Crystal Structure of Tetrameric Homoisocitrate ...the hanging-drop vapor diffusion method (28). Three microliters of 10-mg/ml puriﬁed TtHICDH was added to an equal volume of the

JOURNAL OF BACTERIOLOGY, Oct. 2005, p. 6779–6788 Vol. 187, No. 190021-9193/05/$08.00�0 doi:10.1128/JB.187.19.6779–6788.2005Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Crystal Structure of Tetrameric Homoisocitrate Dehydrogenase froman Extreme Thermophile, Thermus thermophilus: Involvement of

Hydrophobic Dimer-Dimer Interaction in ExtremelyHigh Thermotolerance

Junichi Miyazaki,1 Kuniko Asada,1 Shinya Fushinobu,2 Tomohisa Kuzuyama,1and Makoto Nishiyama1*

Biotechnology Research Center1 and Department of Biotechnology,2 The University of Tokyo,1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

Received 28 May 2005/Accepted 18 July 2005

The crystal structure of homoisocitrate dehydrogenase involved in lysine biosynthesis from Thermus ther-mophilus (TtHICDH) was determined at 1.85-Å resolution. Arg85, which was shown to be a determinant forsubstrate specificity in our previous study, is positioned close to the putative substrate binding site andinteracts with Glu122. Glu122 is highly conserved in the equivalent position in the primary sequence of ICDHand archaeal 3-isopropylmalate dehydrogenase (IPMDH) but interacts with main- and side-chain atoms in thesame domain in those paralogs. In addition, a conserved Tyr residue (Tyr125 in TtHICDH) which extends itsside chain toward a substrate and thus has a catalytic function in the related �-decarboxylating dehydroge-nases, is flipped out of the substrate-binding site. These results suggest the possibility that the conformationof the region containing Glu122-Tyr125 is changed upon substrate binding in TtHICDH. The crystal structureof TtHICDH also reveals that the arm region is involved in tetramer formation via hydrophobic interactionsand might be responsible for the high thermotolerance. Mutation of Val135, located in the dimer-dimerinterface and involved in the hydrophobic interaction, to Met alters the enzyme to a dimer (probably due tosteric perturbation) and markedly decreases the thermal inactivation temperature. Both the crystal structureand the mutation analysis indicate that tetramer formation is involved in the extremely high thermotoleranceof TtHICDH.

Homoisocitrate dehydrogenase (HICDH) is the third en-zyme involved in lysine biosynthesis through �-aminoadipate(19) and is a member of a family of �-decarboxylating dehy-drogenases that includes isocitrate dehydrogenase (ICDH) inthe tricarboxylic acid cycle, 3-isopropylmalate dehydrogenase(IPMDH) in leucine biosynthesis, and tartrate dehydrogenasein vitamin production (2). It has been suggested that theseenzymes diverged from a common ancestral �-decarboxylatingdehydrogenase (4, 9, 34).

We have shown that in an extremely thermophilic bacte-rium, Thermus thermophilus HB27, lysine is synthesizedthrough �-aminoadipate, although lysine is synthesizedthrough diaminopimelate in most bacteria (13, 18, 20, 22, 23,31). We have previously characterized HICDH from T. ther-mophilus HB27 (TtHICDH) in detail and have shown that theenzyme has four unique features (19). The first is its substratespecificity. Although TtHICDH is essential for lysine biosyn-thesis in the bacterium, TtHICDH can catalyze the reactionwith isocitrate as a substrate; isocitrate is the substrate ofICDH at a 20-fold-higher efficiency than toward the nativesubstrate, homoisocitrate. This feature contrasts with HICDHfrom Saccharomyces cerevisiae, which utilizes only homoisoci-trate as a substrate (19). Moreover, the substrate specificity is

easily altered by a single mutation, Arg85Val, to lose activityfor isocitrate but to acquire activity for 3-isopropylmalate, asubstrate of IPMDH. The second feature is the unique subunitorganization. TtHICDH is a homotetramer, while most �-de-carboxylating dehydrogenases are homodimers. Although afew members of �-decarboxylating dehydrogenases are re-ported to be tetramers (26, 27, 33), the interactions necessaryfor tetramer formation have not been elucidated at all. Thethird characteristic is the thermotolerance of TtHICDH, whichis substantially higher than for IPMDH and ICDH from T.thermophilus. The fourth is the phylogenetic location of thisenzyme. TtHICDH is positioned in the deepest branch in thephylogenetic tree constructed for �-decarboxylating dehydro-genases, suggesting that the enzyme may possess features ofthe ancestral enzyme. Thus, the crystal structure of TtHICDHwill provide a better understanding of a wide variety of bio-chemical and evolutionary characteristics.

