Thiamine Pyrophosphate Binding Research Articles

Structural Characterization of the Cotranscriptional Folding of the Thiamin Pyrophosphate Sensing thiC Riboswitch in Escherichia coli.

Riboswitches are RNA-regulating elements that mostly rely on structural changes to modulate gene expression at various levels. Recent studies have revealed that riboswitches may control several regulatory mechanisms cotranscriptionally, i.e., during the transcription elongation of the riboswitch or early in the coding region of the regulated gene. Here, we study the structure of the nascent thiamin pyrophosphate (TPP)-sensing thiC riboswitch in Escherichia coli by using biochemical and enzymatic conventional probing approaches. Our chemical (in-line and lead probing) and enzymatic (nucleases S1, A, T1, and RNase H) probing data provide a comprehensive model of how TPP binding modulates the structure of the thiC riboswitch. Furthermore, by using transcriptional roadblocks along the riboswitch sequence, we find that a certain portion of nascent RNA is needed to sense TPP that coincides with the formation of the P5 stem loop. Together, our data suggest that conventional techniques may readily be used to study cotranscriptional folding of nascent RNAs.

The protein family of pyruvate:quinone oxidoreductases: Amino acid sequence conservation and taxonomic distribution

Pyruvate:quinone oxidoreductases (PQOs) catalyse the oxidative decarboxylation of pyruvate to acetate and concomitant reduction of quinone to quinol with the release of CO2. They are thiamine pyrophosphate (TPP) and flavin-adenine dinucleotide (FAD) containing enzymes, which interact with the membrane in a monotopic way. PQOs are considered as part of alternatives to most recognized pyruvate catabolizing pathways, and little is known about their taxonomic distribution and structural/functional relationship.In this bioinformatics work we tackled these gaps in PQO knowledge. We used the KEGG database to identify PQO coding genes, performed a multiple sequence analysis which allowed us to study the amino acid conservation on these enzymes, and looked at their possible cellular function. We observed that PQOS are enzymes exclusively present in prokaryotes with most of the sequences identified in bacteria. Regarding the amino acid sequence conservation, we found that 75 amino acid residues (out of 570, on average) have a conservation over 90 %, and that the most conserved regions in the protein are observed around the TPP and FAD binding sites. We systematized the presence of conserved features involved in Mg2+, TPP and FAD binding, as well as residues directly linked to the catalytic mechanism. We also established the presence of a new motif named “HEH lock”, possibly involved in the dimerization process. The results here obtained for the PQO protein family contribute to a better understanding of the biochemistry of these respiratory enzymes.

Thiamine-responsive pyruvate dehydrogenase complex deficiency presenting as recurrent muscle weakness: Identification of a novel mutation (p.T111I) in the PDHA1 gene.

Thiamine-responsive pyruvate dehydrogenase complex deficiency presenting as recurrent muscle weakness: Identification of a novel mutation (p.T111I) in the PDHA1 gene.

Reassessing the evolution of the 1-deoxy-D-xylulose 5-phosphate synthase family suggests a possible novel function for the DXS class 3 proteins

The methylerythritol 4-phosphate (MEP) pathway is of paramount importance for generating plastidial isoprenoids. The first enzyme of the MEP pathway, 1-deoxy-D-xylulose-5-phosphate synthase (DXS), catalyzes a flux-controlling step. In plants the DXS gene family is composed of three distinct classes with non-redundant functions. Although the DXS1 and DXS2 subfamilies have been well characterized, the DXS3 subfamily has been considerably understudied. Here, we carried out in silico and functional analyses to better understand the DXS3 class. Our phylogenetic analysis showed high variation in copy number among the different DXS classes, with the apparent absence of DXS1 class in some species. We found that DXS3 subfamily emerged later than DXS1 and DXS2 and it is under less intense purifying selection. Furthermore, in the DXS3 subfamily critical amino acids positions in the thiamine pyrophosphate binding pocket are not conserved. We demonstrated that the DXS3 proteins from Arabidopsis, Maize, and Rice lack functional DXS activity. Moreover, the Arabidopsis DXS3 protein displayed distinctive sub-organellar chloroplast localization not observed in any DXS1 or DXS2 proteins. Co-expression analysis of the DXS3 from Arabidopsis showed that, unlike DXS1 and DXS2 proteins, it co-expresses with genes related to post-embryonic development and reproduction and not with primary metabolism and isoprenoid synthesis.

