A rationally designed monomeric variant of anthranilate phosphoribosyltransferase from Sulfolobus solfataricus is as active as the dimeric wild-type enzyme but less thermostable

The anthranilate phosphoribosyltransferase from Sulfolobus solfataricus (ssAnPRT) forms a homodimer with a hydrophobic subunit interface. To elucidate the role of oligomerisation for catalytic activity and thermal stability of the enzyme, we loosened the dimer by replacing two apolar interface residues with negatively charged residues (mutations I36E and M47D). The purified double mutant I36E+M47D formed a monomer with wild-type catalytic activity but reduced thermal stability. The single mutants I36E and M47D were present in a monomer-dimer equilibrium with dissociation constants of about 1 μM and 20 μM, respectively, which were calculated from the concentration-dependence of their heat inactivation kinetics. The monomeric form of M47D, which is populated at low subunit concentrations, was as thermolabile as monomeric I36E+M47D. Likewise, the dimeric form of I36E, which was populated at high subunit concentrations, was as thermostable as dimeric wild-type ssAnPRT. These findings show that the increased stability of wild-type ssAnPRT compared to the I36E+M47D double mutant is not caused by the amino acid exchanges per se but by the higher intrinsic stability of the dimer compared to the monomer. In accordance with the negligible effect of the mutations on catalytic activity and stability, the X-ray structure of M47D contains only minor local perturbations at the dimer interface. We conclude that the monomeric double mutant resembles the individual wild-type subunits, and that ssAnPRT is a dimer for stability but not for activity reasons.     

Localisation of Plasmodium falciparum uroporphyrinogen III decarboxylase of the heme-biosynthetic pathway in the apicoplast and characterisation of its catalytic properties

Uroporphyrinogen decarboxylase (UROD) is a key enzyme in the heme-biosynthetic pathway and in Plasmodium falciparum it occupies a strategic position in the proposed hybrid pathway for heme biosynthesis involving shuttling of intermediates between different subcellular compartments in the parasite. In the present study, we demonstrate that an N-terminally truncated recombinant P. falciparum UROD (r(Δ)PfUROD) over-expressed and purified from Escherichia coli cells, as well as the native enzyme from the parasite were catalytically less efficient compared with the host enzyme, although they were similar in other enzyme parameters. Molecular modeling of PfUROD based on the known crystal structure of the human enzyme indicated that the protein manifests a distorted triose phosphate isomerase (TIM) barrel fold which is conserved in all the known structures of UROD. The parasite enzyme shares all the conserved or invariant amino acid residues at the active and substrate binding sites, but is rich in lysine residues compared with the host enzyme. Mutation of specific lysine residues corresponding to residues at the dimer interface in human UROD enhanced the catalytic efficiency of the enzyme and dimer stability indicating that the lysine rich nature and weak dimer interface of the wild-type PfUROD could be responsible for its low catalytic efficiency. PfUROD was localised to the apicoplast, indicating the requirement of additional mechanisms for transport of the product coproporphyrinogen to other subcellular sites for its further conversion and ultimate heme formation.     

Crystal structure of dimeric FabZ of Plasmodium falciparum reveals conformational switching to active hexamers by peptide flips

The crystal structure of beta-hydroxyacyl acyl carrier protein dehydratase of Plasmodium falciparum (PfFabZ) has been determined at a resolution of 2.4 A. PfFabZ has been found to exist as a homodimer (d-PfFabZ) in the crystals of the present study in contrast to the reported hexameric form (h-PfFabZ) which is a trimer of dimers crystallized in a different condition. The catalytic sites of this enzyme are located in deep narrow tunnel-shaped pockets formed at the dimer interface. A histidine residue from one subunit of the dimer and a glutamate residue from the other subunit lining the tunnel form the catalytic dyad in the reported crystal structures. While the position of glutamate remains unaltered in the crystal structure of d-PfFabZ compared to that in h-PfFabZ, the histidine residue takes up an entirely different conformation and moves away from the tunnel leading to a His-Phe cis-trans peptide flip at the histidine residue. In addition, a loop in the vicinity has been observed to undergo a similar flip at a Tyr-Pro peptide bond. These alterations not only prevent the formation of a hexamer but also distort the active site geometry resulting in a dimeric form of FabZ that is incapable of substrate binding. The dimeric state and an altered catalytic site architecture make d-PfFabZ distinctly different from the FabZ structures described so far. Dynamic light scattering and size exclusion chromatographic studies clearly indicate a pH-related switching of the dimers to active hexamers.     