In this paper, we report the crystal structure of TtHICDH.The structural analysis reveals that TtHICDH is stabilized by adimer-dimer interaction, which was confirmed by a single mu-tation weakening the tetramerization. Based on its crystalstructure and mutational analysis, we also describe possiblelocal conformational changes upon substrate binding inTtHICDH.

MATERIALS AND METHODS

Crystallization of TtHICDH. Overexpression and purification of TtHICDHwere performed as described previously (19). Crystallization was carried out by

* Corresponding author. Mailing address: Biotechnology ResearchCenter, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. Phone: 81-3-5841-3074. Fax: 81-3-5841-8030. E-mail:[email protected].

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the hanging-drop vapor diffusion method (28). Three microliters of 10-mg/mlpurified TtHICDH was added to an equal volume of the reservoir solutioncontaining 24% polyethylene glycol 400 (PEG400) and 0.1 M citrate, pH 4.8.Then, 3 �l of the solution containing 100 mM MgCl2, 5 mM isocitrate orhomoisocitrate, and 1 �l of 50 mM CdCl2 was added to the drop. The mixturewas equilibrated against 500 �l of the reservoir solution at 20°C. Crystals weregrown in 5 days, with a typical dimension of 0.5 mm by 0.5 mm by 0.05 mm.Before data collection, to avoid freezing, the crystals were transferred to acryoprotectant solution containing 25% PEG400, 0.1 M MgCl2, 5 mM CdCl2, 0.1M citrate, and 5 mM isocitrate or homoisocitrate.

Data collection. Data were collected with an ADSC-Q210 charge-coupled-device camera on the NW12 station of the Photon Factory-AR, High EnergyAccelerator Research Organization (KEK), Japan. All data were collected at 100K on a single crystal mounted in a cryoloop. Diffraction images were indexed,integrated, and scaled using the HKL2000 program (24). The crystal ofTtHICDH belongs to the space group C2221, with the following unit cell dimen-sions: a of 60.96 Å, b of 143.24 Å, and c of 177.85 Å. The data collection andprocessing statistics are given in Table 1. The data collected at the wavelength of1.85 Å were used for subsequent molecular replacement and crystallographicrefinement of TtHICDH.

Structure determination and refinement. Molecular replacement was per-formed with MOLREP (29) in the CCP4 program suite, using the three-dimen-sional structure of the Ala172Val mutant of IPMDH from T. thermophilus(Protein Data Bank [PDB] identifier [ID], 1G2U) (25) as a model. Modelcorrection in the electron density map was performed with the XtalView pro-gram suite (16). Refinement was carried out with CNS1.1 (1). The refinementstatistics are given in Table 1. Although electron density for chain A was clearlyseen throughout the polypeptide, the density for chain B was partly ambiguous,especially for the two regions of residues 72 to 76 and 264 to 265. The final modelof the TtHICDH crystal structure contains all the residues of chain A and mostresidues of chain B except for residues 72 to 76 and 264 to 265 in an asymmetricunit.

Construction of the mutants. Val135Met was a mutation obtained during thecourse of a directed evolution screening, which we will publish in the near futureelsewhere. To construct a mutant of TtHICDH containing a single mutation, theVal135Met protein, an hicdh gene resulting in the mutation Val135Met wasdigested with SmaI and EcoRI and the yielded fragment was introduced into thecorresponding portion of the TtHICDH expression plasmid pETtHICDH101(19). The resultant expression plasmid was named pETtHV135M.