Ensemble Switching Unveils a Kinetic Rheostat Mechanism of the Eukaryotic Thiamine Pyrophosphate Riboswitch.

Thiamine pyrophosphate (TPP) riboswitches regulate thiamine metabolism by inhibiting the translation of enzymes essential to thiamine synthesis pathways upon binding to thiamine pyrophosphate in cells across all domains of life. Recent work on the Arabidopsis thaliana TPP riboswitch suggests a multistep TPP binding process involving multiple riboswitch configurational ensembles and Mg2+ dependence underlies the mechanism of TPP recognition and subsequent transition to the expression-inhibiting state of the aptamer domain followed by changes in the expression platform. However, details of the relationship between TPP riboswitch conformational changes and interactions with TPP and Mg2+ in the aptamer domain constituting this mechanism are unknown. Therefore, we integrated single-molecule multiparameter fluorescence and force spectroscopy with atomistic molecular dynamics simulations and found that conformational transitions within the aptamer domain's sensor helices associated with TPP and Mg2+ ligand binding occurred between at least five different ensembles on timescales ranging from µs to ms. These dynamics are orders of magnitude faster than the 10 sec-timescale folding kinetics associated with expression-state switching in the switch helix. Together, our results show that a TPP and Mg2+ dependent mechanism determines dynamic configurational state ensemble switching of the aptamer domain's sensor helices that regulate the switch helix's stability, which ultimately may lead to the expression-inhibiting state of the riboswitch. Additionally, we propose that two pathways exist for ligand recognition and that this mechanism underlies a kinetic rheostat-like behavior of the Arabidopsis thaliana TPP riboswitch.

Structural and functional impact of clinically relevant E1α variants causing pyruvate dehydrogenase complex deficiency

Pyruvate dehydrogenase complex (PDC) catalyzes the oxidative decarboxylation of pyruvate to acetyl-coenzyme A, hinging glycolysis and the tricarboxylic acid cycle. PDC deficiency, an inborn error of metabolism, has a broad phenotypic spectrum. Symptoms range from fatal lactic acidosis or progressive neuromuscular impairment in the neonatal period, to chronic neurodegeneration. Most disease-causing mutations in PDC deficiency affect the PDHA1 gene, encoding the α subunit of the PDC-E1 component. Detailed biophysical analysis of pathogenic protein variants is a challenging approach to support the design of therapies based on improving and correcting protein structure and function. Herein, we report the characterization of clinically relevant PDC-E1α variants identified in Portuguese PDC deficient patients. These variants bear amino acid substitutions in different structural regions of PDC-E1α. The structural and functional analyses of recombinant heterotetrameric (αα’ββ’) PDC-E1 variants, combined with molecular dynamics (MD) simulations, show a limited impact of the amino acid changes on the conformational stability, apart from the increased propensity for aggregation of the p.R253G variant as compared to wild-type PDC-E1. However, all variants presented a functional impairment in terms of lower residual PDC-E1 enzymatic activity and ≈3–100 × lower affinity for the thiamine pyrophosphate (TPP) cofactor, in comparison with wild-type PDC-E1. MD simulations neatly showed generally decreased stability (increased flexibility) of all variants with respect to the WT heterotetramer, particularly in the TPP binding region. These results are discussed in light of disease severity of the patients bearing such mutations and highlight the difficulty of developing chaperone-based therapies for PDC deficiency.

The Two-Species Model of transketolase explains donor substrate-binding, inhibition and heat-activation

