Systematic analysis of the effect of genomic knock-out of non-essential promiscuous HAD-like phosphatases YcsE, YitU and YwtE on flavin and adenylate content in Bacillus subtilis.

Studying the metabolic role of non-essential promiscuous enzymes is a challenging task, as genetic manipulations usually do not reveal at which point(s) of the metabolic network the enzymatic activity of such protein is beneficial for the organism. Each of the HAD-like phosphatases YcsE, YitU and YwtE of Bacillus subtilis catalyzes the dephosphorylation of 5-amino-6-ribitylamino-uracil 5'-phosphate, which is essential in the biosynthesis of riboflavin. Using CRISPR technology, we have found that the deletion of these genes, individually or in all possible combinations failed to cause riboflavin auxotrophy and did not result in significant growth changes. Analysis of flavin and adenylate content in B. subtilis knockout mutants showed that (i) there must be one or several still unidentified phosphatases that can replace the deleted proteins; (ii) such replacements, however, cannot fully restore the intracellular content of any of three flavins studied (riboflavin, FMN, FAD); (iii) whereas bacterial fitness was not significantly compromised by mutations, the intracellular balance of flavins and adenylates did show some significant changes.

Reverse N-Substituted Hydroxamic Acid Derivatives of Fosmidomycin Target a Previously Unknown Subpocket of 1-Deoxy-d-xylulose 5-Phosphate Reductoisomerase (DXR).

Reverse analogs of the phosphonohydroxamic acid antibiotic fosmidomycin are potent inhibitors of the nonmevalonate isoprenoid biosynthesis enzyme 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR, IspC) of Plasmodium falciparum. Some novel analogs with large phenylalkyl substituents at the hydroxamic acid nitrogen exhibit nanomolar PfDXR inhibition and potent in vitro growth inhibition of P. falciparum parasites coupled with good parasite selectivity. X-ray crystallographic studies demonstrated that the N-phenylpropyl substituent of the newly developed lead compound 13e is accommodated in a subpocket within the DXR catalytic domain but does not reach the NADPH binding pocket of the N-terminal domain. As shown for reverse carba and thia analogs, PfDXR selectively binds the S-enantiomer of the new lead compound. In addition, some representatives of the novel inhibitor subclass are nanomolar Escherichia coli DXR inhibitors, whereas the inhibition of Mycobacterium tuberculosis DXR is considerably weaker.

Preparation and Immunochemical Characterization of a Water-Soluble Gluten Peptide Fraction for Improving the Diagnosis of Celiac Disease.

The diagnosis of celiac disease (CD) is complex and requires a multi-step procedure (symptoms, serology, duodenal biopsy, effect of a gluten-free diet, and optional genetic). The aim of the study was to contribute to the improvement of CD diagnosis by preparing a water-soluble gluten peptide fraction (called Solgluten) and by selecting gluten-specific enzyme-linked immunosorbent assays (ELISA) for the detection of gluten immunogenic gluten peptides (GIPs) in urine and blood serum spiked with Solgluten. Food-grade Solgluten was prepared by the extraction of a peptic digest of vital gluten with water, centrifugation, and freeze-drying. The process was relatively easy, repeatable, and cheap. The content of gliadin-derived GIPs was 491 mg/g. Solgluten was used as antigenic material to compare two competitive ELISA kits (R7021 and K3012) and two sandwich ELISA kits (M2114 and R7041) in their quality regarding the quantitation of GIPs in urine and blood serum. The quality parameters were the reactivity, sensitivity, coefficients of variation and determination, and curve shape. The evaluation of the kits showed a number of discrepancies in individual quality parameters measured in urine and serum. Due to the lowest limit of quantitation and the highest coefficient of determination, M2114 may be the first choice, while R7021 appeared to be less suitable because of the high coefficients of variation and unfavorable curve progression. The results set the stage for improving CD diagnosis by supplementing conventional blood tests with oral provocation with Solgluten and subsequent ELISA measurement of GIPs that could support the no-biopsy approach and by better assessing the effect of a gluten-free diet by monitoring adherence to the diet by measuring GIPs in urine and blood.

Elucidating the Signal Transduction Mechanism of the Blue-Light-Regulated Photoreceptor YtvA: From Photoactivation to Downstream Regulation.

