Search

Prenyltransfer is an early-stage carbon-hydrogen bond (C-H) functionalization prevalent in the biosynthesis of a diverse array of biologically active bacterial, fungal, plant, and metazoan diketopiperazine (DKP) alkaloids. Toward the development of a unified strategy for biocatalytic construction of prenylated DKP indole alkaloids, we sought to identify and characterize a substrate-permissive C2 reverse prenyltransferase (PT). As the first tailoring event within the biosynthesis of cytotoxic notoamide metabolites, PT NotF catalyzes C2 reverse prenyltransfer of brevianamide F. Solving a crystal structure of NotF (in complex with native substrate and prenyl donor mimic dimethylallyl S-thiolodiphosphate (DMSPP)) revealed a large, solvent-exposed active site, intimating NotF may possess a significantly broad substrate scope. To assess the substrate selectivity of NotF, we synthesized a panel of 30 sterically and electronically differentiated tryptophanyl DKPs, the majority of which were selectively prenylated by NotF in synthetically useful conversions (2 to >99%). Quantitative representation of this substrate library and development of a descriptive statistical model provided insight into the molecular origins of NotF's substrate promiscuity. This approach enabled the identification of key substrate descriptors (electrophilicity, size, and flexibility) that govern the rate of NotF-catalyzed prenyltransfer, and the development of an "induced fit docking (IFD)-guided" engineering strategy for improved turnover of our largest substrates. We further demonstrated the utility of NotF in tandem with oxidative cyclization using flavin monooxygenase, BvnB. This one-pot, in vitro biocatalytic cascade enabled the first chemoenzymatic synthesis of the marine fungal natural product, (-)-eurotiumin A, in three steps and 60% overall yield.

The enzyme nicotine oxidoreductase (NicA2) is a member of the flavoprotein amine oxidase family that uses a cytochrome c protein (CycN) as its oxidant instead of dioxygen, which is the oxidant used by most other members of this enzyme family. We recently identified a potential binding site for CycN on the surface of NicA2 through rigid body docking [J. Biol. Chem. (2022) 298(8), 102251]. However, this potential binding interface has not been experimentally validated. In this paper, we used unnatural amino acid incorporation to probe the binding interface between NicA2 and CycN. Our results are consistent with a structural model of the NicA2-CycN complex predicted by protein-protein docking and AlphaFold, suggesting that this is the binding site for CycN on NicA2’s surface. Based on additional mutagenesis of potentially redox active residues in NicA2, we propose that electron transfer from NicA2’s flavin to CycN’s heme occurs without the assistance of a protein-derived wire.

The hydrogen bonding network that surrounds the flavin in Blue Light Utilizing FAD (BLUF) photoreceptors plays a crucial role in sensing and communicating the changes in the electronic structure of the flavin to the protein matrix upon light absorption. The network contains a highly conserved tyrosine that is essential for photoactivation. Using time-resolved infrared spectroscopy (TRIR) and unnatural amino acid (UAA) incorporation, we investigated the photoactivation mechanism and the role of the conserved tyrosine (Y6) in the forward reaction of the photoactivated adenylyl cyclase (AC) from Oscillatoria Acuminata (OaPAC). Our work elucidates the direct connection between the photoactivation process in the BLUF domain and the structural and functional implications on the partner protein for the first time. The TRIR results demonstrate formation of FADH● as an intermediate species on the photoactivation pathway which decays to form the signaling state. Using fluorotyrosine analogs to modulate the physical properties of Y6, the TRIR data reveal that a change in the pKa and/or reduction potential of Y6 has a profound effect on the forward reaction, consistent with a mechanism involving proton transfer or proton-coupled electron transfer from Y6 to the electronically excited FAD. Decreasing the pKa from 9.9 to

Flavins are highly versatile redox-active and colored cofactors in a large variety of proteins. These do include photoenzymes and photoreceptors, although the vast majority performs non-light-driven physiological functions. Nevertheless, electron transfer between flavins and specific nearby amino acid residues (in particular tyrosine, tryptophan, and presumably histidine and arginine) takes place upon excitation of flavin in many flavoproteins. For oxidized flavoproteins these reactions potentially have a photoprotective role. In this Perspective, we outline work on the characterization of early reaction intermediates not only in the relatively well-studied resting oxidized forms but also in the fully reduced and the intrinsically unstable semireduced forms, where ultrafast photooxidation of flavin was recently demonstrated. Along different lines, flavoprotein-based novel photocatalysts for biotechnological applications are presently emerging, employing both substrate photooxidation and photoreduction strategies. Deep insight into the fundamental flavin photochemical reactions may help in guiding and optimizing their development and in the exploration of novel photocatalytic approaches.

