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.

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.

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.

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.

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.

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.

Catalysis of an Essential Step in Vitamin†B2 Biosynthesis by a Consortium of Broad Spectrum Hydrolases.

An enzyme catalysing the essential dephosphorylation of the riboflavin precursor, 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione 5'-phosphate (6), was purified about 800-fold from a riboflavin-producing Bacillus subtilis strain, and was assigned as the translation product of the ycsE gene by mass spectrometry. YcsE is a member of the large haloacid dehalogenase (HAD) superfamily. The recombinant protein was expressed in Escherichia coli. It catalyses the hydrolysis of 6 (vmax , 12 µmol mg(-1) min(-1) ; KM , 54 µm) and of FMN (vmax , 25 µmol mg(-1) min(-1) ; KM , 135 µm). A ycsE deletion mutant of B. subtilis was not riboflavin dependent. Two additional proteins (YwtE, YitU) that catalyse the hydrolysis of 6 at appreciable rates were identified by screening 13 putative HAD superfamily members from B. subtilis. The evolutionary processes that have resulted in the handling of an essential step in the biosynthesis of an essential cofactor by a consortium of promiscuous enzymes require further analysis.

Preparation of flavocoenzyme isotopologues by biotransformation of purines.

Isotope-labeled flavins are crucial reporters for many biophysical studies of flavoproteins. A purine-deficient Escherichia coli strain engineered for expression of the ribAGH genes of Bacillus subtilis converts isotope-labeled purine supplements into the riboflavin precursor, 6,7-dimethyl-8-ribityllumazine, with yields up to 40%. The fermentation products can subsequently be converted into isotope-labeled riboflavin and the cognate flavocoenzymes, FMN and FAD, by in vitro biotransformation with better than 90% yield. Using this approach, more than 100 single or multiple (13)C-, (15)N-, (17)O-, and (18)O-labeled isotopologues of these cofactors and ligands become easily accessible, enabling advanced ligand-based spectroscopy of flavoproteins and lumazine receptor proteins at unprecedented resolution.

Atovaquone for Prophylaxis of Toxoplasmosis after Allogeneic Hematopoietic Stem Cell Transplantation.

Toxoplasmosis and infections by other opportunistic agents such as Pneumocystis jirovecii constitute life-threatening risks for patients after allogeneic hematopoietic stem cell transplantation. Trimethoprim/sulfamethoxazole (TMP-SMX) has been well established for post-transplant toxoplasmosis and pneumocystis prophylaxis, but treatment may be limited due to toxicity. We explored atovaquone as an alternative and compared it with TMP-SMX regarding toxicity and efficacy during the first 100 days after transplantation in 155 consecutive adult stem cell recipients. Eight patients with a prior history of TMP-SMX intolerance received atovaquone as first-line prophylaxis. TMP-SMX was used for 141 patients as first-line strategy, but 13 patients (9.2%) were later switched to atovaquone due to TMP-SMX toxicity or gastrointestinal symptoms. No active toxoplasmosis or active P. jirovecii infection developed under continued prophylaxis with either TMP-SMX or atovaquone. However, for reasons of TMP-SMX and/or atovaquone toxicity, 7 patients were unable to tolerate any efficacious toxoplasmosis prophylaxis and therefore obtained inhalative pentamidine as P. jirovecii prophylaxis but no toxoplasmosis prophylaxis. Importantly, 2 of these patients developed severe toxoplasmosis. In summary, atovaquone appears as a valid alternative for at least some post-transplant patients who cannot tolerate TMP-SMX. This should be further confirmed by multicenter trials.

Insights into the binding of pyridines to the iron-sulfur enzyme IspH.