We recently characterised a low-activity form of E. coli transketolase, TKlow, which also binds the cofactor thiamine pyrophosphate (TPP) with an affinity up to two-orders of magnitude lower than the previously known high TPP-affinity and high-activity form, TKhigh, in the presence of Mg2+. We observed previously that partial oxidation was responsible for increased TKhigh activity, while low-activity TKlow was unmodified. In the present study, the fluorescence-based cofactor-binding assay was adapted to detect binding of the β-hydroxypyruvate (HPA) donor substrate to wild-type transketolase and a variant, S385Y/D469T/R520Q, that is active towards aromatic aldehydes. Transketolase HPA affinity again revealed the two distinct forms of transketolase at a TKhigh:TKlow ratio that matched those observed previously via TPP binding to each variant. The HPA dissociation constant of TKlow was comparable to the substrate-inhibition dissociation constant, KiHPA, determined previously. We provide evidence that KiHPA is a convolution of binding to the low-activity TKlow-TKlow dimer, and the TKlow subunit of the partially-active TKhigh-TKlow mixed dimer, where HPA binding to the TKlow subunit of the mixed dimer results in inhibition of the active TKhigh subunit. Heat-activation of transketolase was similarly investigated and found to convert the TKlow subunit of the mixed dimer to have TKhigh-like properties, but without oxidation.

Effect of Mg2+ Ions on TPP Riboswitch Aptamer Folding

Riboswitch is an RNA molecule that can regulate transcription or translation by sensing ligands. In the thiamine pyrophosphate (TPP) riboswitch, the TPP ligand binds to the riboswitch to regulate gene expression. TPP riboswitch has two binding pockets: one for TPP ligand and other for Mg2+ ions. In the folded state, TPP aptamer has two arms composed of five helices. One arm is composed of P2-P3 helices and the other arm is composed of P4-P5 helices. P1 helix acts as base holding both arms from P2 and P4 helices. We have studied the effect of Mg2+ ions on the conformational transitions in the aptamer domain of TPP riboswitch using three-interaction-site (TIS) coarse-grained model and molecular dynamics simulation. In low [Mg2+] (< 4 mM), aptamer adopts unfolded conformation leading to the disruption of the TPP binding pocket, whereas in high [Mg2+] (> 6 mM), it adopts a folded conformation facilitating the formation and stabilization of TPP binding pocket. At intermediate [Mg2+] (4-6 mM), an intermediate is populated, where the P1 and P2 helices form. Formation of these helices allows the aptamer to adopt a Y-shaped conformation. In this concentration range, the aptamer displays transitions between a close arm Y-shaped conformation forming TPP binding pocket and the open arm Y-shaped conformation disrupting TPP binding pocket.

Novel insights into transketolase activation by cofactor binding identifies two native species subpopulations

Transketolase (TK) cofactor binding has been studied extensively over many years, yet certain mysteries remain, such as a lack of consensus on the cooperativity of thiamine pyrophosphate (TPP) binding into the two active sites, in the presence and absence of the divalent cation, Mg2+. Using a novel fluorescence-based assay, we determined directly the dissociation constants and cooperativity of TPP binding and provide the first comprehensive study over a broad range of cofactor concentrations. We confirmed the high-affinity dissociation constants and revealed a dependence of both the affinity and cooperativity of binding on [Mg2+], which explained the previous lack of consensus. A second, discrete and previously uncharacterised low-affinity TPP binding-site was also observed, and hence indicated the existence of two forms of TK with high- (TKhigh) and low-affinity (TKlow). The relative proportions of each dimer were independent of the monomer-dimer transition, as probed by analytical ultracentrifugation at various [TK]. Mass spectrometry revealed that chemical oxidation of TKlow led to the formation of TKhigh, which was 22-fold more active than TKlow. Finally, we propose a two-species model of transketolase activation that describes the interconversions between apo-/holo-TKhigh and TKlow, and the potential to significantly improve biocatalytic activity by populating only the most active form.

Role of a hairpin-stabilized pause in the Escherichia coli thiC riboswitch function

ABSTRACTTranscriptional pauses have been reported in bacterial riboswitches and, in some cases, their specific positioning has been shown to be important for gene regulation. Here, we show that a hairpin structure in the Escherichia coli thiamin pyrophosphate (TPP) thiC riboswitch is involved in transcriptional pausing and ligand sensitivity. Using in vitro transcription kinetic experiments, we show that all three major transcriptional pauses in the thiC riboswitch are affected by NusA, a transcriptional factor known to stimulate hairpin-stabilized pauses. Using a truncated region of the riboswitch, we isolated the hairpin structure responsible for stabilization of the most upstream pause. Destabilization of this structure led to a weaker pause and a decreased NusA effect. In the context of the full-length riboswitch, this same mutation also led to a weaker pause, as well as a decreased TPP binding affinity. Our work suggests that RNA structures involved in transcriptional pausing in riboswitches are important for ligand sensitivity, most likely by increasing the time allowed to the ligand for binding to the riboswitch.