Consensus sequences have the potential to help classify the structure and function of proteins and highlight key regions that may contribute to their biological properties. Often, the level of significance will track with the extent of sequence conservation, but this should not be considered universal. Arg and Lys dominate a position adjacent to the N1 and C2 carbonyl of flavin mononucleotide (FMN) bound in the proteins of the nitroreductase superfamily. Although this placement satisfies expectations for stabilizing the reduced form of FMN, the substitution of these residues in three subfamilies promoting distinct reactions demonstrates their importance to catalysis as only modest. Replacing Arg34 with Lys, Gln, or Glu enhances FMN binding to a flavin destructase (BluB) by twofold and diminishes FMN turnover by no more than 25%. Similarly, replacing Lys14 with Arg, Gln, or Glu in a nitroreductase (NfsB) does not perturb the binding of the substrate nitrofurazone. The catalytic efficiency does decrease by 21-fold for the K14Q variant, but no change in the midpoint potential of FMN was observed with any of the variants. Equivalent substitution at Arg38 in iodotyrosine deiodinase (IYD) affects catalysis even more modestly (10-fold). While the Arg/Lys to Glu substitution inactivates NfsB and IYD, this change also stabilizes one-electron transfer in IYD contrary to predictions based on other classes of flavoproteins. Accordingly, functional correlations developed in certain structural superfamilies may not necessarily translate well to other superfamilies.

Flavoenzymes mediate a multitude of chemical reactions and are catalytically active both in different oxidation states and in covalent adducts with reagents. The transfer of such reactivity to the organic laboratory using simplified molecular flavins is highly desirable, and such applications in (photo)oxidation reactions are already established. However, molecular flavins have not been used for the reduction of organic substrates yet, although this activity is known and well-studied for DNA photolyase enzymes. We report a catalytic method using reduced molecular flavins as photoreductants and γ-terpinene as a sacrificial reductant. Additionally, we present our design for air-stable, reduced flavin catalysts, which is based on a conformational bias strategy and circumvents the otherwise rapid reduction of O

Bacterial luciferase (Lux) catalyzes oxidation of reduced flavin mononucleotide (FMN) and aldehyde to form oxidized FMN and carboxylic acid via molecular oxygen with concomitant light generation. The enzyme is useful for various detection applications in biomedical experiments. Upon reacting with oxygen, the reduced FMN generates C4a-peroxy-FMN (FMNH-C4a-OO

Flavins are central to countless enzymes but display different reactivities depending on their environments. This is understood to reflect modulation of the flavin electronic structure. To understand changes in orbital natures, energies, and correlation over the ring system, we begin by comparing seven flavin variants differing at C8, exploiting their different electronic spectra to validate quantum chemical calculations. Ground state calculations replicate a Hammett trend and reveal the significance of the flavin π-system. Comparison of higher-level theories establishes CC2 and ACD(2) as methods of choice for characterization of electronic transitions. Charge transfer character and electron correlation prove responsive to the identity of the substituent at C8. Indeed, bond length alternation analysis demonstrates extensive conjugation and delocalization from the C8 position throughout the ring system. Moreover, we succeed in replicating a particularly challenging UV/Vis spectrum by implementing hybrid QM/MM in explicit solvents. Our calculations reveal that the presence of nonbonding lone pairs correlates with the change in the UV/Vis spectrum observed when the 8-methyl is replaced by NH

