Structural and functional insights into tRNA recognition by human tRNA guanine transglycosylase.

Eukaryotic tRNA guanine transglycosylase (TGT) is an RNA-modifying enzyme which catalyzes the base exchange of the genetically encoded guanine 34 of tRNAsAsp,Asn,His,Tyr for queuine, a hypermodified 7-deazaguanine derivative. Eukaryotic TGT is a heterodimer comprised of a catalytic and a non-catalytic subunit. While binding of the tRNA anticodon loop to the active site is structurally well understood, the contribution of the non-catalytic subunit to tRNA binding remained enigmatic, as no complex structure with a complete tRNA was available. Here, we report a cryo-EM structure of eukaryotic TGT in complex with a complete tRNA, revealing the crucial role of the non-catalytic subunit in tRNA binding. We decipher the functional significance of these additional tRNA-binding sites, analyze solution state conformation, flexibility, and disorder of apo TGT, and examine conformational transitions upon tRNA binding.

A novel SPE-UPLC-MS/MS-based assay for the selective, simultaneous quantification of xylosyltransferase-I and -II activity.

Xylosyltransferase-I and -II (XT-I, -II) possess a central role during the glycosylation of proteoglycans (PGs). They catalyze the formation of an O-glycosidic bond between the xylosyl residue of uridinediphosphate-xylose and the core protein of a PG. Thereafter, three following glycosyltransferases lead to the generation of a tetrasaccharide linker, which connects the PG core protein to the respective glycosaminoglycan. The selective quantification of XT-I and XT-II activity is of biological and clinical interest due to their association with fibrotic processes and skeletal dysplasia. There is no assay available to date that simultaneously determines the activity of the two XT isoforms. Although an XT-I selective UPLC MS/MS-based assay was published by Fischer et al., in 2021, the determination of XT-II activity can only be performed simultaneously by the improved assay presented here. To establish the assay, two synthetic peptides, selectively xylosylated by the respective isoform, were identified and the associated measurement parameters for the mass spectrometer were optimized. In addition, the quantitative range of the xylosylated peptides were validated, and the incubation time of the enzyme reaction was optimized for cell culture samples and human sera. The specific enzyme kinetics (KM and Vmax) of the respective XT isoform for the two peptides were also determined. Subsequently, a mathematical model was developed, allowing the simultaneous determination of XT-I and XT-II activity from the chromatographic results. Summarized, a mass spectrometric method suitable for the simultaneous analysis of XT-I and XT-II activity in cell culture lysates, supernatants and human sera was successfully developed.

Isolation and Characterization of Engineered Nucleoside Deoxyribosyltransferase with Enhanced Activity Toward 2'-Fluoro-2'-Deoxynucleoside.

Nucleoside deoxyribosyltransferase (NDT) is an enzyme that replaces the purine or pyrimidine base of 2'-deoxyribonucleoside. This enzyme is generally used in the nucleotide salvage pathway in vivo and synthesizes many nucleoside analogs in vitro for various biotechnological purposes. Since NDT is known to exhibit relatively low reactivity toward nucleoside analogs such as 2'-fluoro-2'-deoxynucleoside, it is necessary to develop an enhanced NDT mutant enzyme suitable for nucleoside analogs. In this study, molecular evolution strategy via error-prone PCR was performed with ndt gene derived from Lactobacillus leichmannii as a template to obtain an engineered NDT with higher substrate specificity to 2FDU (2'-fluoro-2'-deoxyuridine). A mutant library of 214 ndt genes with different sequences was obtained and performed for the conversion of 2FDU to 2FDA (2'-fluoro-2'-deoxyadenosine). The E. coli containing a mutant NDT, named NDTL59Q, showed 1.7-fold (at 40°C) and 4.4-fold (at 50°C) higher 2FDU-to-2FDA conversions compared to the NDTWT, respectively. Subsequently, both NDTWT and NDTL59Q enzymes were over-expressed and purified using a His-tag system in E. coli. Characterization and enzyme kinetics revealed that the NDTL59Q mutant enzyme containing a single point mutation of leucine to glutamine at the 59th position exhibited superior thermal stability with enhanced substrate specificity to 2FDU.