Co-Transcriptional Molecular Assembly Results in a Kinetically Controlled Irreversible RNA Conformational Switch.

Co-transcriptional RNA folding results in dynamic behavior of RNA transcripts. Extra stimuli can perturb the stability of the nascent RNA and alter its conformation and function. Here, an artificial RNA conformational switch, which consists of a 5' aptamer unit for sensing thiamine pyrophosphate (TPP) and a 3' working unit (TAR RNA) that binds Tat peptide connected by a switching sequence, was designed and cotranscriptionally functionalized. Co-transcriptional binding of TPP induced an RNA conformational change that allowed interaction of Tat peptide and the TAR RNA unit. Two conformational states, which are mutually exclusive depending on the TPP binding, did not show post-transcriptional interconversion. The irreversible RNA conformational switch demonstrated here, which kinetically coupled co-transcriptional RNA folding and molecular assembly in nonequilibrium biological systems, will enable efficient switching of RNA functions applicable for constructing intracellular molecular sensors.

Conformational Dynamics of thiM Riboswitch To Understand the Gene Regulation Mechanism Using Markov State Modeling and the Residual Fluctuation Network Approach.

Thiamine pyrophosphate (TPP) riboswitch is a cis-regulatory element in the noncoding region of mRNA. The aptamer domain of TPP riboswitch detects the high abundance of coenzyme thiamine pyrophosphate (TPP) and modulates the gene expression for thiamine synthetic gene. The mechanistic understanding in recognition of TPP in aptamer domain and ligand-induced compactness for folding of expression platform are most important to designing novel modulators. To understand the dynamic behavior of TPP riboswitch upon TPP binding, molecular dynamics simulations were performed for 400 ns in both apo and TPP bound forms of thiM riboswitch from E. coli and analyzed in terms of eRMSD-based Markov state modeling and residual fluctuation network. Markov state models show good correlations in transition probability among metastable states from simulated trajectory and generated models. Structural compactness in TPP bound form is observed which is correlated with SAXS experiment. The importance of junction of P4 and P5 is evident during dynamics, which correlates with FRET analysis. The dynamic nature of two sensor forearms is due to the flexible P1 helix, which is its intrinsic property. The transient state in TPP-bound form was observed in the Markov state model, along with stable states. We believe that this transient state is responsible to assist the influx and outflux of ligand molecule by creating a solvent channel around the junction region of P4 and P5 and such a structure was anticipated in FRET analysis. The dynamic nature of riboswitch is dependent on the interaction between residues on distal loops L3 and L5/P3 and junction P4 and P5, J3/2 which stabilize the J2/4. It helps in the transfer of allosteric information between J2/4 and P3/L5 tertiary docking region through the active site residues. Understanding such information flow will benefit in highlighting crucial residues in highly dynamic and kinetic systems. Here, we report the residues and segments in riboswitch that play vital roles in providing stability and this can be exploited in designing inhibitors to regulate the functioning of riboswitches.

Lighting up riboswitching

RNA Folding RNAs fold as they are synthesized, and this folding is required for function. Uhm et al. describe a single-molecule fluorescence energy transfer assay to monitor cotranscriptional RNA folding. This approach revealed folding in the thiamine pyrophosphate (TPP) riboswitch that regulates translation of genes involved in the synthesis of thiamine, an essential vitamin. The riboswitch folds into the “off” conformation, in which translation is inhibited, even in the absence of the TPP ligand. If TPP is not bound to this off conformation, it can switch to the “on” conformation when transcription pauses near the translation start codon, and this allows translation to start. TPP binding stabilizes the off conformation and prevents the switch. The assay will allow investigation of other cases in which transcriptional speed and pausing affect RNA folding. Proc. Natl. Acad. Sci. U.S.A. 10.1073/pnas.1712983115 (2017).