N-hydroxylating monooxygenases (NMOs) are a subclass of flavin-dependent enzymes that hydroxylate nitrogen atoms. Recently, unique NMOs that perform multiple reactions on one substrate molecule have been identified. Fosfazinomycin M (FzmM) is one such NMO, forming nitrosuccinate from aspartate (Asp) in the fosfazinomycin biosynthetic pathway in someStreptomycessp. This work details the biochemical and kinetic analysis of FzmM. Steady-state kinetic investigation shows that FzmM performs a coupled reaction with Asp (kcat, 3.0 ± 0.01 s-1) forming nitrosuccinate, which can be converted to fumarate and nitrite by the action of FzmL. FzmM displays a 70-fold higherkcat/KMvalue for NADPH compared to NADH and has a narrow optimal pH range (7.5-8.0). Contrary to other NMOs where thekredis rate-limiting, FzmM exhibits a very fastkred(50 ± 0.01 s-1at 4 °C) with NADPH. NADPH binds at aKDvalue of ∼400 μM, and hydride transfer occurs withpro-Rstereochemistry. Oxidation of FzmM in the absence of Asp exhibits a spectrum with a shoulder at ∼370 nm, consistent with the formation of a C(4a)-hydroperoxyflavin intermediate, which decays into oxidized flavin and hydrogen peroxide at a rate 100-fold slower than thekcat. This reaction is enhanced in the presence of Asp with a slightly fasterkoxthan thekcat, suggesting that flavin dehydration or Asp oxidation is partially rate limiting. Multiple sequence analyses of FzmM to NMOs identified conserved residues involved in flavin binding but not for NADPH. Additional sequence analysis to related monooxygenases suggests that FzmM shares sequence motifs absent in other NMOs. Accepted version

Plant cryptochromes are central blue light receptors in land plants and algae. Photoreduction of the flavin bound to the photolyase homology region (PHR) causes a dissociation of the C-terminal extension (CCT) as effector via an unclear pathway. We applied the recently developed in-cell infrared difference (ICIRD) spectroscopy to study the response of the full-length pCRY from Chlamydomonas reinhardtii in living bacterial cells, because the receptor degraded upon isolation. We demonstrate a stabilization of the flavin neutral radical as photoproduct and of the resulting β-sheet reorganization by binding of cellular ATP. Comparison between light-induced structural responses of full-length pCRY and PHR reveals a downshift in frequency of the β-sheet signal, implying an association of the CCT close to the only β-sheet of the PHR in the dark. We provide a missing link in activation of plant cryptochromes after flavin photoreduction by indicating that β-sheet reorganization causes the CCT release and restructuring.

The native function of dihydropyrimidine dehydrogenase (DPD) is to reduce the 5,6-vinylic bond of pyrimidines uracil and thymine with electrons obtained from NADPH. NADPH and pyrimidines bind at separate active sites separated by ∼60 A that are bridged by four Fe4S4 centers. We have shown that DPD undergoes reductive activation, taking up two electrons from NADPH [Beaupre, B. A., et al. (2020) Biochemistry 59, 2419-2431]. pH studies indicate that the rate of turnover is not controlled by the protonation state of the general acid, cysteine 671. The activation of the C671 variants is delineated into two phases particularly at low pH values. Spectral deconvolution of the delineated reductive activation reaction reveals that the initial phase results in the accumulation of charge transfer absorption added to the binding difference spectrum for NADPH. The second phase results in reduction of one of the two flavins. X-ray crystal structure analysis of the C671S variant soaked with NADPH and the slow substrate, thymine, in a low-oxygen atmosphere resolved the presumed activated form of the enzyme that has the FMN cofactor reduced. These data reveal that charge transfer arises from the proximity of the NADPH and FAD bases and that the ensuing flavin is a result of rapid transfer of electrons to the FMN without accumulation of reduced forms of the FAD or Fe4S4 centers. These data suggest that the slow rate of turnover of DPD is governed by the movement of a mobile structural feature that carries the C671 residue.

Non-natural photoenzymatic reactions reported to date have depended on the excitation of electron donor-acceptor complexes formed between substrates and cofactors within protein active sites to facilitate electron transfer. While this mechanism has unlocked new reactivity, it limits the types of substrates that can be involved in this area of catalysis. Here we demonstrate that direct excitation of flavin hydroquinone within "ene"-reductase active sites enables new substrates to participate in photoenzymatic reactions. We found that by using photoexcitation these enzymes gain the ability to reduce acrylamides through a single electron transfer mechanism.