The Tyr125Ala mutant was constructed as follows. Two independent PCRsusing 19HICN (5�-CCCCAAGCTTCCGGAGGTGAACATATGGCGTACCG

GATCTGCTTGATT-3�) and Y125A-1 (5�-GCGCCTTTCCTGCTCCACCGCAAGCCCTTC-3�) and Y125A-2 (5�-GAAGGGCTTGCGGTGGAGCAGGAAAGGCGC-3�) and HICDHCt (5�-TTTGAATTCTTACAGGCTCTTGAGCGCCTC-3�) as primer pairs were performed. PCR conditions used were as follows:(step 1) 94°C for 2 min, (step 2) 94°C for 1 min, (step 3) 65°C for 1 min, (step 4)72°C for 1 min, and (step 5) 72°C for 7 min; steps 2 to 4 were repeated 30 times.A portion of each reaction mixture was mixed and subjected to additional PCRusing 19HICN and HICDHCt as primers. The PCR conditions were as follows:(step 1) 94°C for 10 min, (step 2) 94°C for 1 min, (step 3) 65°C for 1 min, (step4) 72°C for 2 min, and (step 5) 72°C for 7 min; steps 2 to 4 were repeated 30times. The amplified fragment was digested with HindIII and EcoRI and clonedinto pUC119. After verification of the sequence, the NdeI- plus EcoRI-digestedfragment of the plasmid was ligated into pET26b (�) (Novagen, Darmstadt,Germany). The resulting plasmid, pETtHY125A, was used for the expression ofthe Tyr125Ala mutant.

Expression, purification, and determination of quaternary structures ofTtHICDH and the mutant. Overexpression and purification of the mutant werecarried out by the method for wild-type TtHICDH as described previously (19)except for the heat treatment step, which was carried out at 70°C in place of 85°Cfor wild-type TtHICDH. Sedimentation equilibrium analysis was carried outusing 100 �l of 1-mg/ml enzymes with a Beckman Optima XL-A analyticalultracentrifuge fitted with a Beckman An-60Ti analytical rotor. The subunitorganization of each enzyme was analyzed according to the procedure of VanHolde and Baldwin (30) using Origin 2.8 scientific graphing and data analysis andXL-A 2.01 data analysis on a Windows operating system. Partial specific volumesfor the wild-type, Val135Met, and Tyr125Ala proteins were calculated to be0.745, 0.744, and 0.745, respectively, from the amino acid compositions. Molec-ular mass calibration by gel filtration was carried out using a Superdex200prepgrade column (Amersham Bioscience, Tokyo, Japan) at a 2.5-ml/min flowrate.

Thermal inactivation of enzymes. The thermal-inactivation temperatures ofthe enzymes were analyzed as follows. The purified enzymes (0.5 mg/ml) dis-solved in buffer (20 mM potassium phosphate, pH 7.5, 0.5 mM EDTA, 0.2 MNaCl) were incubated at various temperatures for 20 min and immediatelychilled in ice water. Then, aggregated proteins were removed by centrifugation(23,500 � g for 5 min), and the remaining activity in the supernatant wasmeasured. One microliter of the enzyme solution was added to the reactionmixture (50 mM HEPES, pH 8.0, 200 mM KCl, 5 mM MgCl2, 1 mM NAD�, and400 �M isocitrate) that was preincubated at 60°C for 5 min. The reaction wasmonitored at 60°C by following the increase in absorbance at 340 nm.

Kinetic analysis of enzymes. In the kinetic analysis, we used isocitrate insteadof homoisocitrate as the substrate for TtHICDH because homoisocitrate is notcommercially available at present. Five microliters of enzymes (0.1 mg/ml) wasadded to 1 ml of assay buffer (50 mM HEPES, pH 7.5, 200 mM KCl, 5 mMMgCl2, 1 mM NAD�, and 0.1 to 4 mM isocitrate), which had been preincubatedat 60°C for 5 min. The reaction was monitored at 60°C by following the increasein absorbance at 340 nm. Data were analyzed by Cleland’s initial velocity pro-gram Hyper (3).

Graphics manipulation. All of Fig. 1, 2, and 3 were generated using theprograms MOLSCIPT (14), RASTER3D (17), and XtalView (16).

Protein data bank accession number. The atomic coordinates and structurefactors of TtHICDH have been deposited in the RCSB Protein Data Bank withaccession number 1X0L.