Dimer–Dimer Interaction of the Bacterial Selenocysteine Synthase SelA Promotes Functional Active-Site Formation and Catalytic Specificity

The 21st amino acid, selenocysteine (Sec), is incorporated translationally into proteins and is synthesized on its specific tRNA (tRNA^Sec). In Bacteria, the selenocysteine synthase SelA converts Ser-tRNA^Sec, formed by seryl-tRNA synthetase, to Sec-tRNA^Sec. SelA, a member of the fold-type-I pyridoxal 5′-phosphate-dependent enzyme superfamily, has an exceptional homodecameric quaternary structure with a molecular mass of about 500 kDa. Our previously determined crystal structures of Aquifex aeolicus SelA complexed with tRNA^Sec revealed that the ring-shaped decamer is composed of pentamerized SelA dimers, with two SelA dimers arranged to collaboratively interact with one Ser-tRNA^Sec. The SelA catalytic site is close to the dimer–dimer interface, but the significance of the dimer pentamerization in the catalytic site formation remained elusive. In the present study, we examined the quaternary interactions and demonstrated their importance for SelA activity by systematic mutagenesis. Furthermore, we determined the crystal structures of “depentamerized” SelA variants with mutations at the dimer–dimer interface that prevent pentamerization. These dimeric SelA variants formed a distorted and inactivated catalytic site and confirmed that the pentamer interactions are essential for productive catalytic site formation. Intriguingly, the conformation of the non-functional active site of dimeric SelA shares structural features with other fold-type-I pyridoxal 5′-phosphate-dependent enzymes with native dimer or tetramer (dimer-of-dimers) quaternary structures.     

An efficient method to express GPI-anchor proteins in insect cells

Glycosylphosphatidylinositols (GPIs) constitute a class of glycolipids that have various functions, the most basic being to attach proteins to the surface of eukaryotic cells. GPIs have to be taken into account, when expressing surface antigens from parasitic protozoa in heterologous systems. The synthesis of the GPI-anchors was previously reported to be drastically decreased to almost background level following baculovirus infection. Here we describe a new method to express GPI-anchor proteins in insect cells relying on using of a supplementary baculovirus construct that overexpresses the N-acetylglucosaminyl phosphatidylinositol de-N-acetylase, the enzyme catalyzing the second step in the GPI biosynthetic pathway.     

Structural Basis of Heme Binding in the Cu,Zn Superoxide Dismutase from Haemophilus ducreyi

The Cu,Zn superoxide dismutase from Haemophilus ducreyi is characterized by the unique ability to bind heme at its dimer interface. Here we report the high-resolution crystal structures of this protein in the heme-loaded (holo) and heme-free (apo) forms. Heme is asymmetrically bound between the two enzyme subunits, where heme iron is coordinated by two histidine residues, His64 and His 124, provided by the two subunits. Moreover, the binding of heme to the protein is ensured by stabilizing contacts between the prosthetic group and a limited number of other residues, most of which are not present in other bacterial enzyme variants. We show that the introduction of only three mutations at the dimer interface of the enzyme from Haemophilus parainfluenzae, a closely related bacterial species, is sufficient to induce heme-binding ability by this enzyme variant. Heme binding does not alter protein activity. Moreover, the binding of the prosthetic group does not induce any significant structural perturbation at the subunit level and requires only limited local structural rearrangements that widen the cleft at the dimer interface and cause a limited shift in the relative orientation between the subunits. The presence of a preformed heme-binding pocket and the significant solvent exposure of the cofactor to the solvent are compatible with the suggested protective role of the enzyme against heme toxicity or with its involvement in heme trafficking in the periplasmic space.     

Proinflammatory responses by glycosylphosphatidylinositols (GPIs) of Plasmodium falciparum are mainly mediated through the recognition of TLR2/TLR1

The glycosylphosphatidylinositols (GPIs) of Plasmodium falciparum have been shown to activate macrophages and produce inflammatory responses. The activation of macrophages by malarial GPIs involves engagement of Toll like receptor 2 (TLR2) resulting in the intracellular signaling and production of cytokines. In the present study, we investigated the requirement of TLR1 and TLR6 for the TLR2 mediated cell signaling and proinflammatory cytokine production by macrophages. The data demonstrate that malarial GPIs, which contain three fatty acid substituents, preferentially engage TLR2–TLR1 dimeric pair than TLR2–TLR6, whereas their derivatives, sn-2 lyso GPIs, that contain two fatty acid substituents recognize TLR2–TLR6 with slightly higher selectivity as compared to TLR2–TLR1 heteromeric pair. These results are analogous to the recognition of triacylated bacterial and diacylated mycoplasmal lipoproteins, respectively, by TLR2–TLR1 and TLR2–TLR6 dimers, suggesting that the lipid portions of the microbial GPI ligands play essential role in determining their TLR recognition specificity.     