Structural and Biochemical Investigation of the Heterodimeric Murine tRNA-Guanine Transglycosylase.

In tRNAAsp, tRNAAsn, tRNATyr, and tRNAHis of most bacteria and eukaryotes, the anticodon wobble position may be occupied by the modified nucleoside queuosine, which affects the speed and the accuracy of translation. Since eukaryotes are not able to synthesize queuosine de novo, they have to salvage queuine (the queuosine base) as a micronutrient from food and/or the gut microbiome. The heterodimeric Zn2+ containing enzyme tRNA-guanine transglycosylase (TGT) catalyzes the insertion of queuine into the above-named tRNAs in exchange for the genetically encoded guanine. This enzyme has attracted medical interest since it was shown to be potentially useful for the treatment of multiple sclerosis. In addition, TGT inactivation via gene knockout leads to the suppressed cell proliferation and migration of certain breast cancer cells, which may render this enzyme a potential target for the design of compounds supporting breast cancer therapy. As a prerequisite to fully exploit the medical potential of eukaryotic TGT, we have determined and analyzed a number of crystal structures of the functional murine TGT with and without bound queuine. In addition, we have investigated the importance of two residues of its non-catalytic subunit on dimer stability and determined the Michaelis-Menten parameters of murine TGT with respect to tRNA and several natural and artificial nucleobase substrates. Ultimately, on the basis of available TGT crystal structures, we provide an entirely conclusive reaction mechanism for this enzyme, which in detail explains why the TGT-catalyzed insertion of some nucleobases into tRNA occurs reversibly while that of others is irreversible.

19F-NMR Unveils the Ligand-Induced Conformation of a Catalytically Inactive Twisted Homodimer of tRNA-Guanine Transglycosylase.

Understanding the structural arrangements of protein oligomers can support the design of ligands that interfere with their function in order to develop new therapeutic concepts for disease treatment. Recent crystallographic studies have elucidated a novel twisted and functionally inactive form of the homodimeric enzyme tRNA-guanine transglycosylase (TGT), a putative target in the fight against shigellosis. Active-site ligands have been identified that stimulate the rearrangement of one monomeric subunit by 130° against the other one to form an inactive twisted homodimer state. To assess whether the crystallographic observations also reflect the conformation in solution and rule out effects from crystal packing, we performed 19F-NMR spectroscopy with the introduction of 5-fluorotryptophans at four sites in TGT. The inhibitor-induced conformation of TGT in solution was assessed based on 19F-NMR chemical shift perturbations. We investigated the effect of C(4) substituted lin-benzoguanine ligands and identified a correlation between dynamic protein rearrangements and ligand-binding features in the corresponding crystal structures. These involve the destabilization of a helix next to the active site and the integrity of a flexible loop-helix motif. Ligands that either completely lack an attached C(4) substituent or use it to stabilize the geometry of the functionally competent dimer state do not indicate the presence of the twisted dimer form in the NMR spectra. The perturbation of crucial structural motifs in the inhibitors correlates with an increasing formation of the inactive twisted dimer state, suggesting these ligands are able to shift a conformational equilibrium from active C2-symmetric to inactive twisted dimer conformations. These findings suggest a novel concept for the design of drug candidates for further development.

Vanadate as a new substrate for nucleoside phosphorylases.

Orthovanadate was shown to serve as a substrate for nucleoside phosphorylases from Escherichia coli, Shewanella oneidensis, Geobacillus stearothermophilus, and Halomonas chromatireducens AGD 8-3. An exception is thymidine phosphorylase from the extremophilic haloalkaliphilic bacterium Halomonas chromatireducens AGD 8-3, which cannot catalyze the vanadolysis of nucleosides. The kinetic parameters of nucleoside vanadolysis were evaluated.

Uniport, Not Proton-Symport, in a Non-Mammalian SLC23 Transporter.