The blue-light photoreceptor YtvA from Bacillus subtilis has an N-terminal flavin mononucleotide (FMN)-binding light-oxygen-voltage (LOV) domain that is fused to a C-terminal sulfate transporter and anti-σ factor antagonist (STAS) output domain. To interrogate the signal transduction pathway that leads to photoactivation, the STAS domain was replaced with a histidine kinase, so that photoexcitation of the flavin could be directly correlated with biological activity. N94, a conserved Asn that is hydrogen bonded to the FMN C2═O group, was replaced with Ala, Asp, and Ser residues to explore the role of this residue in triggering the structural dynamics that activate the output domain. Femtosecond to millisecond time-resolved multiple probe spectroscopy coupled with a fluorescence polarization assay revealed that the loss of the hydrogen bond between N94 and the C2═O group decoupled changes in the protein structure from photoexcitation. In addition, alterations in N94 also decreased the stability of the Cys-FMN adduct formed in the light-activated state by up to a factor of ∼25. Collectively, these studies shed light on the role of the hydrogen bonding network in the LOV ß-scaffold in signal transduction.

Shining a Spotlight on Methyl Groups: Photochemically Induced Dynamic Nuclear Polarization Spectroscopy of 5-Deazariboflavin and Its Nor Analogs.

5-Deazaflavins are analogs of naturally occurring flavin cofactors. They serve as substitutes for natural flavin cofactors to investigate and modify the reaction pathways of flavoproteins. Demethylated 5-deazaflavins are potential candidates for artificial cofactors, allowing us to fine-tune the reaction kinetics and absorption characteristics of flavoproteins. In this contribution, demethylated 5-deazariboflavin radicals are investigated (1) to assess the influence of the methyl groups on the electronic structure of the 5-deazaflavin radical and (2) to explore their photophysical properties with regard to their potential as artificial cofactors. We determined the proton hyperfine structure of demethylated 5-deazariboflavins using photochemically induced dynamic nuclear polarization (photo-CIDNP) spectroscopy, as well as density functional theory (DFT). To provide context, we compare our findings to a study of flavin mononucleotide (FMN) derivatives. We found a significant influence of the methylation pattern on the absorption properties, as well as on the proton hyperfine coupling ratios of the xylene moiety, which appears to be solvent-dependent. This effect is enhanced by the replacement of N5 by C5-H in 5-deazaflavin derivatives compared to their respective flavin counterparts.

Exploring the Translational Gap of a Novel Class of Escherichia coli IspE Inhibitors.

Discovery of novel antibiotics needs multidisciplinary approaches to gain target enzyme and bacterial activities while aiming for selectivity over mammalian cells. Here, we report a multiparameter optimisation of a fragment-like hit that was identified through a structure-based virtual-screening campaign on Escherichia coli IspE crystal structure. Subsequent medicinal-chemistry design resulted in a novel class of E. coli IspE inhibitors, exhibiting activity also against the more pathogenic bacteria Pseudomonas aeruginosa and Acinetobacter baumannii. While cytotoxicity remains a challenge for the series, it provides new insights on the molecular properties for balancing enzymatic target and bacterial activities simultaneously as well as new starting points for the development of IspE inhibitors with a predicted new mode of action.

Expanding Reaction Horizons: Evidence of the 5-Deazaflavin Radical Through Photochemically Induced Dynamic Nuclear Polarization.

Deazaflavins are important analogues of the naturally occurring flavins: riboflavin, flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD). The use of 5-deazaflavin as a replacement coenzyme in a number of flavoproteins has proven particularly valuable in unraveling and manipulating their reaction mechanisms. It was frequently reported that one-electron-transfer reactions in flavoproteins are impeded with 5-deazaflavin as the cofactor. Based on these findings, it was concluded that the 5-deazaflavin radical is significantly less stable compared to the respective flavin semiquinone and quickly re-oxidizes or undergoes disproportionation. The long-standing paradigm of 5-deazaflavin being solely a two-electron/hydride acceptor/donor-"a nicotinamide in flavin clothing"-needs to be re-evaluated now with the indirect observation of a one-electron-reduced (paramagnetic) species using photochemically induced dynamic nuclear polarization (photo-CIDNP) 1 H nuclear magnetic resonance (NMR) under biologically relevant conditions.

Discovery of novel drug-like antitubercular hits targeting the MEP pathway enzyme DXPS by strategic application of ligand-based virtual screening.