The C-terminal of CysM from Mycobacterium tuberculosis protects the aminoacrylate intermediate and is involved in sulfur donor selectivity

A new crystal structure of the dimeric cysteine synthase CysM from Mycobacterium tuberculosis reveals an open and a closed conformation of the enzyme. In the closed conformation the five carboxy-terminal amino acid residues are inserted into the active site cleft. Removal of this segment results in a decreased lifetime of the α-aminoacrylate reaction intermediate, an increased sensitivity to oxidants such as hydrogen peroxide, and loss of substrate selectivity with respect to the sulfur carrier thiocarboxylated CysO. These results highlight features of CysM that might be of particular importance for cysteine biosynthesis under oxidative stress in M. tuberculosis.     

First structures of an active bacterial tyrosinase reveal copper plasticity

Tyrosinase is a member of the type 3 copper enzyme family that is involved in the production of melanin in a wide range of organisms. The crystal structures of a tyrosinase from Bacillus megaterium were determined at a resolution of 2.0-2.3 Å. The enzyme crystallized as a dimer in the asymmetric unit and was shown to be active in crystal. The overall monomeric structure is similar to that of the monomer of the previously determined tyrosinase from Streptomyces castaneoglobisporus, but it does not contain an accessory Cu-binding “caddie” protein. Two Cu(II) ions, serving as the major cofactors within the active site, are coordinated by six conserved histidine residues. However, determination of structures under different conditions shows varying occupancies and positions of the copper ions. This apparent mobility in copper binding modes indicates that there is a pathway by which copper is accumulated or lost by the enzyme. Additionally, we suggest that residues R209 and V218, situated in a second shell of residues surrounding the active site, play a role in substrate binding orientation based on their flexibility and position. The determination of a structure with the inhibitor kojic acid, the first tyrosinase structure with a bound ligand, revealed additional residues involved in the positioning of substrates in the active site. Comparison of wild-type structures with the structure of the site-specific variant R209H, which possesses a higher monophenolase/diphenolase activity ratio, lends further support to a previously suggested mechanism by which monophenolic substrates dock mainly to CuA.     

Kinetics and thermodynamics of ligand binding to a molten globular enzyme and its native counterpart

An engineered monomeric chorismate mutase (mMjCM) has been found to combine high catalytic activity with the characteristics of a molten globule. To gain insight into the dramatic structural changes that accompany binding of a transition-state analog, we examined mMjCM by isothermal calorimetry and compared it with its dimeric parent protein, MjCM (CM from Methanococcus jannaschii), a thermostable and conventionally folded enzyme. As expected for a ligand-induced ordering process, there is a large entropic penalty for binding to the monomer relative to the dimer (− TΔΔS = 5.1 ± 0.5 kcal/mol, at 20 °C). However, this unfavorable entropy term is largely offset by enthalpic gains (ΔΔH = − 3.5 ± 0.4 kcal/mol), presumably arising from tightening of non-covalent interactions throughout the monomeric complex. Stopped-flow kinetic measurements further reveal that the catalytic molten globule binds and releases ligands significantly faster than its natural counterpart, demonstrating that partial structural disorder can speed up molecular recognition. These results illustrate how structural plasticity may strongly perturb the thermodynamics and kinetics of transition-state recognition while negligibly affecting catalytic efficiency.     

Crystal Structure Determination and Functional Characterization of the Metallochaperone SlyD from Thermus thermophilus