SLC23 family members are transporters of either nucleobases or ascorbate. While the mammalian SLC23 ascorbate transporters are sodium-coupled, the non-mammalian nucleobase transporters have been proposed, but not formally shown, to be proton-coupled symporters. This assignment is exclusively based on in vivo transport assays using protonophores. Here, by establishing the first in vitro transport assay for this protein family, we demonstrate that a representative member of the SLC23 nucleobase transporters operates as a uniporter instead. We explain these conflicting assignments by identifying a critical role of uracil phosphoribosyltransferase, the enzyme converting uracil to UMP, in driving uracil uptake in vivo. Detailed characterization of uracil phosphoribosyltransferase reveals that the sharp reduction of uracil uptake in whole cells in presence of protonophores is caused by acidification-induced enzyme inactivation. The SLC23 family therefore consists of both uniporters and symporters in line with the structurally related SLC4 and SLC26 families that have previously been demonstrated to accommodate both transport modes as well.

Biochemical and structural studies of two tetrameric nucleoside 2'-deoxyribosyltransferases from psychrophilic and mesophilic bacteria: Insights into cold-adaptation.

Nucleoside 2'-deoxyribosyltransferases (NDTs) catalyze the cleavage of glycosidic bonds of 2'-deoxynucleosides and the following transfer of the 2'-deoxyribose moiety to acceptor nucleobases. Here, we report the crystal structures and biochemical properties of the first tetrameric NDTs: the type I NDT from the mesophilic bacterium Enterococcus faecalis V583 (EfPDT) and the type II NDT from the bacterium Desulfotalea psychrophila (DpNDT), the first psychrophilic NDT. This novel structural and biochemical data permitted an exhaustive comparative analysis aimed to shed light into the basis of the high global stability of the psychrophilic DpNDT, which has a higher melting temperature than EfPDT (58.5 °C versus 54.4 °C) or other mesophilic NDTs. DpNDT possesses a combination of unusual structural motifs not present neither in EfPDT nor any other NDT that most probably contribute to its global stability, in particular, a large aliphatic isoleucine-leucine-valine (ILV) bundle accompanied by a vicinal disulfide bridge and also an intersubunit disulfide bridge, the first described for an NDT. The functional and structural features of DpNDT do not fit the standard features of psychrophilic enzymes, which lead us to consider the implication of (sub)cellular levels together with the protein level in the adaptation of enzymatic activity to low temperatures.

Unraveling a Ligand-Induced Twist of a Homodimeric Enzyme by Pulsed Electron-Electron Double Resonance.

Mechanistic insights into protein-ligand interactions can yield chemical tools for modulating protein function and enable their use for therapeutic purposes. For the homodimeric enzyme tRNA-guanine transglycosylase (TGT), a putative virulence target of shigellosis, ligand binding has been shown by crystallography to transform the functional dimer geometry into an incompetent twisted one. However, crystallographic observation of both end states does neither verify the ligand-induced transformation of one dimer into the other in solution nor does it shed light on the underlying transformation mechanism. We addressed these questions in an approach that combines site-directed spin labeling (SDSL) with distance measurements based on pulsed electron-electron double resonance (PELDOR or DEER) spectroscopy. We observed an equilibrium between the functional and twisted dimer that depends on the type of ligand, with a pyranose-substituted ligand being the most potent one in shifting the equilibrium toward the twisted dimer. Our experiments suggest a dissociation-association mechanism for the formation of the twisted dimer upon ligand binding.

Structural and functional insights into human tRNA guanine transgylcosylase.

The eukaryotic tRNA guanine transglycosylase (TGT) is an RNA modifying enzyme incorporating queuine, a hypermodified guanine derivative, into the tRNAsAsp,Asn,His,Tyr. While both subunits of the functional heterodimer have been crystallized individually, much of our understanding of its dimer interface or recognition of a target RNA has been inferred from its more thoroughly studied bacterial homolog. However, since bacterial TGT, by incorporating queuine precursor preQ1, deviates not only in function, but as a homodimer, also in its subunit architecture, any inferences regarding the subunit association of the eukaryotic heterodimer or the significance of its unique catalytically inactive subunit are based on unstable footing. Here, we report the crystal structure of human TGT in its heterodimeric form and in complex with a 25-mer stem loop RNA, enabling detailed analysis of its dimer interface and interaction with a minimal substrate RNA. Based on a model of bound tRNA, we addressed a potential functional role of the catalytically inactive subunit QTRT2 by UV-crosslinking and mutagenesis experiments, identifying the two-stranded ßEßF-sheet of the QTRT2 subunit as an additional RNA-binding motif.