In the present manuscript, we describe how we successfully used ligand-based virtual screening (LBVS) to identify two small-molecule, drug-like hit classes with excellent ADMET profiles against the difficult to address microbial enzyme 1-deoxy-d-xylulose-5-phosphate synthase (DXPS). In the fight against antimicrobial resistance (AMR), it has become increasingly important to address novel targets such as DXPS, the first enzyme of the 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway, which affords the universal isoprenoid precursors. This pathway is absent in humans but essential for pathogens such as Mycobacterium tuberculosis, making it a rich source of drug targets for the development of novel anti-infectives. Standard computer-aided drug-design tools, frequently applied in other areas of drug development, often fail for targets with large, hydrophilic binding sites such as DXPS. Therefore, we introduce the concept of pseudo-inhibitors, combining the benefits of pseudo-ligands (defining a pharmacophore) and pseudo-receptors (defining anchor points in the binding site), for providing the basis to perform a LBVS against M. tuberculosis DXPS. Starting from a diverse set of reference ligands showing weak inhibition of the orthologue from Deinococcus radiodurans DXPS, we identified three structurally unrelated classes with promising in vitro (against M. tuberculosis DXPS) and whole-cell activity including extensively drug-resistant strains of M. tuberculosis. The hits were validated to be specific inhibitors of DXPS and to have a unique mechanism of inhibition. Furthermore, two of the hits have a balanced profile in terms of metabolic and plasma stability and display a low frequency of resistance development, making them ideal starting points for hit-to-lead optimization of antibiotics with an unprecedented mode of action.

Completing the Picture: Determination of 13C Hyperfine Coupling Constants of Flavin Semiquinone Radicals by Photochemically Induced Dynamic Nuclear Polarization Spectroscopy.

We investigate the electronic structure of flavin semiquinone radicals in terms of their 13C hyperfine coupling constants. Photochemically induced dynamic nuclear polarization (photo-CIDNP) spectroscopy was used to study both the neutral and anionic radical species of flavin mononucleotide (FMN) in bulk aqueous solution. Apart from universally 13C-labeled FMN, partially labeled isotopologues are used to increase sensitivity for nuclei exhibiting very small hyperfine couplings and to cope with spectral overlap. In addition, experimental findings are supported by quantum chemical calculations, and implications for the spin density distribution in free flavin radicals are discussed.

Targeting the IspD Enzyme in the MEP Pathway: Identification of a Novel Fragment Class.

The enzymes of the 2-C-methylerythritol-d-erythritol 4-phosphate (MEP) pathway (MEP pathway or non-mevalonate pathway) are responsible for the synthesis of universal precursors of the large and structurally diverse family of isoprenoids. This pathway is absent in humans, but present in many pathogenic organisms and plants, making it an attractive source of drug targets. Here, we present a high-throughput screening approach that led to the discovery of a novel fragment hit active against the third enzyme of the MEP pathway, PfIspD. A systematic SAR investigation afforded a novel chemical structure with a balanced activity-stability profile (16). Using a homology model of PfIspD, we proposed a putative binding mode for our newly identified inhibitors that sets the stage for structure-guided optimization.

Selective 13C labelling reveals the electronic structure of flavocoenzyme radicals.

Flavocoenzymes are nearly ubiquitous cofactors that are involved in the catalysis and regulation of a wide range of biological processes including some light-induced ones, such as the photolyase-mediated DNA repair, magnetoreception of migratory birds, and the blue-light driven phototropism in plants. One of the factors that enable versatile flavin-coenzyme biochemistry and biophysics is the fine-tuning of the cofactor's frontier orbital by interactions with the protein environment. Probing the singly-occupied molecular orbital (SOMO) of the intermediate radical state of flavins is therefore a prerequisite for a thorough understanding of the diverse functions of the flavoprotein family. This may be ultimately achieved by unravelling the hyperfine structure of a flavin by electron paramagnetic resonance. In this contribution we present a rigorous approach to obtaining a hyperfine map of the flavin's chromophoric 7,8-dimethyl isoalloxazine unit at an as yet unprecedented level of resolution and accuracy. We combine powerful high-microwave-frequency/high-magnetic-field electron-nuclear double resonance (ENDOR) with 13C isotopologue editing as well as spectral simulations and density functional theory calculations to measure and analyse 13C hyperfine couplings of the flavin cofactor in DNA photolyase. Our data will provide the basis for electronic structure considerations for a number of flavin radical intermediates occurring in blue-light photoreceptor proteins.

Assessment of the rules related to gaining activity against Gram-negative bacteria.

In the search for new antibacterial compounds, we repositioned an antimalarial compound class by derivatising it based on the so-called "eNTRy" rules for enhanced accumulation into Gram-negative bacteria. We designed, synthesised and evaluated a small library of amino acid modified compounds together with the respective Boc-protected analogues, leading to no substantial improvement in antibacterial activity against Escherichia coli wild-type K12, whereas more distinct activity differences were observed in E. coli mutant strains ΔtolC, D22, ΔacrB and BL21(DE3)omp8. A comparison of the activity results of the E. coli mutants with respect to the known rules related to enhanced activity against Gram-negative bacteria revealed that applicability of the rules is not always ensured. Out of the four amino acids used in this study, glycine derivatives showed highest antibacterial activity, although still suffering from efflux issues.