RESULTS

Crystal structure of TtHICDH. TtHICDH crystallizes usingPEG400 as the precipitant in the space group C2221. There aretwo monomers in the crystallographic asymmetric unit (Table1; Fig. 1A). The crystal diffracts to 1.85 Å. The three-dimen-sional structure of IPMDH from T. thermophilus HB8 with theAla172Val mutation (25), which has 44% identity in aminoacid sequence to TtHICDH, was used as the search model inmolecular replacement. The final structure contains all the sidechains in subunit A, but the side chains of the regions withresidues 72 to 76 and 264 to 265 in subunit B are omittedbecause of the low electron density due to disorder in theseregions. In addition, we could not find the substrate and Mg2�

ion that were added to the crystallization solution and cryo-

TABLE 1. Data collection and refinement statistics

Statistics Parameter Determination

Data collection Beamline NW12Space group C2221Cell dimensions

a (Å) 60.963b (Å) 143.243c (Å) 177.846

Resolution limit (Å) 50.0–1.85 (1.92–1.85)Total no. of reflections 272,815No. of unique reflections 66,618Redundancy 4.1I/�(I) 29.7 (4.2)Completeness (%) 99.9 (100)Rmerge(%) 5.2 (25.9)Overall B factor from Wilson 22.2

Refinement Resolution (Å) 47.44–1.85R factor (%) 21.6Rfree(%) 24.8No. of protein atoms 4,983No. of water atoms 378RMSD from ideal values

Bond length (Å) 0.010Bond angles (°) 1.4Dihedral angles (°) 22.5

Averaged B factor (Å2) 27.4Chain A (Å2) 21.8Chain B (Å2) 32.2Solvent atoms (Å2) 33.6

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protectant in the electron density map. Three hundred seventy-eight water molecules are contained in the asymmetric unit ofthe cell of TtHICDH. The final R factor of refined structure ofTtHICDH is 21.6%, and free R is 24.8%.

The structure of the TtHICDH monomer is composed oftwo domains separated by the substrate-binding pocket and isvery similar to those of IPMDH from T. thermophilus HB8(TtIPMDH; PDB ID, 1IPD) (8), IPMDH from Thiobacillusferrooxidans (TfIPMDH; PDB ID, 1A05) (7), and ICDH fromEscherichia coli (EcICDH; PDB ID, 1AI2) (6), with root meansquare deviations (RMSD) of 1.69 Å, 1.39 Å, and 1.65 Å forthe �-carbons, respectively (Fig. 1B). Although the crystalstructure of TtHICDH contains neither isocitrate nor ho-moisocitrate in the putative substrate-binding site, the confor-mation of TtHICDH is more similar to that of substrate-bound

TfIPMDH in a closed conformation (RMSD of 1.40 Å) than tothat of TtIPMDH in an open conformation (RMSD of 1.72 Å)(Fig. 1B and C). This indicates that TtHICDH adopts a closed-like conformation even without bound substrate.

Substrate-binding site. Because of the better quality, wedescribe the structural detail only for the A subunit ofTtHICDH. The closed-like conformation of the crystal struc-ture of TtHICDH allowed us to elucidate the structural basisfor the substrate specificity in TtHICDH by comparison of itsstructure with those of TfIPMDH and EcICDH, whose struc-tures were determined in the closed forms. Homoisocitrate,isocitrate, and 3-isopropylmalate, which are the substrates ofHICDH, ICDH, and IPMDH, respectively, contain a commonmalate moiety in their structures. The amino acid residues thatrecognize the moiety are conserved Arg88, Arg98, Arg118,

FIG. 1. Crystal structure of TtHICDH. A, TtHICDH subunits A and B colored by hot pink and pale pink, respectively, in an asymmetric unit.B, Superposition of two subunits colored by hot pink and pale pink in an asymmetric unit. C, Superposition of TtHICDH (hot and pale pink) onan open-form IPMDH dimer (TtIPMDH, gray) from T. thermophilus. D, Superposition of TtHICDH and closed-form IPMDH dimer (TfIPMDH,green) from T. ferrooxidans. Putative active-site pockets are marked with “active site” only for subunit A, except in panel B.

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Lys171, Asp204, Asp228, and Asp232 in TtHICDH and occupyspatial positions similar to those in EcICDH and TfIPMDH(Fig. 2B and C) (6, 7). Substrate-bound complex structures ofICDH and IPMDH revealed that the substrate specificity isdetermined by the residues in the loop between strand 3 andhelix 4 (2, 5, 32). We previously reported that Arg85 is one ofthe most important residues for substrate recognition inTtHICDH (19). The crystal structure reveals that Arg85 islocated in the substrate-binding pocket but extends its sidechain toward Glu122 in another domain of the same subunit(Fig. 2A to C). The roles of Arg85 and Glu122 are described indetail in Discussion.

