Elucidating Dynamics of Adenylate Kinase from Enzyme Opening to Ligand Release.

This study explores ligand-driven conformational changes in adenylate kinase (AK), which is known for its open-to-close conformational transitions upon ligand binding and release. By utilizing string free energy simulations, we determine the free energy profiles for both enzyme opening and ligand release and compare them with profiles from the apoenzyme. Results reveal a three-step ligand release process, which initiates with the opening of the adenosine triphosphate-binding subdomain (ATP lid), followed by ligand release and concomitant opening of the adenosine monophosphate-binding subdomain (AMP lid). The ligands then transition to nonspecific positions before complete dissociation. In these processes, the first step is energetically driven by ATP lid opening, whereas the second step is driven by ATP release. In contrast, the AMP lid opening and its ligand release make minor contributions to the total free energy for enzyme opening. Regarding the ligand binding mechanism, our results suggest that AMP lid closure occurs via an induced-fit mechanism triggered by AMP binding, whereas ATP lid closure follows conformational selection. This difference in the closure mechanisms provides an explanation with implications for the debate on ligand-driven conformational changes of AK. Additionally, we determine an X-ray structure of an AK variant that exhibits significant rearrangements in the stacking of catalytic arginines, explaining its reduced catalytic activity. In the context of apoenzyme opening, the sequence of events is different. Here, the AMP lid opens first while the ATP lid remains closed, and the free energy associated with ATP lid opening varies with orientation, aligning with the reported AK opening and closing rate heterogeneity. Finally, this study, in conjunction with our previous research, provides a comprehensive view of the intricate interplay between various structural elements, ligands, and catalytic residues that collectively contribute to the robust catalytic power of the enzyme.

1-deoxy-D-xylulose-5-phosphate synthase from Pseudomonas aeruginosa and Klebsiella pneumoniae reveals conformational changes upon cofactor binding.

The ESKAPE bacteria are the six highly virulent and antibiotic-resistant pathogens that require the most urgent attention for the development of novel antibiotics. Detailed knowledge of target proteins specific to bacteria is essential to develop novel treatment options. The methylerythritol-phosphate (MEP) pathway, which is absent in humans, represents a potentially valuable target for the development of novel antibiotics. Within the MEP pathway, the enzyme 1-deoxy-D-xylulose-5-phosphate synthase (DXPS) catalyzes a crucial, rate-limiting first step and a branch point in the biosynthesis of the vitamins B1 and B6. We report the high-resolution crystal structures of DXPS from the important ESKAPE pathogens Pseudomonas aeruginosa and Klebsiella pneumoniae in both the co-factor-bound and the apo forms. We demonstrate that the absence of the cofactor thiamine diphosphate results in conformational changes that lead to disordered loops close to the active site that might be important for the design of potent DXPS inhibitors. Collectively, our results provide important structural details that aid in the assessment of DXPS as a potential target in the ongoing efforts to combat antibiotic resistance.

The K+-Dependent and -Independent Pyruvate Kinases Acquire the Active Conformation by Different Mechanisms.

Eukarya pyruvate kinases possess glutamate at position 117 (numbering of rabbit muscle enzyme), whereas bacteria have either glutamate or lysine. Those with E117 are K+-dependent, whereas those with K117 are K+-independent. In a phylogenetic tree, 80% of the sequences with E117 are occupied by T113/K114/T120 and 77% of those with K117 possess L113/Q114/(L,I,V)120. This work aims to understand these residues' contribution to the K+-independent pyruvate kinases using the K+-dependent rabbit muscle enzyme. Residues 117 and 120 are crucial in the differences between the K+-dependent and -independent mutants. K+-independent activity increased with L113 and Q114 to K117, but L120 induced structural differences that inactivated the enzyme. T120 appears to be key in folding the protein and closure of the lid of the active site to acquire its active conformation in the K+-dependent enzymes. E117K mutant was K+-independent and the enzyme acquired the active conformation by a different mechanism. In the K+-independent apoenzyme of Mycobacterium tuberculosis, K72 (K117) flips out of the active site; in the holoenzyme, K72 faces toward the active site bridging the substrates through water molecules. The results provide evidence that two different mechanisms have evolved for the catalysis of this reaction.

Structure, mechanism and crystallographic fragment screening of the SARS-CoV-2 NSP13 helicase.