Search for the Active Ingredients from a 2-Aminothiazole DMSO Stock Solution with Antimalarial Activity.

Chemical decomposition of DMSO stock solutions is a common incident that can mislead biological screening campaigns. Here, we share our case study of 2-aminothiazole 1, originating from an antimalarial class that undergoes chemical decomposition in DMSO at room temperature. As previously measured biological activities observed against Plasmodium falciparum NF54 and for the target enzyme PfIspE were not reproducible for a fresh batch, we tackled the challenge to understand where the activity originated from. Solvent- and temperature-dependent studies using HRMS and NMR spectroscopy to monitor the decomposition led to the isolation and in vitro evaluation of several fractions against PfIspE. After four days of decomposition, we successfully isolated the oxygenated and dimerised compounds using SFC purification and correlated the observed activities to them. Due to the unstable nature of the two isolates, it is likely that they undergo further decomposition contributing to the overall instability of the compound.

The crystal structure of mycobacterial epoxide hydrolase A.

The human pathogen Mycobacterium tuberculosis is the causative agent of tuberculosis resulting in over 1 million fatalities every year, despite decades of research into the development of new anti-TB compounds. Unlike most other organisms M. tuberculosis has six putative genes for epoxide hydrolases (EH) of the α/ß-hydrolase family with little known about their individual substrates, suggesting functional significance for these genes to the organism. Due to their role in detoxification, M. tuberculosis EH's have been identified as potential drug targets. Here, we demonstrate epoxide hydrolase activity of M. thermoresistibile epoxide hydrolase A (Mth-EphA) and report its crystal structure in complex with the inhibitor 1,3-diphenylurea at 2.0 Å resolution. Mth-EphA displays high sequence similarity to its orthologue from M. tuberculosis and generally high structural similarity to α/ß-hydrolase EHs. The structure of the inhibitor bound complex reveals the geometry of the catalytic residues and the conformation of the inhibitor. Comparison to other EHs from mycobacteria allows insight into the active site plasticity with respect to substrate specificity. We speculate that mycobacterial EHs may have a narrow substrate specificity providing a potential explanation for the genetic repertoire of epoxide hydrolase genes in M. tuberculosis.

Excited State Vibrations of Isotopically Labeled FMN Free and Bound to a Light-Oxygen-Voltage (LOV) Protein.

Flavoproteins are important blue light sensors in photobiology and play a key role in optogenetics. The characterization of their excited state structure and dynamics is thus an important objective. Here, we present a detailed study of excited state vibrational spectra of flavin mononucleotide (FMN), in solution and bound to the LOV-2 (Light-Oxygen-Voltage) domain of Avena sativa phototropin. Vibrational frequencies are determined for the optically excited singlet state and the reactive triplet state, through resonant ultrafast femtosecond stimulated Raman spectroscopy (FSRS). To assign the observed spectra, vibrational frequencies of the excited states are calculated using density functional theory, and both measurement and theory are applied to four different isotopologues of FMN. Excited state mode assignments are refined in both states, and their sensitivity to deuteration and protein environment are investigated. We show that resonant FSRS provides a useful tool for characterizing photoactive flavoproteins and is able to highlight chromophore localized modes and to record hydrogen/deuterium exchange.

Coupled Methyl Group Rotation in FMN Radicals Revealed by Selective Deuterium Labeling.

Flavin semiquinones are common intermediate redox states in flavoproteins, and thus, knowledge of their electronic structure is essential for fully understanding their chemistry and chemical versatility. In this contribution, we use a combination of high-field electron nuclear double resonance spectroscopy and selective deuterium labeling of flavin mononucleotide (FMN) with subsequent incorporation as cofactor into a variant Avena sativa LOV domain to extract missing traits of the electronic structure of a protein-bound FMN radical. From these experiments, precise values of small proton hyperfine and deuterium nuclear quadrupole couplings could be extracted. Specifically, isotropic hyperfine couplings of -3.34, -0.11, and +0.91 MHz were obtained for the protons H(6), H(9), and H(7α), respectively. These values are discussed in the light of specific protein-cofactor interactions. Furthermore, the temperature behavior of the H(7α) methyl-group rotation elicited by its energy landscape was analyzed in greater detail. Pronounced interplay between the two methyl groups at C(7) and C(8) of FMN could be revealed. Most strikingly, this rotational behavior could be modulated by selective deuterium editing.