Herein, we report an aerobic oxidative C–N bond-forming process that enables the facile synthesis of imidazo[1,2-a]pyridines and takes advantage of a coupled organocatalytic system that uses flavin and iodine. Furthermore, the dual catalytic system can be applied to the one-pot, three-step synthesis of 3-thioimidazo[1,2-a]pyridines from aminopyridines, ketones, and thiols.

Flavin-mediated electron transfer is an important pathway for Fe(III) reduction by dissimilatory iron-reducing bacteria. Although the mechanisms and kinetics of Fe(III) reduction by reduced flavins have been widely studied, the reaction between Fe(II) and oxidized flavins is rarely investigated. Results of this study show that under anoxic conditions, Fe(II) can be oxidized by the oxidized forms of riboflavin (RBF) and flavin mononucleotide (FMN) at pH 7-9. For instance, at pH 9, 73% of 17.8 μM Fe(II) was oxidized by 10 μM RBF within 20 min. Both the rate and extent of oxidation increased with increasing concentrations of oxidized flavins and increasing solution pH. Thermodynamic calculations and kinetic analyses implied that the oxidation of Fe(II) proceeded predominantly via the autodecomposition of Fe2+-RBF- and Fe2+-FMN- complexes, along with minor contributions from direct oxidation of Fe(II) by flavins and flavin radicals. Our findings suggest that the reoxidation of Fe(II) by oxidized flavins may be a rate-controlling factor in microbial Fe(III) reduction via flavin-mediated electron transfer.

Protein motions and enzyme catalysis are often linked. It is hypothesized that ultrafast vibrations (femtosecond–picosecond) enhance the rate of hydride transfer catalyzed by members of the old yellow enzyme (OYE) family of ene-reductases. Here, we use time-resolved infrared (TRIR) spectroscopy in combination with stable “heavy” isotopic labeling (2H, 13C, 15N) of protein and/or cofactor to probe the vibrational energy transfer (VET) between pentaerythritol tetranitrate reductase (a member of the OYE family) and its noncovalently bound flavin mononucleotide (FMN) cofactor. We show that when the FMN cofactor is photoexcited with visible light, vibrational energy is transferred from the flavin to the surrounding protein environment on the picosecond timescale. This finding expands the scope of VET investigation in proteins, which are limited by suitable intrinsic probes, and may have implications in the understanding of the mechanism of recently discovered photoactive flavoenzymes.

Cryptochromes are ubiquitous flavin-binding light sensors closely related to DNA-repairing photolyases. The animal-like cryptochrome CraCRY from the green alga Chlamydomonas reinhardtii challenges the paradigm of cryptochromes as pure blue-light receptors by acting as a (6-4) photolyase, using 8-hydroxy-5-deazaflavin (8-HDF) as a light-harvesting antenna with a 17.4 A distance to flavin and showing spectral sensitivity up to 680 nm. The expanded action spectrum is attributed to the presence of the flavin neutral radical (FADH•) in the dark, despite a rapid FADH• decay observed in vitro in samples exclusively carrying flavin. Herein, the red-light response of CraCRY carrying flavin and 8-HDF was studied, revealing a 3-fold prolongation of the FADH• lifetime in the presence of 8-HDF. Millisecond time-resolved ultraviolet-visible spectroscopy showed the red-light-induced formation and decay of an absorbance band at 458 nm concomitant with flavin reduction. Time-resolved Fourier transform infrared (FTIR) spectroscopy and density functional theory attributed these changes to the deprotonation of 8-HDF, challenging the paradigm of 8-HDF being permanently deprotonated in photolyases. FTIR spectra showed changes in the hydrogen bonding network of asparagine 395, a residue suggested to indirectly control flavin protonation, indicating the involvement of N395 in the stabilization of FADH•. Fluorescence spectroscopy revealed a decrease in the energy transfer efficiency of 8-HDF upon flavin reduction, possibly linked to 8-HDF deprotonation. The discovery of the interdependence of flavin and 8-HDF beyond energy transfer processes highlights the essential role of the antenna, introducing a new concept enabling CraCRY and possibly other bifunctional cryptochromes to fulfill their dual function.