Subunit interface of TtHICDH. TtHICDH is a homotet-rameric �-decarboxylating dehydrogenase which is differentfrom the generally homodimeric �-decarboxylating dehydro-genases. In an asymmetric unit of the TtHICDH crystal, thereare only two subunits. Considering the symmetry of the crys-tals, we could easily identify another intersubunit interaction(Fig. 3A). In the tetramer, two dimers have head-to-head in-teraction, with their arm regions composed of four �-strandsfrom a dimer. Interestingly, only a small number of hydrogen-bonding interactions contributes to the tetramer formation. Onthe other hand, the tetramer formation is highly stabilized byhydrophobic interaction. the Val135 mutant interacts withTyr125 and Val141 mutants from a different subunit of thefunctional dimer and Tyr132 and Leu133 from another dimer(Fig. 3B and C).

Effects of mutation affecting the interdimeric interaction onthe quaternary structure of TtHICDH. We isolated a mutantcontaining Val135Met during the course of a different exper-iment of directed evolution (we will report the results else-where in the near future). Since the mutant contained multipleamino acid substitutions, we constructed a mutant containingonly a single Val135Met mutation. The quaternary structuresof wild-type TtHICDH and the mutant were analyzed by twodifferent methods, gel filtration and sedimentation equilibriumcentrifugation. We first carried out molecular size calibrationusing a Hi-load Superdex200 prepgrade gel filtration column(Fig. 4). Elution volumes of TtHICDH and the Val135Metenzyme were 172.3 ml and 194.6 ml, respectively (Fig. 4A),corresponding to 3.21 and 1.74 subunits, respectively, in asingle molecule (Fig. 4B). To confirm the quaternary structurefurther, we also analyzed the structure by sedimentation equil-ibration. Data were fitted best with the dimer-tetramer equi-librium for both wild-type TtHICDH and the Val135Met mu-tant (Table 2). However, TtHICDH is present mostly as atetramer, and the Val135Met mutant is a dimer at 1.0 mg/ml.These results indicate that Val135 in TtHICDH plays a role intetramerization in TtHICDH.

Heat tolerance of TtHICDH and the mutants. Since thedetermined three-dimensional structure of TtHICDH suggeststhat tetramerization may contribute to its substantially higher

thermal inactivation temperature, the thermotolerance of themutant was analyzed by measuring the remaining activity afterthe purified enzymes were incubated at various temperaturesfor 20 min. Distinct activity (10%) remained even after incu-bation at 100°C for wild-type TtHICDH. On the other hand,the Val135Met mutant completely lost the activity after theincubation at 90°C (Fig. 5). Thermal-inactivation temperaturesto show half of the initial activities were estimated to be ap-proximately 97.6°C and 84.0°C for wild-type TtHICDH and theVal135Met mutant, respectively. These results indicate thattetramer formation plays a critical role in the extreme thermal-inactivation temperature.

Kinetic properties of TtHICDH and the mutants. Becauseof the shortage of homoisocitrate, which is not commerciallyavailable now, in our reagent stock, we carried out the kineticassay of the enzymes using isocitrate as a substrate. The Km

values of TtHICDH and the Val135Met mutant for isocitratewere 521 � 42 �M and 161 � 18 �M, respectively, and the kcat

values were 438 � 14 s1 and 211 � 6 s1 for TtHICDH andthe Val135Met mutant, respectively (Table 3). When calcu-lated per monomer, the kcat values for the wild type and mu-tant were 110 and 106, respectively, indicating that the catalyticmechanism in TtHICDH is the same in the Val135Met mutant.The kcat/Km values, showing the catalytic efficiency, for isocit-rate are 0.84 s1 M1 and 1.3 s1 M1, indicating that theactivity is enhanced by 1.6-fold through tetramer-to-dimerstructural transition generated by the Val135Met mutant andthat the improvement is attributed to the decrease in Km forthe substrate.