(E)-1-Hydroxy-2-methylbut-2-enyl 4-diphosphate reductase (IspH) is a [Fe4S4] cluster-containing enzyme involved in isoprenoid biosynthesis in many bacteria as well as in malaria parasites and is an important drug target. Several inhibitors including amino and thiol substrate analogues, as well as acetylene and pyridine diphosphates, have been reported. Here, we investigate the mode of binding of four pyridine diphosphates to Escherichia coli IspH by using X-ray crystallography. In three cases, one of the iron atoms in the cluster is absent, but in the structure with (pyridin-3-yl)methyl diphosphate, the most potent pyridine-analogue inhibitor reported previously, the fourth iron of the [Fe4S4] cluster is present and interacts with the pyridine ring of the ligand. Based on the results of quantum chemical calculations together with the crystallographic results we propose a side-on η(2) coordination of the nitrogen and the carbon in the 2-position of the pyridine ring to the unique fourth iron in the cluster, which is in the reduced state. The X-ray structure enables excellent predictions using density functional theory of the (14)N hyperfine coupling and quadrupole coupling constants reported previously using HYSCORE spectroscopy, as well as providing a further example of the ability of such [Fe4S4]-containing proteins to form organometallic complexes.

Isotopologue profiling of triterpene formation under physiological conditions. Biosynthesis of lupeol-3-(3'-R-hydroxy)-stearate in Pentalinon andrieuxii.

The biosynthesis of lupeol-3-(3'R-hydroxy)-stearate (procrim b, 1) was investigated in the Mexican medicinal plant Pentalinon andrieuxii by (13)CO2 pulse-chase experiments. NMR analyses revealed positional enrichments of (13)C2-isotopologues in both the triterpenoid and the hydroxystearate moieties of 1. Five of the six isoprene units reflected a pattern with [1,2-(13)C2]- and [3,5-(13)C2]-isotopologues from the respective C5-precursors, IPP and DMAPP, whereas one isoprene unit in the ring E of 1 showed only the [3,5-(13)C2]-connectivity of the original C5-precursor, due to rearrangement of the dammarenyl cation intermediate during the cyclization process. The presence of (13)C2-isotopologues was indicative of [(13)C2]acetyl-CoA being the precursor units in the formation of the fatty acid moiety and of the triterpene via the mevalonate route. The observed labeling pattern was in agreement with a chair-chair-chair-boat conformation of the (S)-2,3-oxidosqualene precursor during the cyclization process, suggesting that the lupeol synthase from P. andrieuxii is of the same type as that from Olea europea and Taraxacum officinale, but different from that of Arabidopsis thaliana. The study shows that (13)CO2 pulse-chase experiments are powerful in elucidating, under in vivo conditions and in a single experiment, the biosynthesis of complex plant products including higher terpenes.

The formation of pyrroline and tetrahydropyridine rings in amino acids catalyzed by pyrrolysine synthase (PylD).

The dehydrogenase PylD catalyzes the ultimate step of the pyrrolysine pathway by converting the isopeptide L-lysine-Nε-3R-methyl-D-ornithine to the 22nd proteinogenic amino acid. In this study, we demonstrate how PylD can be harnessed to oxidize various isopeptides to novel amino acids by combining chemical synthesis with enzyme kinetics and X-ray crystallography. The data enable a detailed description of the PylD reaction trajectory for the biosynthesis of pyrroline and tetrahydropyridine rings as constituents of pyrrolysine analogues.

Deorphaning pyrrolopyrazines as potent multi-target antimalarial agents.

The discovery of pyrrolopyrazines as potent antimalarial agents is presented, with the most effective compounds exhibiting EC50 values in the low nanomolar range against asexual blood stages of Plasmodium falciparum in human red blood cells, and Plasmodium berghei liver schizonts, with negligible HepG2 cytotoxicity. Their potential mode of action is uncovered by predicting macromolecular targets through avant-garde computer modeling. The consensus prediction method suggested a functional resemblance between ligand binding sites in non-homologous target proteins, linking the observed parasite elimination to IspD, an enzyme from the non-mevalonate pathway of isoprenoid biosynthesis, and multi-kinase inhibition. Further computational analysis suggested essential P. falciparum kinases as likely targets of our lead compound. The results obtained validate our methodology for ligand- and structure-based target prediction, expand the bioinformatics toolbox for proteome mining, and provide unique access to deciphering polypharmacological effects of bioactive chemical agents.