Ligand Recognition Mechanism of Thiamine Pyrophosphate Riboswitch Aptamer

Riboswitches regulate gene expression by coupling ligand binding to a structural transition of the riboswitch, but the coupling mechanism is still controversial. We addressed this issue by characterizing both the ligand‐free state of the Escherichia coli thiamine pyrophosphate (TPP) riboswitch aptamer and its structural transition upon ligand binding using single‐molecule fluorescence resonance energy transfer (FRET). Our results reveal that the apo‐aptamer dynamically samples a partially closed form resembling the holo‐aptamer, but TPP binding occurs in both the open and partially closed forms with the same efficiency. Mutation studies reveal that the preformation of the aptamer secondary structure is critical for TPP binding, and that tertiary interaction is established after TPP binding.

Transcriptional pausing at the translation start site operates as a critical checkpoint for riboswitch regulation

On the basis of nascent transcript sequencing, it has been postulated but never demonstrated that transcriptional pausing at translation start sites is important for gene regulation. Here we show that the Escherichia coli thiamin pyrophosphate (TPP) thiC riboswitch contains a regulatory pause site in the translation initiation region that acts as a checkpoint for thiC expression. By biochemically probing nascent transcription complexes halted at defined positions, we find a narrow transcriptional window for metabolite binding, in which the downstream boundary is delimited by the checkpoint. We show that transcription complexes at the regulatory pause site favour the formation of a riboswitch intramolecular lock that strongly prevents TPP binding. In contrast, cotranscriptional metabolite binding increases RNA polymerase pausing and induces Rho-dependent transcription termination at the checkpoint. Early transcriptional pausing may provide a general mechanism, whereby transient transcriptional windows directly coordinate the sensing of environmental cues and bacterial mRNA regulation.

Quantitative and predictive model of kinetic regulation by E. coli TPP riboswitches

ABSTRACTRiboswitches are non-coding elements upstream or downstream of mRNAs that, upon binding of a specific ligand, regulate transcription and/or translation initiation in bacteria, or alternative splicing in plants and fungi. We have studied thiamine pyrophosphate (TPP) riboswitches regulating translation of thiM operon and transcription and translation of thiC operon in E. coli, and that of THIC in the plant A. thaliana. For all, we ascertained an induced-fit mechanism involving initial binding of the TPP followed by a conformational change leading to a higher-affinity complex. The experimental values obtained for all kinetic and thermodynamic parameters of TPP binding imply that the regulation by A. thaliana riboswitch is governed by mass-action law, whereas it is of kinetic nature for the two bacterial riboswitches. Kinetic regulation requires that the RNA polymerase pauses after synthesis of each riboswitch aptamer to leave time for TPP binding, but only when its concentration is sufficient. A quantitative model of regulation highlighted how the pausing time has to be linked to the kinetic rates of initial TPP binding to obtain an ON/OFF switch in the correct concentration range of TPP. We verified the existence of these pauses and the model prediction on their duration. Our analysis also led to quantitative estimates of the respective efficiency of kinetic and thermodynamic regulations, which shows that kinetically regulated riboswitches react more sharply to concentration variation of their ligand than thermodynamically regulated riboswitches. This rationalizes the interest of kinetic regulation and confirms empirical observations that were obtained by numerical simulations.

In Vitro Selection for Small-Molecule-Triggered Strand Displacement and Riboswitch Activity.

An in vitro selection method for ligand-responsive RNA sensors was developed that exploited strand displacement reactions. The RNA library was based on the thiamine pyrophosphate (TPP) riboswitch, and RNA sequences capable of hybridizing to a target duplex DNA in a TPP regulated manner were identified. After three rounds of selection, RNA molecules that mediated a strand exchange reaction upon TPP binding were enriched. The enriched sequences also showed riboswitch activity. Our results demonstrated that small-molecule-responsive nucleic acid sensors can be selected to control the activity of target nucleic acid circuitry.

