+-NADP+, putative initial ES Michaelis complex, by superimposing domain I of the NADP+ complex on that of the substrate complex and then adding the NADP+ molecule to the AvIDH-isocitrate-Mn2+ complex.

A schematic diagram showing the interactions of the cofactor NADP+, isocitrate-Mn2+, and nearby residues is given in Fig. 6. The initiation of the catalytic reaction is the proton abstraction of the C2 hydroxyl of isocitrate. In the structure of the EcIDH, Asp283 was suggested as a proton acceptor (15). In the case of the AvIDH, the residue corresponding to the Asp283 of the EcIDH is Asp350. However, this aspartate residue cannot be considered as a proton acceptor, because this carboxyl oxygen already coordinated with the Mn2+. The most likely proton acceptor in the AvIDH structure is a water molecule, w843 (the number is given by the PDB file of 1ITW [PDB] ), by forming a hydrogen bond to the C2 hydroxyl of isocitrate with a proper angle. The w843 hydrogen-bonded to two waters, w190 and w1378, and the w190 further formed a hydrogen bond with another carboxyl oxygen of Asp350. The well ordered hydrogen-bonding network of water molecules must relay the proton to the carboxyl of Asp350 or to the solvent region. The manner by which this hydrogen-bonding network is constructed resembles that of the eukaryotic PcIDH (25).

Following the formation of the ES Michaelis complex and the abstraction of C2 hydroxyl proton, stereospecific hydride transfer occurs from C2 of isocitrate to C4 of NADP+ nicotinamide ring with direct trajectory overlapping (18, 46, 48). The putative model of the ES Michaelis complex showed that the distance between C2 of isocitrate and C4 of nicotinamide ring was 4.2 Å, and that the angle of N-C4-C2 was 121.8°. These values are consistent with those obtained from the theoretical calculation (49, 50) and various structural analyses of the hydride-transferring enzyme in complex with NAD(P)+ (21, 51-54).

Because the hydride-transferring and decarboxylating steps proceed very rapidly, the stability of the ES Michaelis complex is considered to be one of the most important factors to dominate the turnover rate of IDHs. Until today, three structures of the EcIDH complexed with NADP+ have been deposited in PDB (PDB accession codes: 1AI2 [PDB] , 1IDE [PDB] , and 9ICD [PDB] ). The structure 9ICD [PDB] is the enzyme-NADP+ binary complex, the 1AI2 [PDB] is the enzyme-isocitrate-Ca2+-NADP+ pseudo-Michaelis complex, and the 1IDE [PDB] is a steady-state Y160F mutant complexed with isocitrate-Mg2+ and NADP+ by the Laue determination. In the structure of 9ICD [PDB] , the electron density corresponding to the nicotinamide mononucleotide of the NADP+ was completely disordered (15), whereas in structures of 1AI2 [PDB] and 1IDE [PDB] , the overall model of NADP+ was established (16, 21). These results demonstrated that the conformation of the positively charged nicotinamide ring moiety must be dependent on the presence of the negatively charged {gamma}-carboxyl of isocitrate. In contrast to the EcIDH, the clear electron density, corresponding to the whole NADP+ including the nicotinamide mononucleotide, could be detected in the present analysis. The structure revealed that the Ser87, Ser585, and Asn85 reliably interact with the nicotinamide ribose by the hydrogen bonds. Of these three residues, Ser87 and Asn85 are located at the loop between {beta}3 and {alpha}4, which is not present in the EcIDH structure. These interactions must stabilize the conformation of the nicotinamide mononucleotide moiety and fix the reaction point, even in the absence of substrate. Hence the hydride transfer reaction could occur immediately after the substrate is bound. This scheme of the immediate hydride transfer explains the exceptionally high turnover rate of the AvIDH.

Molecular Evolution—The {beta}-decarboxylating dehydrogenases are ubiquitous enzymes that have evolved divergently from the shared ancestral protein. So far, phylogenetic analysis has revealed that IDHs could be classified into three phylogenetic subfamilies (55-57). Subfamily I primarily comprises archaeal and eubacterial dimeric NADP-IDHs, subfamily II comprises eukaryotic dimeric NADP-IDHs, and subfamily III comprises eukaryotic hetero-oligomeric NAD-IDHs. In this analysis, however, the monomeric IDHs were excluded because a significant sequence similarity was not observed. We have recently shown that the monomeric IDHs have evolved from the ancestor that forms stable homodimers like present-day dimeric IDHs (34). Since the monomeric IDH has arisen through partial gene duplication, the cumulative substitutions, insertions, and deletions occurred to preserve its catalytic ability and to restore the thermodynamically stable folding. Hence the significant sequence alignment between monomeric and dimeric IDHs cannot be performed with the exception of a few narrow regions including substrate and metal ion binding residues.