There is currently a lack of effective drugs to treat people infected with SARS-CoV-2, the cause of the global COVID-19 pandemic. The SARS-CoV-2 Non-structural protein 13 (NSP13) has been identified as a target for anti-virals due to its high sequence conservation and essential role in viral replication. Structural analysis reveals two "druggable" pockets on NSP13 that are among the most conserved sites in the entire SARS-CoV-2 proteome. Here we present crystal structures of SARS-CoV-2 NSP13 solved in the APO form and in the presence of both phosphate and a non-hydrolysable ATP analog. Comparisons of these structures reveal details of conformational changes that provide insights into the helicase mechanism and possible modes of inhibition. To identify starting points for drug development we have performed a crystallographic fragment screen against NSP13. The screen reveals 65 fragment hits across 52 datasets opening the way to structure guided development of novel antiviral agents.

Comparative protein structure network analysis on 3CLpro from SARS-CoV-1 and SARS-CoV-2.

The main protease Mpro , 3CLpro is an important target from coronaviruses. In spite of having 96% sequence identity among Mpros from SARS-CoV-1 and SARS-CoV-2; the inhibitors used to block the activity of SARS-CoV-1 Mpro so far, were found to have differential inhibitory effect on Mpro of SARS-CoV-2. The possible reason could be due to the difference of few amino acids among the peptidases. Since, overall 3-D crystallographic structure of Mpro from SARS-CoV-1 and SARS-CoV-2 is quite similar and mapping a subtle structural variation is seemingly impossible. Hence, we have attempted to study a structural comparison of SARS-CoV-1 and SARS-CoV-2 Mpro in apo and inhibitor bound states using protein structure network (PSN) based approach at contacts level. The comparative PSNs analysis of apo Mpros from SARS-CoV-1 and SARS-CoV-2 uncovers small but significant local changes occurring near the active site region and distributed throughout the structure. Additionally, we have shown how inhibitor binding perturbs the PSG and the communication pathways in Mpros . Moreover, we have also investigated the network connectivity on the quaternary structure of Mpro and identified critical residue pairs for complex formation using three centrality measurement parameters along with the modularity analysis. Taken together, these results on the comparative PSN provide an insight into conformational changes that may be used as an additional guidance towards specific drug development.

Structural basis for substrate recognition of glucose-6-phosphate dehydrogenase from Kluyveromyces lactis.

Glucose-6-phosphate dehydrogenase is the first enzyme in the pentose phosphate pathway. The reaction catalyzed by the enzyme is considered to be the main source of reducing power for nicotinamide adenine dinucleotide phosphate (NADPH) and is a precursor of 5-carbon sugar used by cells. To uncover the structural features of the enzyme, we determined the crystal structures of glucose-6-phosphate dehydrogenase from Kluyveromyces lactis (KlG6PD) in both the apo form and a binary complex with its substrate glucose-6-phosphate. KlG6PD contains a Rossman-like domain for cofactor NADPH binding; it also presents a typical antiparallel ß sheet at the C-terminal domain with relatively the same pattern as those of other homologous structures. Moreover, our structural and biochemical analyses revealed that Lys153 contributes significantly to substrate G6P recognition. This study may provide insights into the structural variation and catalytic features of the G6PD enzyme.

Structural insights into a new substrate binding mode of a histidine acid phosphatase from Legionella pneumophila.

MapA is a histidine acid phosphatase (HAP) from Legionella pneumophila that catalyzes the hydroxylation of a phosphoryl group from phosphomonoesters by an active-site histidine. Several structures of HAPs, including MapA, in complex with the inhibitor tartrate have been solved and the substrate binding tunnel identified; however, the substrate recognition mechanism remains unknown. To gain insight into the mechanism of substrate recognition, the crystal structures of apo-MapA and the MapAD281A mutant in complex with 5'-AMP were solved at 2.2 and 2.6 Å resolution, respectively. The structure of the MapAD281A/5'-AMP complex reveals that the 5'-AMP fits fully into the substrate binding tunnel, with the 2'-hydroxyl group of the ribose moiety stabilized by Glu201 and the adenine moiety sandwiched between His205 and Phe237. This is the second structure of a HAP/AMP complex solved with 5'-AMP binding in a unique manner in the active site. The structure presents a new substrate recognition mechanism of HAPs.

Structural insight into arenavirus replication machinery.

