Design of thiamine analogues for inhibition of thiamine diphosphate (ThDP)-dependent enzymes: Systematic investigation through Scaffold-Hopping and C2-Functionalisation.

Thiamine diphosphate (ThDP), the bioactive form of vitamin B1, is an essential coenzyme needed for processes of cellular metabolism in all organisms. ThDP-dependent enzymes all require ThDP as a coenzyme for catalytic activity, although individual enzymes vary significantly in substrate preferences and biochemical reactions. A popular way to study the role of these enzymes through chemical inhibition is to use thiamine/ThDP analogues, which typically feature a neutral aromatic ring in place of the positively charged thiazolium ring of ThDP. While ThDP analogues have aided work in understanding the structural and mechanistic aspects of the enzyme family, at least two key questions regarding the ligand design strategy remain unresolved: 1) which is the best aromatic ring? and 2) how can we achieve selectivity towards a given ThDP-dependent enzyme? In this work, we synthesise derivatives of these analogues covering all central aromatic rings used in the past decade and make a head-to-head comparison of all the compounds as inhibitors of several ThDP-dependent enzymes. Thus, we establish the relationship between the nature of the central ring and the inhibitory profile of these ThDP-competitive enzyme inhibitors. We also demonstrate that introducing a C2-substituent onto the central ring to explore the unique substrate-binding pocket can further improve both potency and selectivity.

Crystal structure of Escherichia coli thiamine pyrophosphate-sensing riboswitch in the apo state.

The thiamine pyrophosphate (TPP)-sensing riboswitch is one of the earliest discovered and most widespread riboswitches. Numerous structural studies have been reported for this riboswitch bound with various ligands. However, the ligand-free (apo) structure remains unknown. Here, we report a 3.1 Å resolution crystal structure of Escherichia coli TPP riboswitch in the apo state, which exhibits an extended, Y-shaped conformation further supported by small-angle X-ray scattering data and driven molecular dynamics simulations. The loss of ligand interactions results in helical uncoiling of P5 and disruption of the key tertiary interaction between the sensory domains. Opening of the aptamer propagates to the gene-regulatory P1 helix and generates the key conformational flexibility needed for the switching behavior. Much of the ligand-binding site at the three-way junction is unaltered, thereby maintaining a partially preformed pocket. Together, these results paint a dynamic picture of the ligand-induced conformational changes in TPP riboswitches that confer conditional gene regulation.

Ambient temperature structure of phosphoketolase from Bifidobacterium longum determined by serial femtosecond X-ray crystallography.

Phosphoketolase and transketolase are thiamine diphosphate-dependent enzymes and play a central role in the primary metabolism of bifidobacteria: the bifid shunt. The enzymes both catalyze phosphorolytic cleavage of xylulose 5-phosphate or fructose 6-phosphate in the first reaction step, but possess different substrate specificity in the second reaction step, where phosphoketolase and transketolase utilize inorganic phosphate (Pi) and D-ribose 5-phosphate, respectively, as the acceptor substrate. Structures of Bifidobacterium longum phosphoketolase holoenzyme and its complex with a putative inhibitor, phosphoenolpyruvate, were determined at 2.5 Šresolution by serial femtosecond crystallography using an X-ray free-electron laser. In the complex structure, phosphoenolpyruvate was present at the entrance to the active-site pocket and plugged the channel to thiamine diphosphate. The phosphate-group position of phosphoenolpyruvate coincided well with those of xylulose 5-phosphate and fructose 6-phosphate in the structures of their complexes with transketolase. The most striking structural change was observed in a loop consisting of Gln546-Asp547-His548-Asn549 (the QN-loop) at the entrance to the active-site pocket. Contrary to the conformation of the QN-loop that partially covers the entrance to the active-site pocket (`closed form') in the known crystal structures, including the phosphoketolase holoenzyme and its complexes with reaction intermediates, the QN-loop in the current ambient structures showed a more compact conformation with a widened entrance to the active-site pocket (`open form'). In the phosphoketolase reaction, the `open form' QN-loop may play a role in providing the binding site for xylulose 5-phosphate or fructose 6-phosphate in the first step, and the `closed form' QN-loop may help confer specificity for Pi in the second step.

Exploring the structure, function of thiamine pyrophosphate riboswitch, and designing small molecules for antibacterial activity.