Methyl groups matter: Photo-CIDNP characterizations of the semiquinone radicals of FMN and demethylated FMN analogs.

In this contribution, the relative hyperfine couplings are determined for the 1H nuclei of the flavin mononucleotide (FMN) radical in an aqueous environment. In addition, three structural analogs with different methylation patterns are characterized and the influence of the substituents at the isoalloxazine moiety on the electronic structure of the radicals is explored. By exploiting nuclear hyperpolarization generated via the photo-CIDNP (chemically induced dynamic nuclear polarization) effect, it is possible to study the short-lived radical species generated by in situ light excitation. Experimental data are extracted by least-squares fitting and supported by quantum chemical calculations and published values from electron paramagnetic resonance and electron-nuclear double resonance. Furthermore, mechanistic details of the photoreaction of the investigated flavin analogs with l-tryptophan are derived from the photo-CIDNP spectra recorded at different pH values. Thereby, the neutral and anionic radicals of FMN and three structural analogs are, for the first time, characterized in terms of their electronic structure in an aqueous environment.

Structure of GTP cyclohydrolase I from Listeria monocytogenes, a potential anti-infective drug target.

A putative open reading frame encoding GTP cyclohydrolase I from Listeria monocytogenes was expressed in a recombinant Escherichia coli strain. The recombinant protein was purified and was confirmed to convert GTP to dihydroneopterin triphosphate (Km = 53 µM; vmax = 180 nmol mg-1 min-1). The protein was crystallized from 1.3 M sodium citrate pH 7.3 and the crystal structure was solved at a resolution of 2.4 Š(Rfree = 0.226) by molecular replacement using human GTP cyclohydrolase I as a template. The protein is a D5-symmetric decamer with ten topologically equivalent active sites. Screening a small library of about 9000 compounds afforded several inhibitors with IC50 values in the low-micromolar range. Several inhibitors had significant selectivity with regard to human GTP cyclohydrolase I. Hence, GTP cyclohydrolase I may be a potential target for novel drugs directed at microbial infections, including listeriosis, a rare disease with high mortality.

Novel reverse thia-analogs of fosmidomycin: Synthesis and antiplasmodial activity.

Thia analogs of fosmidomycin are potent inhibitors of the non-mevalonate isoprenoid biosynthesis enzyme 1-deoxy-d-xylulose 5-phosphate reductoisomerase (IspC, Dxr) of Plasmodium falciparum. Several new thioethers displayed antiplasmodial in vitro activity in the low nanomolar range, without apparent cytotoxic effects in HeLa cells. The (S)-(+)-enantiomer of a typical representative selectively inhibited IspC and the growth of P. falciparum in continuous culture. The inhibitor was stable at pH 7.6 and room temperature, and no racemization was observed under these conditions during a period of up to two days. Oxidation of selected thioethers to sulfones reduced antiplasmodial activity and the inhibitory activity against Escherichia coli, Mycobacterium tuberculosis and P. falciparum IspC orthologs.

Long-Lived Hydrated FMN Radicals: EPR Characterization and Implications for Catalytic Variability in Flavoproteins.

Until now, FMN/FAD radicals could not be stabilized in aqueous solution or other protic solvents because of rapid and efficient dismutation reactions. In this contribution, a novel system for stabilizing flavin radicals in aqueous solution is reported. Subsequent to trapping FMN in an agarose matrix, light-generated FMN radicals could be produced that were stable for days even under aerobic conditions, and their concentrations were high enough for extensive EPR characterization. All large hyperfine couplings could be extracted by using a combination of continuous-wave EPR and low-temperature ENDOR spectroscopy. To map differences in the electronic structure of flavin radicals, two exemplary proton hyperfine couplings were compared with published values from various neutral and anionic flavoprotein radicals: C(6)H and C(8α)H 3. It turned out that FMN•- in an aqueous environment shows the largest hyperfine couplings, whereas for FMNH• under similar conditions, hyperfine couplings are at the lower end and the values of both vary by up to 30%. This finding demonstrates that protein-cofactor interactions in neutral and anionic flavoprotein radicals can alter their electron spin density in different directions. With this aqueous system that allows the characterization of flavin radicals without protein interactions and that can be extended by using selective isotope labeling, a powerful tool is now at hand to quantify interactions in flavin radicals that modulate the reactivity in different flavoproteins.