Flavin-dependent halogenases (FDHs) are known for installing halogens on natural products. To date, most reported FDHs are two-component FDHs, which require a flavin reductase as the reaction partner to function. Here, we report the identification of a new halogenated biaryl compound 2-chloro venemycin (1) through constitutive expression of the regulator gene vemR in the vem gene cluster in Streptomyces sp. S006 and media optimization. In addition, we provide biochemical evidence that, in the absence of the flavin reductase, purified FDH VemK catalyzes the regioselective halogenation of the pyrone moiety of venemycin (2). Mutagenesis studies showed that T315 and R317 residues are likely crucial for catalysis and NAD(P)H binding. VemK represents the first characterized single-component FDH from Streptomyces and the first FDH that halogenates a pyrone moiety.

Real-time observation of structure changes associated with protein function remains a major challenge. Ultrafast pump-probe methods record dynamics in light activated proteins, but the assignment of spectroscopic observables to specific structure changes can be difficult. The BLUF (blue light using flavin) domain proteins are an important class of light sensing flavoprotein. Here, we incorporate the unnatural amino acid (UAA) azidophenylalanine (AzPhe) at key positions in the H-bonding environment of the isoalloxazine chromophore of two BLUF domains, namely, PixD and AppABLUF; both proteins retain the red-shift on irradiation characteristic of photoactivity. Steady state and ultrafast time resolved infrared difference measurements of the azido mode reveal site-specific information on the nature and dynamics of light driven structure change. AzPhe dynamics are thus shown to be an effective probe of BLUF domain photoactivation, revealing significant differences between the two proteins and a differential response of the two sites to chromophore excitation.

Flavins are ubiquitous redox-active compounds capable of producing reactive oxygen (O2•-, •OH, and H2O2) and flavin radical species in natural environments, yet their roles in the redox transformations of environmental contaminants, such as arsenic (As), remain to be investigated. Here, we show that reduced flavins can be a source of effective oxidants for As(III) under both oxic and anoxic conditions. For instance, in the presence of 15 μM reduced riboflavin (RBFH2), 22% of 30 μM As(III) is oxidized in aerated solution at pH 7.0. The co-oxidation of As(III) with RBFH2 is pH-dependent, with a faster reaction rate under mildly acidic relative to alkaline conditions. Quencher tests with 2-propanol (for •OH) and catalase (for H2O2) indicate that As(III) oxidation under oxic conditions is likely controlled by flavin-derived •OH at pH 5.2 and 7.0, and by H2O2 at pH 9.0. Kinetic modeling further implies that flavin-derived reactive oxygen species are mainly responsible for As(III) oxidation under oxic conditions, whereas oxidation of As(III) under anoxic conditions at pH 9.0 is attributed to riboflavin radicals (RBFH•) generated from co-existing oxidized and reduced riboflavin. The demonstrated ability of flavins to catalyze As(III) oxidation has potential implications for As redox cycling in the environment.

Topoisomerase II (TOP2) poisons as anticancer drugs work by trapping TOP2 cleavage complexes (TOP2cc) to generate DNA damage. Repair of such damage by tyrosyl DNA phosphodiesterase 2 (TDP2) could render cancer cells resistant to TOP2 poisons. Inhibiting TDP2 thus represents an attractive mechanism-based chemosensitization approach. Currently known TDP2 inhibitors lack cellular potency and / or permeability. We report herein two novel subtypes of the deazaflavin TDP2 inhibitor core. By introducing an additional phenyl ring to the N-10 phenyl ring (subtype 11), or to the N-3 site of the deazaflavin scaffold (subtype 12) we have generated novel analogues with considerably improved biochemical potency and / or permeability. Importantly, many analogues of both subtypes, particularly compounds 11a, 11e, 12a, 12b and 12h, exhibited much stronger cancer cell sensitizing effect than the best reported previous analogue 4a toward the treatment with etoposide (ETP), suggesting that these analogues could serve as effective cellular probes.