Arenaviruses can cause severe haemorrhagic fever and neurological diseases in humans and other animals, exemplified by Lassa mammarenavirus, Machupo mammarenavirus and lymphocytic choriomeningitis virus, posing great threats to public health1-4. These viruses encode a large multi-domain RNA-dependent RNA polymerase for transcription and replication of the viral genome5. Viral polymerases are one of the leading antiviral therapeutic targets. However, the structure of arenavirus polymerase is not yet known. Here we report the near-atomic resolution structures of Lassa and Machupo virus polymerases in both apo and promoter-bound forms. These structures display a similar overall architecture to influenza virus and bunyavirus polymerases but possess unique local features, including an arenavirus-specific insertion domain that regulates the polymerase activity. Notably, the ordered active site of arenavirus polymerase is inherently switched on, without the requirement for allosteric activation by 5'-viral RNA, which is a necessity for both influenza virus and bunyavirus polymerases6,7. Moreover, dimerization could facilitate the polymerase activity. These findings advance our understanding of the mechanism of arenavirus replication and provide an important basis for developing antiviral therapeutics.

Adsorption of unfolded Cu/Zn superoxide dismutase onto hydrophobic surfaces catalyzes its formation of amyloid fibrils.

Intracellular aggregates of superoxide dismutase 1 (SOD1) are associated with amyotrophic lateral sclerosis. In vivo, aggregation occurs in a complex and dense molecular environment with chemically heterogeneous surfaces. To investigate how SOD1 fibril formation is affected by surfaces, we used an in vitro model system enabling us to vary the molecular features of both SOD1 and the surfaces, as well as the surface area. We compared fibril formation in hydrophilic and hydrophobic sample wells, as a function of denaturant concentration and extraneous hydrophobic surface area. In the presence of hydrophobic surfaces, SOD1 unfolding promotes fibril nucleation. By contrast, in the presence of hydrophilic surfaces, increasing denaturant concentration retards the onset of fibril formation. We conclude that the mechanism of fibril formation depends on the surrounding surfaces and that the nucleating species might correspond to different conformational states of SOD1 depending on the nature of these surfaces.

Structures of a RAG-like transposase during cut-and-paste transposition.

Transposons have had a pivotal role in genome evolution1 and are believed to be the evolutionary progenitors of the RAG1-RAG2 recombinase2, an essential component of the adaptive immune system in jawed vertebrates3. Here we report one crystal structure and five cryo-electron microscopy structures of Transib4,5, a RAG1-like transposase from Helicoverpa zea, that capture the entire transposition process from the apo enzyme to the terminal strand transfer complex with transposon ends covalently joined to target DNA, at resolutions of 3.0-4.6 Å. These structures reveal a butterfly-shaped complex that undergoes two cycles of marked conformational changes in which the 'wings' of the transposase unfurl to bind substrate DNA, close to execute cleavage, open to release the flanking DNA and close again to capture and attack target DNA. Transib possesses unique structural elements that compensate for the absence of a RAG2 partner, including a loop that interacts with the transposition target site and an accordion-like C-terminal tail that elongates and contracts to help to control the opening and closing of the enzyme and assembly of the active site. Our findings reveal the detailed reaction pathway of a eukaryotic cut-and-paste transposase and illuminate some of the earliest steps in the evolution of the RAG recombinase.

Low-resolution SAXS and structural dynamics analysis on M. tuberculosis GmhB enzyme involved in GDP-heptose biosynthetic pathway.

The M. tuberculosis GmhB protein converts the d-glycero-α-d-manno-heptose 1,7-bisphosphate (GMB) intermediate into d-glycero-α-d-manno-heptose 1-phosphate by removing the phosphate group at the C-7 position. To understand the structure and substrate binding mechanism, the MtbGmhB was purified which elutes as monomer on gel filtration column. The small angle x-ray scattering analysis shows that MtbGmhB forms fully folded monomer with shape profile similar to its modeled structure. The circular dichroism analysis shows 38% α-helix, 15% ß-sheets and 47% random coil structures in MtbGmhB, similar to haloalkanoic acid dehalogenase (HAD) phosphohydrolase enzymes. The modeled MtbGmhB structure shows the catalytic site, which forms a concave, semicircular surface using the three loops around GMB substrate binding site. Dynamic simulation analysis on (i) Apo (ii) GMB bound (iii) GMB + Mg2+ bound (iv) Zn2+ +GMB + Mg2+ bound MtbGmhB structures show that Zn2+ as well as Mg2+ ions stabilize the loop conformation and trigger the changes in GMB substrate binding to active site of MtbGmhB. Upon demetallization, the large conformational changes occurred in ions binding loops, and leads to difference in GMB substrate binding to MtbGmhB. Our study provides information about structure and substrate binding of MtbGmhB, which may contribute in therapeutic development against M. tuberculosis.