Ile209 of Leishmania donovani xanthine phosphoribosyltransferase plays a key role in determining its purine base specificity.

Xanthine phosphoribosyltransferase (XPRT) and hypoxanthine-guanine phosphoribosyltransferase (HGPRT) are purine salvaging enzymes of Leishmania donovani with distinct 6-oxopurine specificities. LdXPRT phosphoribosylates xanthine, hypoxanthine, and guanine, with preference toward xanthine, whereas LdHGPRT phosphoribosylates only hypoxanthine and guanine. In our study, LdXPRT was used as a model to understand these purine base specificities. Mutating I209 to V, the conserved residue found in HGPRTs, reduced the affinity of LdXPRT for xanthine, converting it to an HGXPRT-like enzyme. The Y208F mutation in the active site indicated that aromatic residue interactions with the purine ring are limited to pi-pi binding forces and do not impart purine base specificity. Deleting the unique motif (L55-Y82) of LdXPRT affected enzyme activity. Our studies established I209 as a key residue determining the 6-oxopurine specificity of LdXPRT.

Targeting a Cryptic Pocket in a Protein-Protein Contact by Disulfide-Induced Rupture of a Homodimeric Interface.

Interference with protein-protein interfaces represents an attractive as well as challenging option for therapeutic intervention and drug design. The enzyme tRNA-guanine transglycosylase, a target to fight Shigellosis, is only functional as a homodimer. Although we previously produced monomeric variants by site-directed mutagenesis, we only crystallized the functional dimer, simply because upon crystallization the local protein concentration increases and favors formation of the dimer interface, which represents an optimal and highly stable packing of the protein in the solid state. Unfortunately, this prevents access to structural information about the interface geometry in its monomeric state and complicates the development of modulators that can interfere with and prevent dimer formation. Here, we report on a cysteine-containing protein variant in which, under oxidizing conditions, a disulfide linkage is formed. This reinforces a novel packing geometry of the enzyme. In this captured quasi-monomeric state, the monomer units arrange in a completely different way and, thus, expose a loop-helix motif, originally embedded into the old interface, now to the surface. The motif adopts a geometry incompatible with the original dimer formation. Via the soaking of fragments into the crystals, we identified several hits accommodating a cryptic binding site next to the loop-helix motif and modulated its structural features. Our study demonstrates the druggability of the interface by breaking up the homodimeric protein using an introduced disulfide cross-link. By rational concepts, we increased the potency of these fragments to a level where we confirmed their binding by NMR to a nondisulfide-linked TGT variant. The idea of intermediately introducing a disulfide linkage may serve as a general concept of how to transform a homodimer interface into a quasi-monomeric state and give access to essential structural and design information.

pH-Independent Heat Capacity Changes during Phosphorolysis Catalyzed by the Pyrimidine Nucleoside Phosphorylase from Geobacillus thermoglucosidasius.

Enzyme-catalyzed reactions sometimes display curvature in their Eyring plots in the absence of denaturation, indicative of a change in activation heat capacity. However, the effects of pH and (de)protonation on this phenomenon have remained unexplored. Herein, we report a kinetic characterization of the thermophilic pyrimidine nucleoside phosphorylase from Geobacillus thermoglucosidasius across a two-dimensional working space covering 35 °C and 3 pH units with two substrates displaying different pKa values. Our analysis revealed the presence of a measurable activation heat capacity change ΔCp⧧ in this reaction system, which showed no significant dependence on medium pH or substrate charge. Our results further describe the remarkable effects of a single halide substitution that has a minor influence on ΔCp⧧ but conveys a significant kinetic effect by decreasing the activation enthalpy, causing a >10-fold rate increase. Collectively, our results present an important piece in the understanding of enzymatic systems across multidimensional working spaces where the choice of reaction conditions can affect the rate, affinity, and thermodynamic phenomena independently of one another.

Helicobacter pylori Xanthine-Guanine-Hypoxanthine Phosphoribosyltransferase-A Putative Target for Drug Discovery against Gastrointestinal Tract Infections.