Effect of mutation at Tyr125 on catalytic activity and tet-ramer formation. The Tyr residues Tyr140 and Tyr160 inTfIPMDH and EcICDH, respectively, are conserved amongrelated �-decarboxylating dehydrogenases. In the crystal struc-tures of TfIPMDH and EcICDH, the Tyr residue extends itsside chain toward the substrate to perform dehydrogenation ofthe substrate and/or protonation of the substrate after decar-boxylation (11). A Tyr residue (Tyr125) is conserved at thecorresponding position in the primary sequence of TtHICDH.However, the Tyr residue extends its side chain in the oppositedirection (Fig. 2B and C), contributing to the hydrophobicinteraction necessary for tetramer formation (Fig. 3). To elu-cidate the role of Tyr125 in catalytic function, tetramer forma-tion, and thermostability in TtHICDH, we constructed themutant TtHICDH with replacement of Tyr125 by Ala. Kineticanalysis showed that the mutation significantly decreased thekcat value (0.1 s1) by eightfold but that the Km value forisocitrate slightly increased (707 � 97 �M) (Table 3). Theresult suggests that Tyr125 is involved in the catalytic processof this enzyme. On the other hand, analysis of the quaternarystructure of the mutant by both gel filtration and sedimenta-tion equilibrium analysis (Fig. 4; Table 2) showed that themutant is a homotetramer since the elution volume was 164.5

FIG. 2. Substrate-binding site of TtHICDH. A, Stereoview of the substrate-binding site of TtHICDH with electron density map. B, Superpo-sition of the substrate-binding site of TtHICDH (hot pink) with isocitrate-bound ICDH from E. coli (cyan; PDB ID, 1AI2). Amino acid residuesof TtHICDH and ICDH are indicated by bold and light letters, respectively. C, Superposition of the substrate-binding site of TtHICDH with3-isopropylmalate-bound IPMDH from T. ferrooxidans (green; PDB ID, 1A05). Amino acid residues of TtHICDH and IPMDH are indicated bybold and light letters, respectively. Strand 3 and helix 4 are marked in panels B and C.


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ml, corresponding to 4.00 subunits organized in a single mol-ecule and since only a monomer-tetramer model fit with thesedimentation equilibrium. The mutant showed a thermal in-activation curve similar to that of wild-type TtHICDH (Fig. 5).These results indicate that the mutation affected neither theoligomeric state nor the thermotolerance of TtHICDH butaffected catalytic function.

DISCUSSION

In our previous study, we showed that the substrate speci-ficity of TtHICDH was dramatically altered by the Arg85Valmutation (19). The Arg85Val mutant lost activity towards iso-citrate but interestingly showed activity towards 3-isopropyl-malate, the substrate of IPMDH that is not normally recog-nized by TtHICDH. Superposition of the structure ofTtHICDH with those of EcICDH and TfIPMDH indicatesthat Arg85 is found at a position close to the -moiety of thesubstrates, occupies a position similar to that of the corre-sponding Leu92 in TfIPMDH, and is positioned 1.6 Å (for

�-carbons) away from Val116, the counterpart of Arg85, inEcICDH (Fig. 2B and C). Therefore, we assume that replace-ment of Arg85 with Val may increase hydrophobicity in theregion, which may facilitate binding of 3-isopropylmalate andprevent isocitrate from binding.

The crystal structure of TtHICDH indicates that the guani-dyl group of the Arg85 side chain interacts with Glu122 fromanother domain in the same subunit. Glu122 is highly con-served in the primary structures of most HICDHs, ICDHs, andarchaeal IPMDHs, although its function has not yet been char-acterized. In EcICDH, the equivalent residue, Glu157, inter-acts with the main-chain nitrogen of Phe190 in the same do-main and is not directed towards the substrate-binding site inboth apo and holo forms of EcICDH and TtIPMDH (8, 11)(Fig. 2B and C). Therefore, it is unlikely that Glu157 inEcICDH is involved in catalysis. In TtHICDH, however,Glu122 is flipped towards the substrate-binding site to interactwith Arg85 electrostatically (Fig. 2A to C).

Another feature characteristic of the TtHICDH structure is

FIG. 4. Gel filtration analysis of TtHICDHs. A, Elution profiles of TtHICDH, the Tyr125Ala mutant, and the Val135Met mutant in aSuperdex200 prepgrade column; B, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of TtHICDH and the mutants. Lane 1, molecularmass markers; lane 2, purified TtHICDH; lane 3, purified Tyr125Ala protein; lane 3, purified Val135Met protein. Molecular mass markers usedwere phospholyrase b (97 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (30 kDa), and soybean trypsin inhibitor(20 kDa). C, Molecular size calibration using Superdex200 prepgrade gel filtration with a marker protein; 1, aldolase (158 kDa); 2, bovine serumalbumin (67 kDa); 3, ovalbumin (43 kDa); 4, RNase A (13.7 kDa).