The present structure of the AvIDH-NADP+ complex together with the previously reported structure of the AvIDH-isocitrate-Mn2+ complex demonstrated all of the residues that participate in the substrate and cofactor recognition as well as the overall folding topology, allowing us to compare the IDHs in subfamily I (EcIDH and Bacillus subtilis IDH (BsIDH) (58)) and in subfamily II (PcIDH) whose structures have already been determined. Surprisingly, several structural features suggested that the AvIDH is more closely related to the eukaryotic PcIDH rather than the bacterial EcIDH or BsIDH. First, the conformation of the bound substrate and the spatial configuration of the substrate-binding residues and the peripheral water molecules are remarkably similar between AvIDH and PcIDH. In contrast, the structure of the substrate and substrate-binding residues in the EcIDH and BsIDH cannot be very well superimposed on that in the AvIDH. Second, the {alpha}-helix ({alpha}20) that plays a partial role in NADP+ recognition is also conserved in the PcIDH while it is alternated with a short loop in the EcIDH and BsIDH. Third, the intertwining two Greek key motifs are completely conserved in both the AvIDH and PcIDH structures. Structure-based sequence alignment shows that several residues in the Greek key motif regions are conserved between AvIDH and PcIDH. In the cases of EcIDH and BsIDH, this motif is partly replaced by {alpha}-helices and is named the clasp-like domain (Fig. 7). A DALI structural similarity search (59) also revealed that the AvIDH is more structurally similar to the PcIDH (PDB code 1LWD [PDB] ; Z score = 21.7; r.m.s.d. = 2.2 Å for 255 C{alpha} atoms) than the EcIDH (PDB code 1ISO [PDB] ; Z score = 16.2; r.m.s.d. = 14.0 Å for 405 C{alpha} atoms). These findings imply that the monomeric IDHs might have branched off from the ancestor of the present-day eukaryotic NADP-IDHs.



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  		FIG. 7.
Structural comparison among AvIDH, PcIDH, and EcIDH. A, clasp region of AvIDH, PcIDH, and EcIDH. In both AvIDH and PcIDH structures, the clasp region is formed with symmetrically intertwining two Greek key motifs. The structures colored red represent another subunit. B, structure-based sequence alignment between Greek key motif regions of the PcIDH and AvIDH. The identical residues are colored red. The alignment was partly performed using the program ClustalW (63). C, the NADP+ recognition {alpha}-helix of the AvIDH superimposed on that of the PcIDH and EcIDH. The AvIDH is colored green, PcIDH is colored red, and EcIDH is colored blue. In the EcIDH structure, the {alpha}-helix is replaced with a short loop. The panels A and C are prepared using the program MOLSCRIPT (60) and Raster3D (61).

 


   FOOTNOTES
 
The atomic coordinates and structure factors (code 1j1w) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

* This work was supported in part by a grant-in-aid for Scientific Research and a research grant from the National Project on Protein Structural and Functional Analyses from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Present address: Institute of Advanced Energy, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan. Back

§ To whom correspondence should be addressed. Tel.: 81-11-706-3221; Fax: 81-11-706-4905; E-mail: tanaka{at}castor.sci.hokudai.ac.jp.

1 The abbreviations used are: IDH, isocitrate dehydrogenase; IPMDH, 3-isopropylmalate dehydrogenase; NADP-IDH, NADP+-dependent isocitrate dehydrogenase; NAD-IDH, NAD+-dependent isocitrate dehydrogenase; AvIDH, A. vinelandii IDH; EcIDH, E. coli IDH; PcIDH, porcine mitochondrial IDH; TtIPMDH, T. thermophilus IPMDH; BsIDH, B. subtilis IDH; PDB, Protein Data Bank; r.m.s.d., root-mean-squared deviation; w, water; ES, enzyme-substrate. Back

2 M. Yao, Y. Yasutake, and I. Tanaka, manuscript in preparation. Back


   ACKNOWLEDGMENTS
 
We thank M. Kawamoto and H. Sakai of the Japan Synchrotron Radiation Research Institute (JASRI) for their kind help in the x-ray diffraction experiment at the beamline BL41XU, SPring-8.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
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