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.

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.

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.

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.

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.

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.

Non-classical disproportionation revealed by photo-chemically induced dynamic nuclear polarization NMR.

Photo-chemically induced dynamic nuclear polarization (photo-CIDNP) was used to observe the light-induced disproportionation reaction of 6,7,8-trimethyllumazine starting out from its triplet state to generate a pair of radicals comprising a one-electron reduced and a one-electron oxidized species. Our evidence is based on the measurement of two marker proton hyperfine couplings, Aiso(H(6α)) and Aiso(H(8α)), which we correlated to predictions from density functional theory. The ratio of these two hyperfine couplings is reversed in the oxidized and the reduced radical species. Observation of the dismutation reaction is facilitated by the exceptional C-H acidity of the methyl group at position 7 of 6,7,8-trimethyllumazine and the slow proton exchange associated with it, which leads to NMR-distinguishable anionic (TML-) and neutral (TMLH) protonation forms.

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.

Influence of the cofactor structure on the photophysical processes initiating signal transduction in a phototropin-derived LOV domain.

Due to their biological importance, the photochemistry of blue-light photoreceptor proteins has been studied extensively over the last few decades. Most blue-light photoreceptors, such as cryptochromes and phototropins, utilize flavin chromophores as their cofactors. After irradiation with light, the chromophore undergoes electron transfer with nearby redox-active amino-acid residues within the protein, whereby this first step of signal transduction may be initiated either from the flavin's excited singlet or triplet state. Despite the collective effort of theoreticians and experimentalists to characterize and understand the photochemistry of flavoproteins, the mechanistic details of the excited state processes initiating signal transduction are yet to be revealed. Here, we use a light-oxygen-voltage-sensing domain from Avena sativa phototropin to get additional insight into the excited state photochemistry of flavoproteins. The influence of structural variations of the cofactor flavin mononucleotide (FMN) is explored by varying the methyl substitution pattern in positions 7 and 8 of the flavin core. The photophysical properties of the FMN derivatives, in the absence and presence of the protein environment, are investigated by UV-vis absorption, fluorescence, and electron paramagnetic resonance spectroscopies as well as cyclic voltammetry. The comparison of the properties of the modified flavin cofactors with those of FMN shows that the rates of the different excited state reactions, and therefore also the singlet/triplet yields, can be modulated substantially by only minor structural modifications of the flavin core.

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.

Aryl bis-sulfonamides bind to the active site of a homotrimeric isoprenoid biosynthesis enzyme IspF and extract the essential divalent metal cation cofactor.

Characterizing the mode of action of non-covalent inhibitors in multisubunit enzymes often presents a great challenge. Most of the conventionally used methods are based on ensemble measurements of protein-ligand binding in bulk solution. They often fail to accurately describe multiple binding processes occurring in such systems. Native electrospray ionization mass spectrometry (ESI-MS) of intact protein complexes is a direct, label-free approach that can render the entire distribution of ligand-bound states in multimeric protein complexes. Here we apply native ESI-MS to comprehensively characterize the isoprenoid biosynthesis enzyme IspF from Arabidopsis thaliana, an example of a homomeric protein complex with multiple binding sites for several types of ligands, including a metal cofactor and a synthetic inhibitor. While standard biophysical techniques failed to reveal the mode of action of recently discovered aryl-sulfonamide-based inhibitors of AtIspF, direct native ESI-MS titrations of the protein with the ligands and ligand competition assays allowed us to accurately capture the solution-phase protein-ligand binding equilibria in full complexity and detail. Based on these combined with computational modeling, we propose a mechanism of AtIspF inhibition by aryl bis-sulfonamides that involves both the competition with the substrate for the ligand-binding pocket and the extraction of Zn2+ from the enzyme active site. This inhibition mode is therefore mixed competitive and non-competitive, the latter exerting a key inhibitory effect on the enzyme activity. The results of our study deliver a profound insight into the mechanisms of AtIspF action and inhibition, open new perspectives for designing inhibitors of this important drug target, and demonstrate the applicability and value of the native ESI-MS approach for deep analysis of complex biomolecular binding equilibria.

Variation in LOV Photoreceptor Activation Dynamics Probed by Time-Resolved Infrared Spectroscopy.