Investigation of the role of central metal ion of Cyathus bulleri laccase 1 using guanidinium chloride-induced denaturation.

The structure and folding/unfolding kinetics of Cyathus bulleri laccase 1 (Lcc1), expressed in Pichia pastoris, were analyzed by spectroscopic methods to understand the role of central metal ion. Far UV CD structure analysis revealed major ß-sheet and minor α helical segments present in the Lcc1. A significant change in the spectrum of Lcc1, indicative of unfolding of secondary structures, was observed with increasing concentrations of guanidinium chloride (GdnHCl) during Trp fluorescence, absorption and CD measurements. A similar trend was also noticed for enzyme activity with respect to GdnHCl concentrations. To establish the role of copper ion in the catalytic activity of laccase, a complete removal of copper was carried out and addition of copper was found to restore the structure and activity during the refolding process. The apo form was also reconstituted by addition of zinc ion which restored nearly 84% of enzyme activity, indicating non-essential role of copper in maintaining conformation and activity. Structural studies and inductively coupled plasma mass spectrometry data supported these observations.

Allostery in Coagulation Factor VIIa Revealed by Ensemble Refinement of Crystallographic Structures.

A critical step in injury-induced initiation of blood coagulation is the formation of the complex between the trypsin-like protease coagulation factor VIIa (FVIIa) and its cofactor tissue factor (TF), which converts FVIIa from an intrinsically poor enzyme to an active protease capable of activating zymogens of downstream coagulation proteases. Unlike its constitutively active ancestor trypsin, FVIIa is allosterically activated (by TF). Here, ensemble refinement of crystallographic structures, which uses multiple copies of the entire structure as a means of representing structural flexibility, is applied to explore the impacts of inhibitor binding to trypsin and FVIIa, as well as cofactor binding to FVIIa. To assess the conformational flexibility and its role in allosteric pathways in these proteases, main-chain hydrogen bond networks are analyzed by calculating the hydrogen-bond propensity. Mapping pairwise propensity differences between relevant structures shows that binding of the inhibitor benzamidine to trypsin has a minor influence on the protease flexibility. For FVIIa, in contrast, the protease domain is "locked" into the catalytically competent trypsin-like configuration upon benzamidine binding as indicated by the stabilization of key structural features: the nonprime binding cleft and the oxyanion hole are stabilized, and the effect propagates from the active site region to the calcium-binding site and to the vicinity of the disulphide bridge connecting with the light chain. TF binding to FVIIa furthermore results in stabilization of the 170 loop, which in turn propagates an allosteric signal from the TF-binding region to the active site. Analyses of disulphide bridge energy and flexibility reflect the striking stability difference between the unregulated enzyme and the allosterically activated form after inhibitor or cofactor binding. The ensemble refinement analyses show directly, for the first time to our knowledge, whole-domain structural footprints of TF-induced allosteric networks present in x-ray crystallographic structures of FVIIa, which previously only have been hypothesized or indirectly inferred.

Development of Recombinant Methioninase for Cancer Treatment.