Helicobacter pylori (Hp) is a human pathogen that lives in the gastric mucosa of approximately 50% of the world's population causing gastritis, peptic ulcers, and gastric cancer. An increase in resistance to current drugs has sparked the search for new Hp drug targets and therapeutics. One target is the disruption of nucleic acid production, which can be achieved by impeding the synthesis of 6-oxopurine nucleoside monophosphates, the precursors of DNA and RNA. These metabolites are synthesized by Hp xanthine-guanine-hypoxanthine phosphoribosyltransferase (XGHPRT). Here, nucleoside phosphonates have been evaluated, which inhibit the activity of this enzyme with Ki values as low as 200 nM. The prodrugs of these compounds arrest the growth of Hp at a concentration of 50 µM in cell-based assays. The kinetic properties of HpXGHPRT have been determined together with its X-ray crystal structure in the absence and presence of 9-[(N-3-phosphonopropyl)-aminomethyl-9-deazahypoxanthine, providing a basis for new antibiotic development.

Development of a xylosyltransferase-I-selective UPLC MS/MS activity assay using a specific acceptor peptide.

Xylosyltransferases-I and -II (XT-I and -II) play an important role regarding the homeostasis of the extracellular matrix. Both enzymes catalyze the initial step of the proteoglycan (PG) biosynthesis by the transfer of xylose from their natural substrate uridine diphosphate (UDP) -xylose to a PG-core protein. The subsequent addition of further sugars, catalyzed by different glycosyltransferases, leads to the formation of a tetrasaccharide linker, which connects the PG-core protein and glycosaminoglycans. The reason for the appearance of two XT isoforms in all higher organisms is not known and remarkable, as both enzymes are able to initiate PG biosynthesis. The determination of the XT-I activity is of clinical importance because it can be used as a biomarker of several PG-associated fibrotic diseases. Since previous assays did not adequately differentiate between both XT-isoforms, the aim of this study was to develop an XT-I selective mass spectrometric (MS) assay. For this purpose, we initially used isoform-specific supernatants to successfully identify a synthetic acceptor peptide which was xylosylated much more selectively by the XT-I when compared to the XT-II isoform. The assay was further optimized concerning methodical parameters such as the injection volume and the incubation time of the reaction-mixture. By using samples covering a broad XT-activity spectrum, we successfully validated the assay to be used not only for the quantification of cell culture samples but also human serum specimens. Compared to previously used XT-activity assays, our newly developed test is more selective and sensitive, less expensive and easier to perform in high throughput.

The Importance of Charge in Perturbing the Aromatic Glue Stabilizing the Protein-Protein Interface of Homodimeric tRNA-Guanine Transglycosylase.

Bacterial tRNA-guanine transglycosylase (Tgt) is involved in the biosynthesis of the modified tRNA nucleoside queuosine present in the anticodon wobble position of tRNAs specific for aspartate, asparagine, histidine, and tyrosine. Inactivation of the tgt gene leads to decreased pathogenicity of Shigella bacteria. Therefore, Tgt constitutes a putative target for Shigellosis drug therapy. Since it is only active as homodimer, interference with dimer-interface formation may, in addition to active-site inhibition, provide further means to disable this protein. A cluster of four aromatic residues seems important to stabilize the homodimer. We mutated residues of this aromatic cluster and analyzed each mutated variant with respect to the dimer and thermal stability or enzyme activity by applying native mass spectrometry, a thermal shift assay, enzyme kinetics, and X-ray crystallography. Our structural studies indicate a strong influence of pH on the homodimer stability. Apparently, protonation of a histidine within the aromatic cluster supports the collapse of an essential structural motif within the dimer interface at slightly acidic pH.

The first report of two homozygous sequence variants in FKRP and SELENON genes associated with syndromic congenital muscular dystrophy in Iran: Further expansion of the clinical phenotypes.