During the last decade, riboswitches emerged as new small-molecule sensing RNA in bacteria. Thiamine pyrophosphate (TPP) riboswitch is widely distributed and occurs in plants, bacteria, fungi, and archaea. Extensive biochemical, structural, and genetic studies have been carried out to elucidate the recognition mechanism of TPP riboswitches. However, a comprehensive report summarizing all information on recognition principles and newly designed ligands for TPP riboswitch is scarce in the literature. This review gives a comprehensive understanding of the TPP riboswitch's structure, mechanism, and methods applied to design ligands for the TPP riboswitch. The ligand-bound TPP riboswitch was studied with various experimental and theoretical techniques to elucidate the conformational dynamics. The mutation studies shed light on the significance of pyrimidine sensing helix for the binding of ligands. Further, the structure-activity relationship study and fragment-based approach lead to the development of ligands with Kd values at the sub-micromolar level. However, there is a need to design more potent inhibitors for TPP riboswitch for therapeutic applications. The recent advancements in ligand design highlight the TPP riboswitch as a promising target for developing new antibiotics. This article is categorized under: Regulatory RNAs/RNAi/Riboswitches > Riboswitches Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs.

Entropy Driving the Mg2+-Induced Folding of TPP Riboswitch RNA.

Mg2+ is well known to facilitate the structural folding of RNA. However, the thermodynamic and dynamic roles of Mg2+ in RNA folding remain elusive. Here, we exploit single-molecule fluorescence resonance energy transfer (smFRET) and isothermal titration calorimetry (ITC) to study the mechanism of Mg2+ in facilitating the folding of thiamine pyrophosphate (TPP) riboswitch RNA. The results of smFRET identify that the presence of Mg2+ compacts the RNA and enlarges the conformational dispersity among individual RNA molecules, resulting in a large gain of entropy. The compact yet flexible conformations triggered by Mg2+ may help the riboswitch recognize its specific ligand and further fold. This is supported by the ITC experiments, in which the Mg2+-induced RNA folding is driven by entropy (ΔS) instead of enthalpy (ΔH). Our results complement the understanding of the Mg2+-induced RNA folding. The strategy developed in this work can be used to model other RNAs' folding under different conditions.

Frontiers in the enzymology of thiamin diphosphate-dependent enzymes.

Enzymes that use thiamin diphosphate (ThDP), the biologically active derivative of vitamin B1, as a cofactor play important roles in cellular metabolism in all domains of life. The analysis of ThDP enzymes in the past decades have provided a general framework for our understanding of enzyme catalysis of this protein family. In this review, we will discuss recent advances in the field that include the observation of "unusual" reactions and reaction intermediates that highlight the chemical versatility of the thiamin cofactor. Further topics cover the structural basis of cooperativity of ThDP enzymes, novel insights into the mechanism and structure of selected enzymes, and the discovery of "superassemblies" as reported, for example, acetohydroxy acid synthase. Finally, we summarize recent findings in the structural organisation and mode of action of 2-keto acid dehydrogenase multienzyme complexes and discuss future directions of this exciting research field.

SHAPE-enabled fragment-based ligand discovery for RNA.

The transcriptome represents an attractive but underused set of targets for small-molecule ligands. Here, we devise a technology that leverages fragment-based screening and SHAPE-MaP RNA structure probing to discover small-molecule fragments that bind an RNA structure of interest. We identified fragments and cooperatively binding fragment pairs that bind to the thiamine pyrophosphate (TPP) riboswitch with millimolar to micromolar affinities. We then used structure-activity relationship information to efficiently design a linked-fragment ligand, with no resemblance to the native ligand, with high ligand efficiency and druglikeness, that binds to the TPP thiM riboswitch with high nanomolar affinity and that modulates RNA conformation during cotranscriptional folding. Principles from this work are broadly applicable, leveraging cooperativity and multisite binding, for developing high-quality ligands for diverse RNA targets.

TPP Riboswitch Populates Holo-Form-like Structure Even in the Absence of Cognate Ligand at High Mg2+ Concentration.

Riboswitches are noncoding RNA that regulate gene expression by folding into specific three-dimensional structures (holo-form) upon binding by their cognate ligand in the presence of Mg2+. Riboswitch functioning is also hypothesized to be under kinetic control requiring large cognate ligand concentrations. We ask the question under thermodynamic conditions, can the riboswitches populate structures similar to the holo-form only in the presence of Mg2+ and absence of cognate ligand binding. We addressed this question using thiamine pyrophosphate (TPP) riboswitch as a model system and computer simulations using a coarse-grained model for RNA. The folding free energy surface (FES) shows that with the initial increase in Mg2+ concentration ([Mg2+]), the aptamer domain (AD) of TPP riboswitch undergoes a barrierless collapse in its dimensions. On further increase in [Mg2+], intermediates separated by barriers appear on the FES, and one of the intermediates has a TPP ligand-binding competent structure. We show that site-specific binding of the Mg2+ aids in the formation of tertiary contacts. For [Mg2+] greater than physiological concentration, AD folds into a structure similar to the crystal structure of the TPP holo-form even in the absence of the TPP ligand. The folding kinetics shows that TPP AD populates an intermediate due to the misalignment of two arms present in the structure, which acts as a kinetic trap, leading to larger folding timescales. The predictions of the intermediate structures from the simulations are amenable for experimental verification.