Photoreceptor proteins play a vital role in a wide range of light-regulated processes. The formation of the light-adapted state of blue light using flavin (BLUF) photoreceptors is thought to involve rearrangements of hydrogen-bonding networks upon photoexcitation. Free energy simulations with partial charges corresponding to relevant ground and excited states of the Slr1694 BLUF domain characterize conformations prior to and following photoexcitation. The simulations indicate that Trp91 is thermodynamically favored to be in the active site, although it is also able to sample conformations outside the active site. For experimentally observed conformations of Trp91, Gln50 is thermodynamically favored to be oriented for a proton relay bridging Tyr8 and the flavin. When Trp91 is rotated such that it can donate a hydrogen bond to Gln50, as observed in other BLUF domains, the proton relay is not thermodynamically favored in the ground state, providing a possible explanation for the relatively fast photocycle of the Slr1694 BLUF domain. Photoexcitation to the locally excited (LE) state of the flavin induces the formation of the proton relay if it is not already formed. Electrostatically embedded time-dependent density functional theory calculations indicate that the proton relay reduces the energy gap between the LE state and the charge-transfer (CT) state associated with electron transfer from Tyr8 to the flavin. Although the CT state is higher in energy than the LE state prior to photoexcitation, the protein environment can reorganize in a manner that stabilizes the CT state so that it is lower than the LE state, enabling the LE to CT state transition. An electrostatic analysis identifies motions of individual residues, such as Arg65, that stabilize electron transfer from Tyr8 to the flavin. These conformational changes facilitate the critical proton-coupled electron transfer reaction in the BLUF photocycle.

We present a variant of the algebraic diagrammatic construction (ADC) scheme by combining ADC with the polarizable embedding (PE) model. The presented PE-ADC method is implemented through second and third order and is designed with the aim of performing accurate calculations of excited states in large molecular systems. Accuracy and large-scale applicability are demonstrated with three case studies, and we further analyze the importance of both state-specific and linear-response-type corrections to the excitation energies in the presence of the polarizable environment. We demonstrate how our combined method can be readily applied to study photoinduced biochemical processes as we model the charge-transfer (CT) excitation which is key to the photoprotection mechanism in the dodecin protein with PE-ADC(2). Through direct access to state-of-the-art excited state analysis, we find that the polarizable environment plays a decisive role by significantly increasing the CT character of the electronic excitation in dodecin. PE-ADC is thus suited to decipher photoinduced processes in complex, biomolecular systems at high precision and at reasonable computational cost.

The pa1225 gene of Pseudomonas aeruginosa strain PAO1 was cloned, and the resulting enzyme (PA1225) was purified and revealed to be an NADPH:quinone reductase. By using kinetics, fluorescence, and mass spectrometric analyses, PA1225 was shown to utilize FAD to transfer a hydride ion from NADPH to quinones. The enzyme could also use NADH, but with an efficiency that was 40-fold lower than that of NADPH as suggested by the kcat/Km values at pH 6.0. Similar initial rates of reaction were determined with 1,4-benzoquinone and 2,6-dimethoxy-1,4-benzoquinone in the range between 25 and 200 μM, suggesting a low Km value for the quinone-oxidizing substrate. The lack of inhibition by NADP+ versus NADPH at saturating concentrations of 1,4-benzoquinone was consistent with a ping-pong bi-bi mechanism. The reductive half-reaction at pH 6.0 had Kd values of 0.07 mM with NADPH and 1.8 mM with NADH; the kred for flavin reduction was independent of pH with values of ∼10 s–1 with NADPH and ∼5 s–1 with NADH. Thus, the enzyme...

Dodecins are small flavin binding proteins occurring in archaea and bacteria. They are remarkable for binding dimers of flavins with their functional relevant aromatic isoalloxazine rings deeply covered. Bacterial dodecins are widely spread and found in a large variety of pathogens, among them Pseudomonas aeruginosa, Streptococcus pneumonia, Ralstonia solanacearum, and Mycobacterium tuberculosis ( M. tuberculosis). In this work, we seek to understand the function of dodecins from M. tuberculosis dodecin. We describe flavin binding in thermodynamic and kinetic properties and achieve mechanistic insight in dodecin function by applying spectroscopic and electrochemical methods. Intriguingly, we reveal a significant pH dependence in the affinity and specificity of flavin binding. Our data give insight in M. tuberculosis dodecin function and advance the current understanding of dodecins as flavin storage and sequestering proteins. We suggest that the dodecin in M. tuberculosis may specifically be important for flavin homeostasis during the elaborate lifestyle of this organism, which calls for the evaluation of this protein as drug target.