FIG. 3. Subunit interaction of TtHICDH. A, Overall structure of the TtHICDH tetramer. B, Stereoview of the electron density map in theinterface. C, Close-up view of dimer-dimer interface. In panel C, a half of the amino acid residues involved in hydrophobic dimer-dimer interactionare shown. Subunits A, B, C, and D are colored by green, gold, hot pink, and blue, respectively.


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found at Tyr125, a conserved residue among related �-decar-boxylating dehydrogenases. The phenolic side chain of Tyr125occupies a position totally different from those of the corre-sponding Tyr residues in TfIPMDH and EcICDH. Neverthe-less, the Tyr125Ala mutation caused a substantial decrease inthe kcat value, as do corresponding mutations in EcICDH(Tyr160) and TtIPMDH (Tyr140) (11, 15, 21). This resultsuggests that Tyr125 is involved in the catalytic function ofTtHICDH. If the unusual orientation of Glu122 and Tyr125 ispresent under physiological conditions, we speculate that sub-strate binding causes a conformational change of the regioncontaining Glu122 and Tyr125 in TtHICDH as follows, basedon the crystal structure of TtHICDH and information on themutation effect of Tyr125. In this scenario, when a substratewith a negatively charged -moiety of a substrate, such ashomoisocitrate and isocitrate, binds to the substrate-bindingsite, the interaction between Arg85 and Glu122 in the apoform may be lost due to repulsion between the negativecharges of the substrate -moiety and Glu122. The positivecharge of Arg85 may interact with a carboxylate of the sub-strate -moiety. Glu122 then rotates its side chain to form ahydrogen bond, possibly with a main-chain nitrogen in the caseof EcICDH. Accompanied by the Glu122 rotation, Tyr125located on the same loop may be delivered to the substrate-binding site to play the catalytic role. Since the Tyr residuetakes the same position in ICDH and IPMDH even in an apo

form (7, 15), it is unlikely that the Tyr residue changes theorientation upon substrate binding in other �-decarboxylatingdehydrogenases. It should be noted that the pH level used forthe crystallization of TtHICDH was 4.8, which was lower thanthose for other �-decarboxylating dehydrogenases (pHs 5.4 to7.5). This may suggest the possibility that the unusual orienta-tion of Glu122 and Tyr125 may not be seen under physiologicalconditions. In any case, to understand the substrate recogni-tion mechanism, X-ray analysis of substrate-bound complex isobviously required.

TtHICDH is a tetrameric enzyme with extremely high ther-motolerance in comparison with TtICDH and TtIPMDH (12,19). The Val135Met mutation causes a significant decrease inthe thermotolerance, accompanied by the tetramer-to-dimertransition of the quaternary structure (Fig. 5). Therefore, theseresults indicate that the tetramer formation is one of the majorfactors ensuring the extremely high thermotolerance of thisenzyme. Val135 contributes to a hydrophobic environmentwith Tyr125 and Val141 from another subunit of the samedimer and Tyr132 and Leu133 from another dimer (Fig. 3Band C). The Tyr125Ala mutation would also weaken the hy-drophobic interaction in the dimer-dimer interface. However,the mutation apparently does not preclude tetramerization ofthe enzyme (Fig. 4A and C; Table 2). Although the Tyr125Alamutation was introduced at the position close to Val135, themutant still possesses high thermotolerance (Fig. 5). We as-sume that the large side chain of Met introduced at position135 would hinder the correct hydrophobic interaction betweenthe residues and therefore prevented TtHICDH from forminga tetramer, leading to lower thermotolerance. It should benoted that the activity of the dimeric TtHICDH mutant (theVal135Met mutant) is 1.6-fold higher than that of the tet-rameric wild-type TtHICDH because of the improved Km

value for isocitrate (Table 3), indicating that tetramerization ofTtHICDH may sacrifice some activity for obtaining higherthermotolerance.