Molecular mechanism of thiamine pyrophosphate import into mitochondria: a molecular simulation study.

The import of thiamine pyrophosphate (TPP) through both mitochondrial membranes was studied using a total of 3-µs molecular dynamics simulations. Regarding the translocation through the mitochondrial outer membrane, our simulations support the conjecture that TPP uses the voltage-dependent anion channel, the major pore of this membrane, for its passage to the intermembrane space, as its transport presents significant analogies with that used by other metabolites previously studied, in particular with ATP. As far as passing through the mitochondrial inner membrane is concerned, our simulations show that the specific carrier of TPP has a single binding site that becomes accessible, through an alternating access mechanism. The preference of this transporter for TPP can be rationalized mainly by three residues located in the binding site that differ from those identified in the ATP/ADP carrier, the most studied member of the mitochondrial carrier family. The simulated transport mechanism of TPP highlights the essential role, at the energetic level, of the contributions coming from the formation and breakage of two networks of salt bridges, one on the side of the matrix and the other on the side of the intermembrane space, as well as the interactions, mainly of an ionic nature, formed by TPP upon its binding. The energy contribution provided by the cytosolic network establishes a lower barrier than that of the matrix network, which can be explained by the lower interaction energy of TPP on the matrix side or possibly a uniport activity.

Synthetic Enzyme-Catalyzed CO2 Fixation Reactions.

In recent years, (de)carboxylases that catalyze reversible (de)carboxylation have been targeted for application as carboxylation catalysts. This has led to the development of proof-of-concept (bio)synthetic CO2 fixation routes for chemical production. However, further progress towards industrial application has been hampered by the thermodynamic constraint that accompanies fixing CO2 to organic molecules. In this Review, biocatalytic carboxylation methods are discussed with emphases on the diverse strategies devised to alleviate the inherent thermodynamic constraints and their application in synthetic CO2 -fixation cascades.

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'ßß') 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 mechanism of a one-substrate transketolase reaction. Part II.

In a recent paper, we showed the difference between the first stage of the one-substrate and the two-substrate transketolase reactions - the possibility of transfer of glycolaldehyde formed as a result of cleavage of the donor substrate from the thiazole ring of thiamine diphosphate to its aminopyrimidine ring through the tricycle formation stage, which is necessary for binding and splitting the second molecule of donor substrate [O.N. Solovjeva et al., The mechanism of a one-substrate transketolase reaction, Biosci. Rep. 40 (8) (2020) BSR20180246]. Here we show that under the action of the reducing agent a tricycle accumulates in a significant amount. Therefore, a significant decrease in the reaction rate of the one-substrate transketolase reaction compared to the two-substrate reaction is due to the stage of transferring the first glycolaldehyde molecule from the thiazole ring to the aminopyrimidine ring of thiamine diphosphate. Fragmentation of the four-carbon thiamine diphosphate derivatives showed that two glycolaldehyde molecules are bound to both coenzyme rings and the erythrulose molecule is bound to a thiazole ring. It was concluded that in the one-substrate reaction erythrulose is formed on the thiazole ring of thiamine diphosphate from two glycol aldehyde molecules linked to both thiamine diphosphate rings. The kinetic characteristics were determined for the two substrates, fructose 6-phosphate and glycolaldehyde.

Inhibition and Crystal Structure of the Human DHTKD1-Thiamin Diphosphate Complex.

DHTKD1 is the E1 component of the 2-oxoadipate dehydrogenase complex, which is an enzyme involved in the catabolism of (hydroxy-)lysine and tryptophan. Mutations in DHTKD1 have been associated with 2-aminoadipic and 2-oxoadipic aciduria, Charcot-Marie-Tooth disease type 2Q and eosinophilic esophagitis, but the pathophysiology of these clinically distinct disorders remains elusive. Here, we report the identification of adipoylphosphonic acid and tenatoprazole as DHTKD1 inhibitors using targeted and high throughput screening, respectively. We furthermore elucidate the DHTKD1 crystal structure with thiamin diphosphate bound at 2.25 Å. We also report the impact of 10 disease-associated missense mutations on DHTKD1. Whereas the majority of the DHTKD1 variants displayed impaired folding or reduced thermal stability in combination with absent or reduced enzyme activity, three variants showed no abnormalities. Our work provides chemical and structural tools for further understanding of the function of DHTKD1 and its role in several human pathologies.