Interfacial electron transfer between an electroactive biofilm and an electrode is a crucial step for microbial fuel cells (MFCs) and other bio-electrochemical systems. Here, a hierarchically porous nitrogen-doped carbon nanotubes (CNTs)/reduced graphene oxide (rGO) composite with polyaniline as the nitrogen source has been developed for the MFC anode. This composite possesses a nitrogen atom-doped surface for improved flavin redox reaction and a three-dimensional hierarchically porous structure for rich bacterial biofilm growth. The maximum power density achieved with the N-CNTs/rGO anode in S. putrefaciens CN32 MFCs is 1137 mW m–2, which is 8.9 times compared with that of the carbon cloth anode and also higher than those of N-CNTs (731.17 mW m–2), N-rGO (442.26 mW m–2), and the CNTs/rGO (779.9 mW m–2) composite without nitrogen doping. The greatly improved bio-electrocatalysis could be attributed to the enhanced adsorption of flavins on the N-doped surface and the high density of biofilm adhesion for fa...

Extensive effort has been devoted to engineering flavin-dependent halogenases (FDHs) with improved stability, expanded substrate scope, and altered regioselectivity. Here, we show that variants of rebeccamycin halogenase (RebH) catalyze enantioselective desymmetrization of methylenedianilines via halogenation of these substrates distal to their pro-stereogenic center. Structure-guided engineering was used to increase the conversion and selectivity of these reactions, and the synthetic utility of the halogenated products was shown via conversion of to a chiral α-substituted indole. These results constitute the first reported examples of asymmetric catalysis by FDHs.

Analysis of the genome sequence of Streptomyces sp. KCB13F003 showed the presence of a cryptic gene cluster encoding flavin-dependent halogenase and nonribosomal peptide synthetase. Pleiotropic approaches using multiple culture media followed by LC-MS-guided isolation and spectroscopic analysis enabled the identification of two new chlorinated cyclic hexapeptides, ulleungmycins A and B (1 and 2). Their structures, including absolute configurations, were determined by 1D and 2D NMR techniques, advanced Marfey's analysis, and GITC derivatization. The new peptides, featuring unusual amino acids 5-chloro-l-tryptophan and d-homoleucine, exhibited moderate antibacterial activities against Gram-positive pathogenic bacteria including methicillin-resistant and quinolone-resistant Staphylococcus aureus.

We report on novel variants of the flavin-based fluorescent protein iLOV with absorption and emission optical bands shifted to the longer wavelengths relative to their precursor. First attempts (J. Phys. Chem. B 2015, 119, 5176; 2016, 120, 3344) to improve spectral properties of iLOV, a fluorescent marker in living cells, showed that single point mutation Q489K could lead to a promising variant, but its realization was not successful due to unfavorable conformation of the flexible lysine side chain pointing away from the chromophore. The results of molecular simulations presented in this work evidence that location of the charged lysine residue near the chromophore isoalloxazine ring can be fixed by introducing additional mutations in iLOV. Several suggested protein variants are characterized by using classical and QM/MM molecular dynamics simulations followed by optimization of structures in the ground and excited states. Transition energies between the S0 and S1 states are computed by using the advanced...

The primary photochemical reaction of the light, oxygen, and voltage (LOV) domain of the blue-light photosensor YtvA of Bacillus subtilis were investigated using high-level QM(DFT/MRCI)/MM methods. After blue-light excitation, the Sγ atom of the reactive cysteine forms a covalent bond with the C4a of the flavin mononucleotide (FMN) ring. Two conformations for the side chain of reactive cysteine with occupancies of 70% (conf A) and 30% (conf B) are observed in the X-ray crystallographic structures of the YtvA-LOV (Moglich, A.; Moffat, K. J. Mol. Biol. 2007, 373, 112−126). In conf A, the thiol group is directed toward the dimethylbenzene moiety of the FMN ring whereas it is placed directly above the N5 atom of the FMN ring in conf B. Starting from both conformations, the singlet and triplet excited pathways were evaluated. The singlet states excited from conf A decay nonradiatively to the triplet states by intersystem crossing (ISC). After the formation of a neutral biradical, the triplet states cross over ...

HcbA1 is a unique flavoenzyme that catalyzes the first step in the bacterial hexachlorobenzene catabolic pathway. Here we report in vitro reconstitution of the HcbA1-catalyzed reaction. Detailed mechanistic studies provide evidence for nucleophilic aromatic substitution and flavin-N5-oxide formation.