Pseudilins: halogenated, allosteric inhibitors of the non-mevalonate pathway enzyme IspD.

The enzymes of the non-mevalonate pathway for isoprenoid biosynthesis have been identified as attractive targets with novel modes of action for the development of herbicides for crop protection and agents against infectious diseases. This pathway is present in many pathogenic organisms and plants, but absent in mammals. By using high-throughput screening, we identified highly halogenated marine natural products, the pseudilins, to be inhibitors of the third enzyme, IspD, in the pathway. Their activity against the IspD enzymes from Arabidopsis thaliana and Plasmodium vivax was determined in photometric and NMR-based assays. Cocrystal structures revealed that pseudilins bind to an allosteric pocket by using both divalent metal ion coordination and halogen bonding. The allosteric mode of action for preventing cosubstrate (CTP) binding at the active site was elucidated. Pseudilins show herbicidal activity in plant assays and antiplasmodial activity in cell-based assays.

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.

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.

Enzymes from the haloacid dehalogenase (HAD) superfamily catalyse the elusive dephosphorylation step of riboflavin biosynthesis.

The missing link: Studies on the biosynthesis of riboflavin have failed to characterise dephosphorylation of the intermediate 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione 5'-phosphate. We show that this reaction can be catalysed in Escherichia coli by YigB and YbjI and in plant chloroplasts by AtcpFHy1, which are members of the haloacid dehalogenase superfamily.

Structures and reaction mechanisms of GTP cyclohydrolases.

GTP cyclohydrolases generate the first committed intermediates for the biosynthesis of certain vitamins/cofactors (folic acid, riboflavin, deazaflavin, and tetrahydrobiopterin), deazapurine antibiotics, some t-RNA bases (queuosine, archaeosine), and the phytotoxin, toxoflavin. They depend on divalent cations for hydrolytic opening of the imidazole ring of the substrate, guanosine triphosphate (GTP). Surprisingly, the ring opening reaction is not the rate-limiting step for GTP cyclohydrolases I and II whose mechanism have been studied in some detail. GTP cyclohydrolase I, Ib, and II are potential targets for novel anti-infectives. Genetic factors modulating the activity of human GTP cyclohydrolase are highly pleiotropic, since the signal transponders whose biosyntheses require their participation (nitric oxide, catecholamines) impact a very wide range of physiological phenomena. Recent studies suggest that human GTP cyclohydrolase may become an oncology target.

Probing the reaction mechanism of IspH protein by x-ray structure analysis.

Isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) represent the two central intermediates in the biosynthesis of isoprenoids. The recently discovered deoxyxylulose 5-phosphate pathway generates a mixture of IPP and DMAPP in its final step by reductive dehydroxylation of 1-hydroxy-2-methyl-2-butenyl 4-diphosphate. This conversion is catalyzed by IspH protein comprising a central iron-sulfur cluster as electron transfer cofactor in the active site. The five crystal structures of IspH in complex with substrate, converted substrate, products and PP(i) reported in this article provide unique insights into the mechanism of this enzyme. While IspH protein crystallizes with substrate bound to a [4Fe-4S] cluster, crystals of IspH in complex with IPP, DMAPP or inorganic pyrophosphate feature [3Fe-4S] clusters. The IspH:substrate complex reveals a hairpin conformation of the ligand with the C(1) hydroxyl group coordinated to the unique site in a [4Fe-4S] cluster of aconitase type. The resulting alkoxide complex is coupled to a hydrogen-bonding network, which serves as proton reservoir via a Thr167 proton relay. Prolonged x-ray irradiation leads to cleavage of the C(1)-O bond (initiated by reducing photo electrons). The data suggest a reaction mechanism involving a combination of Lewis-acid activation and proton coupled electron transfer. The resulting allyl radical intermediate can acquire a second electron via the iron-sulfur cluster. The reaction may be terminated by the transfer of a proton from the beta-phosphate of the substrate to C(1) (affording DMAPP) or C(3) (affording IPP).