The elevated requirement for methionine (MET) of cancer cells is termed MET dependence. To selectively target the MET dependence of tumors for treatment on a large-scale preclinical and clinical basis, the L-methionine α-deamino-γ-mercaptomethane-lyase (EC 4.4.1.11) (methioninase, [METase]) gene from Pseudomonas putida has been cloned in Escherichia coli using the polymerase chain reaction (PCR). Purification using two DEAE Sepharose FF ion-exchange column and one ActiClean Etox endotoxin-affinity chromatography column has been established. Plasmid pMGLTrc03, which has a trc promoter and a spacing of 12 nucleotides between the Shine-Dalgarno sequence and the ATG translation initiation codon, was selected as the most suitable plasmid. The recombinant bacteria produced rMETase at 43% of the total proteins in soluble fraction by simple batch fermentation using a 500 L fermentor. Crystals were directly obtained from crude enzyme with 87% yield by a crystallization in the presence of 9.0% polyethylene glycol 6000, 3.6% ammonium sulfate, and 0.18 M sodium chloride using a 100 L crystallizer. After recrystallization, the enzyme was purified by anion-exchange column chromatography to remove endotoxins and by gel filtration for polishing. Purified rMETase is stable to lyophilization. In order to prevent immunological reactions which might be produced by multiple dosing of rMETase and to prolong the serum half-life of rMETase, the N-hydroxysuccinimidyl ester of methoxypolyethylene glycol propionic acid (M-SPA-PEG 5000) has been coupled to rMETase. The PEGylated molecules (PEG-rMETase) were purified from unreacted PEG with Amicon 30 K centriprep concentrators or by Sephacryl S-300 HR gel-filtration chromatography. Unreacted rMETase was removed by DEAE Sepharose FF anion-exchange chromatography. The resulting PEG-rMETase subunit, produced from a PEG/rMETase ratio of 30/1 in the synthetic reaction, had a molecular mass of approximately 53 kda determined by matrix-assisted laser desorption/ionization mass spectrometry, indicating the conjugation of two PEG molecules per subunit of rMETase and eight per tetramer. PEG-rMETase molecules obtained from reacting ratios of PEG/rMETase of 30/1 had an enzyme activity of 70% of unmodified rMETase. PEGylation of rMETase increased the serum half-life of the enzyme in rats to approximately 160 min compared to 80 min for unmodified rMETase. PEG-rMETase could deplete serum MET levels to less than 0.1 µM for approximately 8 h compared to 2 h for rMETase in rats. A significant prolongation of in vivo activity and effective MET depletion by the PEG-rMETase were achieved by the simultaneous administration of pyridoxal 5'-phosphate. rMETase was also conjugated with methoxypolyethylene glycol succinimidyl glutarate 5000 (MEGC-PEG). Miniosmotic pumps containing various concentrations of PLP were implanted in BALB-C mice. PLP-infused mice were then injected with a single dose of 4000 or 8000 units/kg PEG-rMETase. Mice infused with 5, 50, 100, 200, and 500 mg/mL PLP-containing miniosmotic pumps increased plasma PLP to 7, 24, 34, 60, and 95 µM, respectively, from the PLP baseline of 0.3 µM. PLP increased the half-life of MEGC-PEG-rMETase holoenzyme in a dose-dependent manner. The extended time of MET depletion by MEGC-PEG-rMETase was due to the maintenance of active MEGC-PEG-rMETase holoenzyme by infused PLP.

Catalytic mechanism for the conversion of salicylate into catechol by the flavin-dependent monooxygenase salicylate hydroxylase.

Salicylate hydroxylase (NahG) is a flavin-dependent monooxygenase that catalyzes the decarboxylative hydroxylation of salicylate into catechol in the naphthalene degradation pathway in Pseudomonas putida G7. We explored the mechanism of action of this enzyme in detail using a combination of structural and biophysical methods. NahG shares many structural and mechanistic features with other versatile flavin-dependent monooxygenases, with potential biocatalytic applications. The crystal structure at 2.0 Šresolution for the apo form of NahG adds a new snapshot preceding the FAD binding in flavin-dependent monooxygenases. The kcat/Km for the salicylate reaction catalyzed by the holo form is >105 M-1 s-1 at pH 8.5 and 25 °C. Hammett plots for Km and kcat using substituted salicylates indicate change in rate-limiting step. Electron-donating groups favor the hydroxylation of salicylate by a peroxyflavin to yield a Wheland-like intermediate, whereas the decarboxylation of this intermediate is faster for electron-withdrawing groups. The mechanism is supported by structural data and kinetic studies at different pHs. The salicylate carboxyl group lies near a hydrophobic region that aids decarboxylation. A conserved histidine residue is proposed to assist the reaction by general base/general acid catalysis.

Evaluating apoenzyme-coenzyme-substrate interactions of methane monooxygenase with an engineered active site for electron harvesting: a computational study.

Low-temperature methane oxidation is one of the greatest challenges in energy research. Although methane monooxygenase (MMO) does this catalysis naturally, how to use this biocatalyst in a fuel cell environment where the electrons generated during the oxidation process is harvested and used for energy generation has not yet been investigated. A key requirement to use this enzyme in a fuel cell is wiring of the active site of the enzyme directly to the supporting electrode. In soluble MMO (sMMO), two cofactors, i.e., nicotinamide adenine di-nucleotide (NAD+) and flavin adenine dinucleotide (FAD) provide opportunities for direct attachment of the enzyme system to a supporting electrode. However, once modified to be compatible with a supporting metal electrode via FeS functionalization, how the two cofactors respond to complex binding phenomena is not yet understood. Using docking and molecular dynamic simulations, modified cofactors interactions with sMMO-reductase (sMMOR) were studied. Studies revealed that FAD modification with FeS did not interfere with binding phenomena. In fact, FeS introduction significantly improved the binding affinity of FAD and NAD+ on sMMOR. The simulations revealed a clear thermodynamically more favorable electron transport path for the enzyme system. This system can be used as a fuel cell and we can use FeS-modified-FAD as the anchoring molecule as opposed to using NAD+. The overall analysis suggests the strong possibility of building a fuel cell that could catalyze methane oxidation using sMMO as the anode biocatalyst.