It is known that ICDH from Thermotoga maritima (26) andIPMDH from Sulfolobus tokodaii 7 (StIPMDH) (27, 33) arealso tetrameric �-decarboxylating dehydrogenases, althoughmost �-decarboxylating dehydrogenases are dimers. Interest-ingly, the tetrameric �-decarboxylating dehydrogenases arefound only in hyperthermophiles and not in mesophiles.ICDHs from two hyperthermophilic archaea, Pyrococcus furio-sus and Aeropyrum pernix, are dimeric enzymes having thermo-stability (melting temperatures, �100°C) higher than that ofthe tetrameric ICDH from Thermotoga maritima, StIPMDH,and TtHICDH (26). In TtHICDH, the arm region forming the�-sheet is responsible for the tetramerization. However, inICDH from Aeropyrum pernix and EcICDH (6, 10), the armregion contains an additional �-helix and interacts with thesame region from another subunit to form a small domainnamed clasp, which may prevent these ICDHs from forming atetramer. Thus, the fact that two hyperthermophilic ICDHs aredimeric indicates that tetramerization is not the only way toacquire elevated thermostability. In tetrameric StIPMDH, thehydrophobic residues that correspond to Val135, Tyr132, andVal141 in TtHICDH are conserved as Val135, Val131, andIle141, respectively, indicating that this enzyme may have thesimilar hydrophobic dimer-dimer interactions to form a tet-ramer (27). The crystal structure of StIPMDH at a 2.8-Å res-

FIG. 5. Thermal inactivation of TtHICDHs. Remaining activity af-ter heat treatment at appropriate temperatures for 20 min. In eachfigure, TtHICDH, the Tyr125Ala mutant, and the Val135Met mutantare indicated by open circles, open diamonds, and open squares, re-spectively.

TABLE 2. Quaternary structure of TtHICDH and mutants

Enzyme

Equilibrium constant% Dimers: %

tetramers (monomer)at 1 mg/ml (%)

Monomer-dimer(M1)

Monomer-tetramer(M3)

TtHICDH 5.06 � 1069 4.16 � 10138 33.4:66.6Tyr125Ala mutanta 4.49 � 102 12.7:87.3Val135Met mutant 1.16 � 1030 1.50 � 1057 96.6:3.4

a The Tyr125Ala mutant is fitted with only the monomer-tetramer equilibrium.


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olution (which has recently been deposited in the RCSB Pro-tein Data Bank as 1WPW) indicates that StIPMDH is alsostabilized by a dimer-dimer interaction like that of TtHICDH.On the other hand, although ICDH from T. maritima is also atetrameric �-decarboxylating dehydrogenase (26), its aminoacid sequence in the arm region does not align well with thoseof TtHICDH and StIPMDH, suggesting that the enzyme has adifferent strategy to form a tetramer. Considering thatTtHICDH possesses ancestral features of �-decarboxylatingdehydrogenases, through billions of years of evolution, the roleof the arm region for tetramerization to enhance thermosta-bility may have been lost to enhance the activity for �-decar-boxylating dehydrogenation, especially in mesophilic organ-isms (Table 3).

In summary, TtHICDH, while structurally similar to othermembers of its family, forms a tetramer that is more thermo-tolerant than the Val135Met mutant, which forms dimers.Tyr125 forming the active site is found in a conformation thatis likely to change upon substrate binding and/or under phys-iological conditions. The observation that the Val135Met mu-tant is catalytically more active at the expense of thermotoler-ance suggests that other related enzymes may have evolvedthat way.

ACKNOWLEDGMENTS

We thank the staff of the Photon Factory for their assistance with thedata collection. The data collection was approved by the Photon Fac-tory Advisory Committee (proposal no. 2003G103). We also thankElinor T. Adman (Washington University School of Medicine) forproviding us useful comments for completing the manuscript.

This work was supported in part by grant-in-aid for scientific re-search from the Ministry of Education, Culture, Sports, Science andTechnology of Japan, from Noda Institute for Scientific Research, andfrom the Nagase Science and Technology Foundation.

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TABLE 3. Kinetic parameters of TtHICDH and mutants

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TtHICDH 521 � 42 440 � 14 110 8.4 � 101

Tyr125Ala mutant 707 � 97 73 � 3 18 1.0 � 101

Val135Met mutant 161 � 18 211 � 6 106 1.3 � 100


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