BACKGROUND: Congenital muscular dystrophy (CMD) refers to hypotonia and delayed motor development that is manifested at or near the birth. Additional presentations have been observed in CMD syndromes. METHODS: Thorough clinical examinations were performed on two unrelated Iranian families with typical symptoms of CMD and uncommon features such as intellectual disability and nephrolithiasis. The genomic DNA of probands were subjected to whole exome sequencing. Following the detection of candidate variants with a bioinformatic pipeline, the familial co-segregation analysis was carried out using polymerase chain reaction-based Sanger sequencing. RESULTS: We identified a missense homozygous variant in the fukutin-related protein (FKRP) gene (c.968G>A, p.Arg323His) related to CMD-dystroglycanopathy type B5 (MDDGB5) and a frameshift homozygous variant in the selenoprotein N (SELENON) gene (c.1446delC, p.Asn483Thrfs*11) associated with congenital rigid-spine muscular dystrophy 1 (RSMD1), which were completely segregated with the phenotypes in the families. These variants were not found in either the 1000 Genomes Project or the Exome Aggregation Consortium. The present study provides the first report of these homozygous sequence variants in Iran. Moreover, our study was the first observation of nephrolithiasis in FKRP-related dystroglycanopathy and intellectual disability in SELENON-related myopathies. Based on in silico studies and molecular docking, these variations induced pathogenic effects on the proteins. CONCLUSIONS: Our findings extend the genetic database of Iranian patients with CMD and, in general, the phenotypical spectrum of syndromic CMD. It is recommended to consider these variants for a more accurate clinical interpretation, prenatal diagnosis and genetic counseling in families with a history of CMD, especially in those combined with cognitive impairments or renal dysfunctions.

Cryo-EM structure of arabinosyltransferase EmbB from Mycobacterium smegmatis.

Arabinosyltransferase B (EmbB) belongs to a family of membrane-bound glycosyltransferases that build the lipidated polysaccharides of the mycobacterial cell envelope, and are targets of anti-tuberculosis drug ethambutol. We present the 3.3 Å resolution single-particle cryo-electron microscopy structure of Mycobacterium smegmatis EmbB, providing insights on substrate binding and reaction mechanism. Mutations that confer ethambutol resistance map mostly around the putative active site, suggesting this to be the location of drug binding.

Fragment Screening Hit Draws Attention to a Novel Transient Pocket Adjacent to the Recognition Site of the tRNA-Modifying Enzyme TGT.

Fragment-based lead discovery was applied to tRNA-guanine transglycosylase, an enzyme modifying post-transcriptionally tRNAs in Shigella, the causative agent of shigellosis. TGT inhibition prevents translation of Shigella's virulence factor VirF, hence reducing pathogenicity. One discovered fragment opens a transient subpocket in the preQ1-recognition site by pushing back an aspartate residue. This step is associated with reorganization of further amino acids structurally transforming a loop adjacent to the recognition site by duplicating the volume of the preQ1-recognition pocket. We synthesized 6-carboxamido-, 6-hydrazido-, and 4-guanidino-benzimidazoles to target the opened pocket, including a dihydro-imidazoquinazoline with a propyn-1-yl exit vector pointing into the transient pocket and displacing a conserved water network. MD simulations and hydration-site analysis suggest water displacement to contribute favorably to ligand binding. A cysteine residue, exclusively present in bacterial TGTs, serves as gatekeeper of the transient subpocket. It becomes accessible upon pocket opening for selective covalent attachment of electrophilic ligands in eubacterial TGTs.

Cryo-EM snapshots of mycobacterial arabinosyltransferase complex EmbB2-AcpM2.

Inhibition of Mycobacterium tuberculosis (Mtb) cell wall assembly is an established strategy for anti-TB chemotherapy. Arabinosyltransferase EmbB, which catalyzes the transfer of arabinose from the donor decaprenyl-phosphate-arabinose (DPA) to its arabinosyl acceptor is an essential enzyme for Mtb cell wall synthesis. Analysis of drug resistance mutations suggests that EmbB is the main target of the front-line anti-TB drug, ethambutol. Herein, we report the cryo-EM structures of Mycobacterium smegmatis EmbB in its "resting state" and DPA-bound "active state". EmbB is a fifteen-transmembrane-spanning protein, assembled as a dimer. Each protomer has an associated acyl-carrier-protein (AcpM) on their cytoplasmic surface. Conformational changes upon DPA binding indicate an asymmetric movement within the EmbB dimer during catalysis. Functional studies have identified critical residues in substrate recognition and catalysis, and demonstrated that ethambutol inhibits transferase activity of EmbB by competing with DPA. The structures represent the first step directed towards a rational approach for anti-TB drug discovery.