Thiamine Mono- and Diphosphate Phosphatases in Bovine Brain Synaptosomes.

Neurodegenerative diseases are accompanied by changes in the activity of thiamine mono- and diphosphate phosphatases, but molecular identification of these mammalian enzymes is incomplete. In this work, the protein fraction of bovine brain synaptosomes displaying phosphatase activity toward thiamine derivatives was subjected to affinity chromatography on thiamine-Sepharose. Protein fractions eluted with thiamine (pH 7.4 or 5.6), NaCl, and urea were assayed for the phosphatase activity against thiamine monophosphate (ThMP), thiamine diphosphate (ThDP), and structurally similar purine nucleotides. Proteins in each fraction were identified by mass spectrometry using the SwissProt database for all organisms because of insufficient annotation of the bovine genome. Peptides of two annotated bacterial phosphatases, alkaline phosphatase L from the DING protein family and exopolyphosphatase, were identified in the acidic thiamine eluate. The abundance of peptides of alkaline phosphatase L and exopolyphosphatase in the eluted fractions correlated with ThMPase and ThDPase activities, respectively. The elution profiles of the ThMPase and ThDPase activities differed from the elution profiles of nucleotide phosphatases, thus indicating the specificity of these enzymes toward thiamine derivatives. The search for mammalian DING phosphatases in the eluates from thiamine-Sepharose revealed X-DING-CD4, mostly eluted by the acidic thiamine solution (pH 5.6). The identified exopolyphosphatase demonstrated structural similarity with apyrases possessing the ThDPase activity. The obtained results demonstrate that mammalian DING proteins and apyrases exhibit ThMPase and ThDPase activity, respectively.

Oxalyl-CoA Decarboxylase Enables Nucleophilic One-Carbon Extension of Aldehydes to Chiral α-Hydroxy Acids.

The synthesis of complex molecules from simple, renewable carbon units is the goal of a sustainable economy. Here we explored the biocatalytic potential of the thiamine-diphosphate-dependent (ThDP) oxalyl-CoA decarboxylase (OXC)/2-hydroxyacyl-CoA lyase (HACL) superfamily that naturally catalyzes the shortening of acyl-CoA thioester substrates through the release of the C1 -unit formyl-CoA. We show that the OXC/HACL superfamily contains promiscuous members that can be reversed to perform nucleophilic C1 -extensions of various aldehydes to yield the corresponding 2-hydroxyacyl-CoA thioesters. We improved the catalytic properties of Methylorubrum extorquens OXC by rational enzyme engineering and combined it with two newly described enzymes-a specific oxalyl-CoA synthetase and a 2-hydroxyacyl-CoA thioesterase. This enzymatic cascade enabled continuous conversion of oxalate and aromatic aldehydes into valuable (S)-α-hydroxy acids with enantiomeric excess up to 99 %.

Competing Protonation and Halide Elimination as a Probe of the Character of Thiamin-Derived Reactive Intermediates.

Decarboxylation reactions from comparable thiamin diphosphate- and thiamin-derived adducts of p-(halomethyl)benzoylformic acids in enzymic and non-enzymic reactions, respectively, reveal critical distinctions in otherwise similar Breslow intermediates. The ratio of protonation to chloride elimination from the Breslow intermediate is 102-fold greater in the enzymic process. This is consistent with a lower intrinsic barrier to proton transfer on the enzyme, implicating formation of a localized tetrahedral (sp3) carbanion that is formed as CO2 is produced. In contrast, slower protonation in solution of the decarboxylated intermediate is consistent with formation of a delocalized planar carbanionic enol/enamine. The proposed structural and reactive character of the enzymic Breslow intermediate is consistent with Warshel's general theory of enzymic catalysis, structural characterization of related intermediates, and the lower kinetic barrier in reactions that occur without changes in hybridization.

Navigating within thiamine diphosphate-dependent decarboxylases: Sequences, structures, functional positions, and binding sites.