SlyD (sensitive to lysis D; product of the slyD gene) is a prolyl isomerase [peptidyl-prolyl cis/trans isomerase (PPIase)] of the FK506 binding protein (FKBP) type with chaperone properties. X-ray structures derived from three different crystal forms reveal that SlyD from Thermus thermophilus consists of two domains representing two functional units. PPIase activity is located in a typical FKBP domain, whereas chaperone function is associated with the autonomously folded insert-in-flap (IF) domain. The two isolated domains are stable and functional in solution, but the presence of the IF domain increases the PPIase catalytic efficiency of the FKBP domain by 2 orders of magnitude, suggesting that the two domains act synergistically to assist the folding of polypeptide chains. The substrate binding surface of SlyD from T. thermophilus was mapped by NMR chemical shift perturbations to hydrophobic residues of the IF domain, which exhibits significantly reduced thermodynamic stability according to NMR hydrogen/deuterium exchange and fluorescence equilibrium transition experiments. Based on structural homologies, we hypothesize that this is due to the absence of a stabilizing β-strand, suggesting in turn a mechanism for chaperone activity by ‘donor-strand complementation.’ Furthermore, we identified a conserved metal (Ni²⁺) binding site at the C-terminal SlyD-specific helical appendix of the FKBP domain, which may play a role in metalloprotein assembly.     

Structural and Functional Characterization of the Clostridium perfringens N-Acetylmannosamine-6-phosphate 2-Epimerase Essential for the Sialic Acid Salvage Pathway

Pathogenic bacteria are endowed with an arsenal of specialized enzymes to convert nutrient compounds from their cell hosts. The essential N-acetylmannosamine-6-phosphate 2-epimerase (NanE) belongs to a convergent glycolytic pathway for utilization of the three amino sugars, GlcNAc, ManNAc, and sialic acid. The crystal structure of ligand-free NanE from Clostridium perfringens reveals a modified triose-phosphate isomerase (β/α)₈ barrel in which a stable dimer is formed by exchanging the C-terminal helix. By retaining catalytic activity in the crystalline state, the structure of the enzyme bound to the GlcNAc-6P product identifies the topology of the active site pocket and points to invariant residues Lys⁶⁶ as a putative single catalyst, supported by the structure of the catalytically inactive K66A mutant in complex with substrate ManNAc-6P. ¹H NMR-based time course assays of native NanE and mutated variants demonstrate the essential role of Lys⁶⁶ for the epimerization reaction with participation of neighboring Arg⁴³, Asp¹²⁶, and Glu¹⁸⁰ residues. These findings unveil a one-base catalytic mechanism of C2 deprotonation/reprotonation via an enolate intermediate and provide the structural basis for the development of new antimicrobial agents against this family of bacterial 2-epimerases.     

Structures of dimeric nonstandard nucleotide triphosphate pyrophosphatase from Pyrococcus horikoshii OT3: functional significance of interprotomer conformational changes

Nonstandard nucleotide triphosphate pyrophosphatase (NTPase) can efficiently hydrolyze nonstandard purine nucleotides in the presence of divalent cations. The crystal structures of the NTPase from Pyrococcus horikoshii OT3 (PhNTPase) have been determined in two unliganded forms and in three liganded forms with inosine 5′-monophosphate (IMP), ITP and Mn²⁺, which visualize the recognition of these ligands unambiguously. The overall structure of PhNTPase is similar to that of previously reported crystal structures of the NTPase from Methanococcus jannaschii and the human ITPase. They share a similar protomer folding with two domains and a similar homodimeric quaternary structure. The dimeric interface of NTPase is well conserved, and the dimeric state might be important to constitute the active site of this enzyme. A conformational analysis of the five snapshots of PhNTPase structures using the multiple superposition method reveals that IMP, ITP and Mn²⁺ bind to the active site without inducing large local conformational changes, indicating that a combination of interdomain and interprotomer rigid-body shifts mainly describes the conformational change of PhNTPase. The interdomain and interprotomer conformations of the ITP liganded form are essentially the same as those observed in the unliganded form 1, indicating that ITP binding to PhNTPase in solution may follow the selection mode in which ITP binds to the subunit that happens to be in the conformation observed in the unliganded form 1. In contrast to the human ITPase inducing a large domain closure upon ITP binding, the interdomain active site cleft is generally closed in PhNTPase and only the IMP binding form shows a remarkable domain opening by 14° only in the B subunit. The interprotomer rigid-body rotation of PhNTPase has a tendency to keep the dimeric 2-fold symmetry, which is also true in human ITPase, thereby suggesting its relevance to the positive cooperativity of the dimeric NTPases. The exception of this rule is observed in the IMP liganded form in which the dimeric 2-fold symmetry is broken by a 3° interprotomer rotation in an unusual direction. A combination of the exceptional interdomain and interprotomer relocations is most likely the reason for the observed asymmetric IMP binding that might be necessary for PhNTPase to release the reaction product IMP.     