Conformational changes on substrate binding revealed by structures of Methylobacterium extorquens malate dehydrogenase.

Three high-resolution X-ray crystal structures of malate dehydrogenase (MDH; EC 1.1.1.37) from the methylotroph Methylobacterium extorquens AM1 are presented. By comparing the structures of apo MDH, a binary complex of MDH and NAD+, and a ternary complex of MDH and oxaloacetate with ADP-ribose occupying the pyridine nucleotide-binding site, conformational changes associated with the formation of the catalytic complex were characterized. While the substrate-binding site is accessible in the enzyme resting state or NAD+-bound forms, the substrate-bound form exhibits a closed conformation. This conformational change involves the transition of an α-helix to a 310-helix, which causes the adjacent loop to close the active site following coenzyme and substrate binding. In the ternary complex, His284 forms a hydrogen bond to the C2 carbonyl of oxaloacetate, placing it in a position to donate a proton in the formation of (2S)-malate.

Structure of glyoxysomal malate dehydrogenase (MDH3) from Saccharomyces cerevisiae.

Malate dehydrogenase (MDH), a carbohydrate and energy metabolism enzyme in eukaryotes, catalyzes the interconversion of malate to oxaloacetate (OAA) in conjunction with that of nicotinamide adenine dinucleotide (NAD+) to NADH. Three isozymes of MDH have been reported in Saccharomyces cerevisiae: MDH1, MDH2 and MDH3. MDH1 is a mitochondrial enzyme and a member of the tricarboxylic acid cycle, whereas MDH2 is a cytosolic enzyme that functions in the glyoxylate cycle. MDH3 is a glyoxysomal enzyme that is involved in the reoxidation of NADH, which is produced during fatty-acid ß-oxidation. The affinity of MDH3 for OAA is lower than those of MDH1 and MDH2. Here, the crystal structures of yeast apo MDH3, the MDH3-NAD+ complex and the MDH3-NAD+-OAA ternary complex were determined. The structure of the ternary complex suggests that the active-site loop is in the open conformation, differing from the closed conformations in mitochondrial and cytosolic malate dehydrogenases.

Resonance assignments for the apo-form of the cellulose-active lytic polysaccharide monooxygenase TaLPMO9A.

The apo-form of the 24.4 kDa AA9 family lytic polysaccharide monooxygenase TaLPMO9A from Thermoascus aurantiacus has been isotopically labeled and recombinantly expressed in Pichia pastoris. In this paper, we report the 1H, 13C, and 15N chemical shift assignments, as well as an analysis of the secondary structure of the protein based on the secondary chemical shifts.

Crystal Structure of the Human tRNA Guanine Transglycosylase Catalytic Subunit QTRT1.

RNA modifications have been implicated in diverse and important roles in all kingdoms of life with over 100 of them present on tRNAs. A prominent modification at the wobble base of four tRNAs is the 7-deaza-guanine derivative queuine which substitutes the guanine at position 34. This exchange is catalyzed by members of the enzyme class of tRNA guanine transglycosylases (TGTs). These enzymes incorporate guanine substituents into tRNAAsp, tRNAAsn tRNAHis, and tRNATyr in all kingdoms of life. In contrast to the homodimeric bacterial TGT, the active eukaryotic TGT is a heterodimer in solution, comprised of a catalytic QTRT1 subunit and a noncatalytic QTRT2 subunit. Bacterial TGT enzymes, that incorporate a queuine precursor, have been identified or proposed as virulence factors for infections by pathogens in humans and therefore are valuable targets for drug design. To date no structure of a eukaryotic catalytic subunit is reported, and differences to its bacterial counterpart have to be deducted from sequence analysis and models. Here we report the first crystal structure of a eukaryotic QTRT1 subunit and compare it to known structures of the bacterial TGT and murine QTRT2. Furthermore, we were able to determine the crystal structure of QTRT1 in complex with the queuine substrate.