Feedback Inhibition of Deoxy-d-xylulose-5-phosphate Synthase Regulates the Methylerythritol 4-Phosphate Pathway

The 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway leads to the biosynthesis of isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP), the precursors for isoprene and higher isoprenoids. Isoprene has significant effects on atmospheric chemistry, whereas other isoprenoids have diverse roles ranging from various biological processes to applications in commercial uses. Understanding the metabolic regulation of the MEP pathway is important considering the numerous applications of this pathway. The 1-deoxy-D-xylulose-5-phosphate synthase (DXS) enzyme was cloned from Populus trichocarpa, and the recombinant protein (PtDXS) was purified from Escherichia coli. The steady-state kinetic parameters were measured by a coupled enzyme assay. An LC-MS/MS-based assay involving the direct quantification of the end product of the enzymatic reaction, 1-deoxy-D-xylulose 5-phosphate (DXP), was developed. The effect of different metabolites of the MEP pathway on PtDXS activity was tested. PtDXS was inhibited by IDP and DMADP. Both of these metabolites compete with thiamine pyrophosphate for binding with the enzyme. An atomic structural model of PtDXS in complex with thiamine pyrophosphate and Mg(2+) was built by homology modeling and refined by molecular dynamics simulations. The refined structure was used to model the binding of IDP and DMADP and indicated that IDP and DMADP might bind with the enzyme in a manner very similar to the binding of thiamine pyrophosphate. The feedback inhibition of PtDXS by IDP and DMADP constitutes an important mechanism of metabolic regulation of the MEP pathway and indicates that thiamine pyrophosphate-dependent enzymes may often be affected by IDP and DMADP.

Observation of Intrinsic Conformational Dynamics of TPP Riboswitch and its Structural Transition Induced by TPP Binding

A riboswitch is a non-coding region of an mRNA which recognizes a specific metabolite and regulates the expression of the mRNA itself. They are composed of two domains: an aptamer domain which recognizes a metabolite, and downstream expression platform. It is believed that metabolite binding to the aptamer domain induces a structural change of the riboswitch, signaling “on” or “off” for the transcription or translation of the mRNA.Recent publications of a number of high-resolution structures of ligand-bound aptamer domains (halo-form) greatly contributed to our understanding of how the riboswitches interact with their natural ligands at the atomic-level. However, little is known about the nature of the unbound form of riboswitches (apo-form), and how conformational changes of riboswitches is induced by binding of a ligand to the aptamer domain.We used fluorescence resonance energy transfer (FRET) to study conformational dynamics of the Escherichia coli thiM TPP (thiamine pyrophosphate) riboswitch. We found that even though the apo-aptamer dynamically samples a closed conformation resembling the halo-aptamer, both the open form and the closed form recognize the ligand with equal preference. And the final structural transition of the riboswitch induced by the ligand is also observed. Both indicate that the ligand is recognized via the induced-fit mechanism. Furthermore, the role of Mg ions on the ligand recognition mechanism will be also discussed.

Examining strategies to facilitate vitamin B1 biofortification of plants by genetic engineering

Thiamin (vitamin B1) is made by plants and microorganisms but is an essential micronutrient in the human diet. All organisms require it as a cofactor in its form as thiamin pyrophosphate (TPP) for the activity of key enzymes of central metabolism. In humans, deficiency is widespread particularly in populations where polished rice is a major component of the diet. Considerable progress has been made on the elucidation of the biosynthesis pathway within the last few years enabling concrete strategies for biofortification purposes to be devised, with a particular focus here on genetic engineering. Furthermore, the vitamin has been shown to play a role in both abiotic and biotic stress responses. The precursors for de novo biosynthesis of thiamin differ between microorganisms and plants. Bacteria use intermediates derived from purine and isoprenoid biosynthesis, whereas the pathway in yeast involves the use of compounds from the vitamin B3 and B6 groups. Plants on the other hand use a combination of the bacterial and yeast pathways and there is subcellular partitioning of the biosynthesis steps. Specifically, thiamin biosynthesis occurs in the chloroplast of plants through the separate formation of the pyrimidine and thiazole moieties, which are then coupled to form thiamin monophosphate (TMP). Phosphorylation of thiamin to form TPP occurs in the cytosol. Therefore, thiamin (or TMP) must be exported from the chloroplast to the cytosol for the latter step to be executed. The regulation of biosynthesis is mediated through riboswitches, where binding of the product TPP to the pre-mRNA of a biosynthetic gene modulates expression. Here we examine and hypothesize on genetic engineering approaches attempting to increase the thiamin content employing knowledge gained with the model plant Arabidopsis thaliana. We will discuss the regulatory steps that need to be taken into consideration and can be used a prerequisite for devising such strategies in crop plants.