Dimeric Crystal Structure of Rabbit l-Gulonate 3-Dehydrogenase/λ-Crystallin: Insights into the Catalytic Mechanism

l-Gulonate 3-dehydrogenase (GDH) is a bifunctional dimeric protein that functions not only as an NAD⁺-dependent enzyme in the uronate cycle but also as a taxon-specific λ-crystallin in rabbit lens. Here we report the first crystal structure of GDH in both apo form and NADH-bound holo form. The GDH protomer consists of two structural domains: the N-terminal domain with a Rossmann fold and the C-terminal domain with a novel helical fold. In the N-terminal domain of the NADH-bound structure, we identified 11 coenzyme-binding residues and found 2 distinct side-chain conformers of Ser124, which is a putative coenzyme/substrate-binding residue. A structural comparison between apo form and holo form and a mutagenesis study with E97Q mutant suggest an induced-fit mechanism upon coenzyme binding; coenzyme binding induces a conformational change in the coenzyme-binding residues Glu97 and Ser124 to switch their activation state from resting to active, which is required for the subsequent substrate recruitment. Subunit dimerization is mediated by numerous intersubunit interactions, including 22 hydrogen bonds and 104 residue pairs of van der Waals interactions, of which those between two cognate C-terminal domains are predominant. From a structure/sequence comparison within GDH homologues, a much greater degree of interprotomer interactions (both polar and hydrophobic) in the rabbit GDH would contribute to its higher thermostability, which may be relevant to the other function of this enzyme as λ-crystallin, a constitutive structural protein in rabbit lens. The present crystal structures and amino acid mutagenesis studies assigned the role of active-site residues: catalytic base for His145 and substrate binding for Ser124, Cys125, Asn196, and Arg231. Notably, Arg231 participates in substrate binding from the other subunit of the GDH dimer, indicating the functional significance of the dimeric state. Proper orientation of the substrate-binding residues for catalysis is likely to be maintained by an interprotomer hydrogen-bonding network of residues Asn196, Gln199, and Arg231, suggesting a network-based substrate recognition of GDH.     

Structural and functional insights into O-methyltransferase from Bacillus cereus

The specific substrates, mechanisms, and structures of the bacterial O-methyltransferases (OMTs) are not as well characterized as those of other OMTs. Recent studies have suggested that bacterial OMTs catalyze regiospecific reactions that might be used to produce novel compounds. In this study, we investigated the structural and functional features of an OMT from Bacillus cereus (BcOMT2). This enzyme catalyzes the O-methylation of flavonoids in vitro in an S-adenosylmethionine-dependent and regiospecific manner. We solved the crystal structures of the BcOMT2 apoenzyme and the BcOMT2-S-adenosylhomocysteine (SAH) co-complex at resolutions of 1.8 and 1.2 Å, respectively. These structures reveal that the overall structure of dimeric BcOMT2 is similar to that of the canonical OMT but that BcOMT2 also has a unique N-terminal helical region that is responsible for dimerization. The binding of SAH causes both local and remote conformational changes in the dimer interface that stabilize the dimerization of BcOMT2. SAH binding also causes ordering of residues Glu171 to Gly186, which are disordered in the apoenzyme structure and are known determinants of substrate specificity, and thus contributes to formation of the substrate binding pocket. Our structural analysis indicated a resemblance between the active site of BcOMT2 and that of metal-dependent OMTs. Using mutational analysis, we confirmed that BcOMT2 is a Mg²⁺-dependent OMT. These results provide structural and functional insights into the dimerization mechanism and substrate specificity of BcOMT2.     

Crystal structure studies of NADP dependent isocitrate dehydrogenase from Thermus thermophilus exhibiting a novel terminal domain

NADP⁺ dependent isocitrate dehydrogenase (IDH) is an enzyme catalyzing oxidative decarboxylation of isocitrate into oxalosuccinate (intermediate) and finally the product α-ketoglutarate. The crystal structure of Thermus thermophilus isocitrate dehydrogenase (TtIDH) ternary complex with citrate and cofactor NADP⁺ was determined using X-ray diffraction method to a resolution of 1.80 Å. The overall fold of this protein was resolved into large domain, small domain and a clasp domain. The monomeric structure reveals a novel terminal domain involved in dimerization, very unique and novel domain when compared to other IDH’s. And, small domain and clasp domain showing significant differences when compared to other IDH’s of the same sub-family. The structure of TtIDH reveals the absence of helix at the clasp domain, which is mainly involved in oligomerization in other IDH’s. Also, helices/beta sheets are absent in the small domain, when compared to other IDH’s of the same sub family. The overall TtIDH structure exhibits closed conformation with catalytic triad residues, Tyr144-Asp248-Lys191 are conserved. Oligomerization of the protein is quantized using interface area and subunit–subunit interactions between protomers. Overall, the TtIDH structure with novel terminal domain may be categorized as a first structure of subfamily of type IV.     