The light, oxygen, voltage (LOV) domain proteins are blue light photoreceptors that utilize a noncovalently bound flavin mononucleotide (FMN) cofactor as the chromophore. The modular nature of these proteins has led to their wide adoption in the emerging fields of optogenetics and optobiology, where the LOV domain has been fused to a variety of output domains leading to novel light-controlled applications. In this work, we extend our studies of the subpicosecond to several hundred microsecond transient infrared spectroscopy of the isolated LOV domain AsLOV2 to three full-length photoreceptors in which the LOV domain is fused to an output domain: the LOV-STAS protein, YtvA, the LOV-HTH transcription factor, EL222, and the LOV-histidine kinase, LovK. Despite differences in tertiary structure, the overall pathway leading to cysteine adduct formation from the FMN triplet state is highly conserved, although there are slight variations in rate. However, significant differences are observed in the vibrational spectra and kinetics after adduct formation, which are directly linked to the specific output function of the LOV domain. While the rate of adduct formation varies by only 3.6-fold among the proteins, the subsequent large-scale structural changes in the full-length LOV photoreceptors occur over the micro- to submillisecond time scales and vary by orders of magnitude depending on the different output function of each LOV domain.

Mechanism of Allosteric Inhibition of the Enzyme IspD by Three Different Classes of Ligands.

Enzymes of the nonmevalonate pathway of isoprenoid biosynthesis are attractive targets for the development of herbicides and drugs against infectious diseases. While this pathway is essential for many pathogens and plants, mammals do not depend on it for the synthesis of isoprenoids. IspD, the third enzyme of the nonmevalonate pathway, is unique in that it has an allosteric regulatory site. We elucidated the binding mode of phenylisoxazoles, a new class of allosteric inhibitors. Allosteric inhibition is effected by large conformational changes of a loop region proximal to the active site. We investigated the different roles of residues in this loop by mutation studies and identified repulsive interactions with Asp291 and Asp292 to be responsible for inhibition. Crystallographic data and the response of mutant enzymes to three different classes of allosteric inhibitors provide an in-depth understanding of the allosteric mechanism. The obtained mutant enzymes show selective resistance to allosteric inhibitors and provide conceptually valuable information for future engineering of herbicide-resistant crops. We found that the isoprenoid precursors IPP and DMAPP are natural inhibitors of Arabidopsis thaliana IspD; however, they do not seem to bind to the allosteric site.

Femtosecond to Millisecond Dynamics of Light Induced Allostery in the Avena sativa LOV Domain.

The rational engineering of photosensor proteins underpins the field of optogenetics, in which light is used for spatiotemporal control of cell signaling. Optogenetic elements function by converting electronic excitation of an embedded chromophore into structural changes on the microseconds to seconds time scale, which then modulate the activity of output domains responsible for biological signaling. Using time-resolved vibrational spectroscopy coupled with isotope labeling, we have mapped the structural evolution of the LOV2 domain of the flavin binding phototropin Avena sativa (AsLOV2) over 10 decades of time, reporting structural dynamics between 100 fs and 1 ms after optical excitation. The transient vibrational spectra contain contributions from both the flavin chromophore and the surrounding protein matrix. These contributions are resolved and assigned through the study of four different isotopically labeled samples. High signal-to-noise data permit the detailed analysis of kinetics associated with the light activated structural evolution. A pathway for the photocycle consistent with the data is proposed. The earliest events occur in the flavin binding pocket, where a subpicosecond perturbation of the protein matrix occurs. In this perturbed environment, the previously characterized reaction between triplet state isoalloxazine and an adjacent cysteine leads to formation of the adduct state; this step is shown to exhibit dispersive kinetics. This reaction promotes coupling of the optical excitation to successive time-dependent structural changes, initially in the ß-sheet and then α-helix regions of the AsLOV2 domain, which ultimately gives rise to Jα-helix unfolding, yielding the signaling state. This model is tested through point mutagenesis, elucidating in particular the key mediating role played by Q513.

Decoding Biosynthetic Pathways in Plants by Pulse-Chase Strategies Using (13)CO2 as a Universal Tracer †.

(13)CO2 pulse-chase experiments monitored by high-resolution NMR spectroscopy and mass spectrometry can provide (13)C-isotopologue compositions in biosynthetic products. Experiments with a variety of plant species have documented that the isotopologue profiles generated with (13)CO2 pulse-chase labeling are directly comparable to those that can be generated by the application of [U-(13)C6]glucose to aseptically growing plants. However, the application of the (13)CO2 labeling technology is not subject to the experimental limitations that one has to take into account for experiments with [U-(13)C6]glucose and can be applied to plants growing under physiological conditions, even in the field. In practical terms, the results of biosynthetic studies with (13)CO2 consist of the detection of pairs, triples and occasionally quadruples of (13)C atoms that have been jointly contributed to the target metabolite, at an abundance that is well above the stochastic occurrence of such multiples. Notably, the connectivities of jointly transferred (13)C multiples can have undergone modification by skeletal rearrangements that can be diagnosed from the isotopologue data. As shown by the examples presented in this review article, the approach turns out to be powerful in decoding the carbon topology of even complex biosynthetic pathways.