Thiamine diphosphate-dependent decarboxylases catalyze both cleavage and formation of CC bonds in various reactions, which have been assigned to different homologous sequence families. This work compares 53 ThDP-dependent decarboxylases with known crystal structures. Both sequence and structural information were analyzed synergistically and data were analyzed for global and local properties by means of statistical approaches (principle component analysis and principal coordinate analysis) enabling complexity reduction. The different results obtained both locally and globally, that is, individual positions compared with the overall protein sequence or structure, revealed challenges in the assignment of separated homologous families. The methods applied herein support the comparison of enzyme families and the identification of functionally relevant positions. The findings for the family of ThDP-dependent decarboxylases underline that global sequence identity alone is not sufficient to distinguish enzyme function. Instead, local sequence similarity, defined by comparisons of structurally equivalent positions, allows for a better navigation within several groups of homologous enzymes. The differentiation between homologous sequences is further enhanced by taking structural information into account, such as BioGPS analysis of the active site properties or pairwise structural superimpositions. The methods applied herein are expected to be transferrable to other enzyme families, to facilitate family assignments for homologous protein sequences.

TPP riboswitch aptamer: Role of Mg2+ ions, ligand unbinding, and allostery.

Riboswitches are non-coding RNAs that regulate gene expression in response to the binding of metabolites. Their abundance in bacteria makes them ideal drug targets. The prokaryotic thiamine pyrophosphate (TPP) riboswitch regulates gene expression in a wide range of bacteria by undergoing conformational changes in response to the binding of TPP. Although an experimental structure for the aptamer domain of the riboswitch is now available, details of the conformational changes that occur during the binding of the ligand, and the factors that govern these conformational changes, are still not clear. This study employs microsecond-scale molecular dynamics simulations to provide insights into the functioning of the riboswitch aptamer in atomistic detail. A mechanism for the transmission of conformational changes from the ligand-binding site to the P1 switch helix is proposed. Mg2+ ions in the binding site play a critical role in anchoring the ligand to the riboswitch. Finally, modeling the egress of TPP from the binding site reveals a two-step mechanism for TPP unbinding. Findings from this study can motivate the design of future studies aimed at modulating the activity of this drug target.

High-yield production of D-1,2,4-butanetriol from lignocellulose-derived xylose by using a synthetic enzyme cascade in a cell-free system.

Approaches using metabolic engineering to produce D-1, 2, 4-butanetriol (BT) from renewable biomass in microbial systems have achieved initial success. However, due to the lack of incomplete understanding of the complex branch pathway, the efficient fermentation system for BT production was difficult to develop. Here we reconstituted a cell-free system in vitro using purified enzymes to produce BT from d-xylose. The factors that influencing the efficiency of cell-free system, including enzyme concentration, reaction buffer, pH, temperature, metal ion additives and cofactors were first identified to define optimal reaction conditions and essential components for the cascade reaction. Meanwhile, a natural cofactor recycling system was found in cell-free system. Finally, we were able to convert 18 g/L xylose to 6.1 g/L BT within 40 h with a yield of 48.0%. The feasibility of cell-free system to produce BT in corncob hydrolysates was also determined.

Structural insights into the mechanism of inhibition of AHAS by herbicides.

Acetohydroxyacid synthase (AHAS), the first enzyme in the branched amino acid biosynthesis pathway, is present only in plants and microorganisms, and it is the target of >50 commercial herbicides. Penoxsulam (PS), which is a highly effective broad-spectrum AHAS-inhibiting herbicide, is used extensively to control weed growth in rice crops. However, the molecular basis for its inhibition of AHAS is poorly understood. This is despite the availability of structural data for all other classes of AHAS-inhibiting herbicides. Here, crystallographic data for Saccharomyces cerevisiae AHAS (2.3 Å) and Arabidopsis thaliana AHAS (2.5 Å) in complex with PS reveal the extraordinary molecular mechanisms that underpin its inhibitory activity. The structures show that inhibition of AHAS by PS triggers expulsion of two molecules of oxygen bound in the active site, releasing them as substrates for an oxygenase side reaction of the enzyme. The structures also show that PS either stabilizes the thiamin diphosphate (ThDP)-peracetate adduct, a product of this oxygenase reaction, or traps within the active site an intact molecule of peracetate in the presence of a degraded form of ThDP: thiamine aminoethenethiol diphosphate. Kinetic analysis shows that PS inhibits AHAS by a combination of events involving FAD oxidation and chemical alteration of ThDP. With the emergence of increasing levels of resistance toward front-line herbicides and the need to optimize the use of arable land, these data suggest strategies for next generation herbicide design.