Structure of N-acetyl-beta-D-glucosaminidase (GcnA) from the endocarditis pathogen Streptococcus gordonii and its complex with the mechanism-based inhibitor NAG-thiazoline

The crystal structure of GcnA, an N-acetyl-β-d-glucosaminidase from Streptococcus gordonii, was solved by multiple wavelength anomalous dispersion phasing using crystals of selenomethionine-substituted protein. GcnA is a homodimer with subunits each comprised of three domains. The structure of the C-terminal α-helical domain has not been observed previously and forms a large dimerisation interface. The fold of the N-terminal domain is observed in all structurally related glycosidases although its function is unknown. The central domain has a canonical (β/α)₈ TIM-barrel fold which harbours the active site. The primary sequence and structure of this central domain identifies the enzyme as a family 20 glycosidase. Key residues implicated in catalysis have different conformations in two different crystal forms, which probably represent active and inactive conformations of the enzyme. The catalytic mechanism for this class of glycoside hydrolase, where the substrate rather than the enzyme provides the cleavage-inducing nucleophile, has been confirmed by the structure of GcnA complexed with a putative reaction intermediate analogue, N-acetyl-β-d-glucosamine-thiazoline. The catalytic mechanism is discussed in light of these and other family 20 structures.     

Mutational analysis of cysteine 328 and cysteine 368 at the interface of Plasmodium falciparum adenylosuccinate synthetase

Plasmodium falciparum adenylosuccinate synthetase, a homodimeric enzyme, contains 10 cysteine residues per subunit. Among these, Cys250, Cys328 and Cys368 lie at the dimer interface and are not conserved across organisms. PfAdSS has a positively charged interface with the crystal structure showing additional electron density around Cys328 and Cys368. Biochemical characterization of site directed mutants followed by equilibrium unfolding studies permits elucidation of the role of interface cysteines and positively charged interface in dimer stability. Mutation of interface cysteines, Cys328 and Cys368 to serine, perturbed the monomer-dimer equilibrium in the protein with a small population of monomer being evident in the double mutant. Introduction of negative charge in the form of C328D mutation resulted in stabilization of protein dimer as evident by size exclusion chromatography at high ionic strength buffer and equilibrium unfolding in the presence of urea. These observations suggest that cysteines at the dimer interface of PfAdSS may indeed be charged and exist as thiolate anion.     

Two Complementary Enzymes for Threonylation of tRNA in Crenarchaeota: Crystal Structure of Aeropyrum pernix Threonyl-tRNA Synthetase Lacking a cis-Editing Domain

In protein synthesis, threonyl-tRNA synthetase (ThrRS) must recognize threonine (Thr) from the 20 kinds of amino acids and the cognate tRNA^Thr from different tRNAs in order to generate Thr-tRNA^Thr. In general, an organism possesses one kind of gene corresponding to ThrRS. However, it has been recently found that some organisms have two different genes for ThrRS in the genome, suggesting that their proteins ThrRS-1 and ThrRS-2 function separately and complement each other in the threonylation of tRNA^Thr, one for catalysis and the other for trans-editing of misacylated Ser-tRNA^Thr. In order to clarify their three-dimensional structures, we performed X-ray analyses of two putatively assigned ThrRSs from Aeropyrum pernix (ApThrRS-1 and ApThrRS-2). These proteins were overexpressed in Escherichia coli, purified, and crystallized. The crystal structure of ApThrRS-1 has been successfully determined at 2.3 Å resolution. ApThrRS-1 is a dimeric enzyme composed of two identical subunits, each containing two domains for the catalytic reaction and for anticodon binding. The essential editing domain is completely missing as expected. These structural features reveal that ThrRS-1 catalyzes only the aminoacylation of the cognate tRNA, suggesting the necessity of the second enzyme ThrRS-2 for trans-editing. Since the N-terminal sequence of ApThrRS-2 is similar to the sequence of the editing domain of ThrRS from Pyrococcus abyssi, ApThrRS-2 has been expected to catalyze deaminoacylation of a misacylated serine moiety at the CCA terminus.