Naturally produced halogenated compounds are ubiquitous across all domains of life where they perform a multitude of biological functions and adopt a diversity of chemical structures. Accordingly, a diverse collection of enzyme catalysts to install and remove halogens from organic scaffolds has evolved in nature. Accounting for the different chemical properties of the four halogen atoms (fluorine, chlorine, bromine, and iodine) and the diversity and chemical reactivity of their organic substrates, enzymes performing biosynthetic and degradative halogenation chemistry utilize numerous mechanistic strategies involving oxidation, reduction, and substitution. Biosynthetic halogenation reactions range from simple aromatic substitutions to stereoselective C-H functionalizations on remote carbon centers and can initiate the formation of simple to complex ring structures. Dehalogenating enzymes, on the other hand, are best known for removing halogen atoms from man-made organohalogens, yet also function naturally, albeit rarely, in metabolic pathways. This review details the scope and mechanism of nature’s halogenation and dehalogenation enzymatic strategies, highlights gaps in our understanding, and posits where new advances in the field might arise in the near future.

A bacterial flavin transferase (ApbE) was recently employed for flavin mononucleotide (FMN) modification on the Na+-translocating NADH:quinone oxidoreductase C (NqrC) protein in the pathogenic Gram-negative bacterium Vibrio cholerae. We employed this unique post-translational modification in mammalian cells and found that the FMN transfer reaction robustly occurred when NqrC and ApbE were genetically targeted in the cytosol of live mammalian cells. Moreover, NqrC expression in the endoplasmic reticulum (NqrC-ER) induced the retro-translocation of NqrC to the cytosol, leading to the proteasome-mediated ER-associated degradation of NqrC, which is considered to be an innate immunological response toward the bacterial protein. This unexpected cellular process of NqrC-ER could be exploited for the construction of an in cellulo proteasome inhibitor screening system, and our proposed approach yielded substantially improved results compared to a previous method. In addition, a truncated version of RnfG (half-RnfG...

The increased proliferation of activated hepatic stellate cells (HSCs) is associated with hepatic fibrosis and excessive extracellular matrix (ECM)-protein production. We examined the inhibitory effects of the Monascus purpureus-fermented metabolites, ankaflavin and monascin (15 and 30 μM), on the Akt/nuclear factor (NF)-κB and p38 mitogen-activated protein kinase (MAPK) signaling pathways in HSC-T6 (activated hepatic stellate cell line). Ankaflavin and monascin (30 μM) induced apoptosis and significantly inhibited cell growth (cell viabilities: 80.2 ± 5.43% and 62.8 ± 8.20%, respectively, versus control cells; P0.05). Apoptosis and G1 phase arrest (G1 phase percentages: 76.1 ± 2.85% and 79.9 ± 1.80%, respectively, versus control cells 65.9 ± 4.94%; P0.05) correlated with increased p53 and p21 levels and caspase 3 activity and decreased cyclin D1 and Bcl-2-family protein levels (P0.05, all cases). The apoptotic effects of ankaflavin and monascin were HSC-T6-specific, suggesting their potential in treating liver fibrosis.

The first structure of nicotine oxidoreductase (NicA2) was determined by X-ray crystallography. Pseudomonas putida has evolved nicotine-degrading activity to provide a source of carbon and nitrogen. The structure establishes NicA2 as a member of the monoamine oxidase family. Residues 1-50 are disordered and may play a role in localization. The nicotine-binding site proximal to the isoalloxazine ring of flavin shows an unusual composition of the classical aromatic cage (W427 and N462). The active site architecture is consistent with the proposed binding of the deprotonated form of the substrate and the flavin-dependent oxidation of the pyrrolidone C-N bond followed by nonenzymatic hydrolysis.

The flavin reductase of the alkanesulfonate monooxygenase system (SsuE) contains a conserved π-helix located at the tetramer interface that originates from the insertion of Tyr118 into helix α4 of SsuE. Although the presence of π-helices provides an evolutionary gain of function, the defined role of these discrete secondary structures remains largely unexplored. The Tyr118 residue that generated the π-helix in SsuE was substituted with Ala to evaluate the functional role of this distinctive structural feature. Interestingly, generation of the Y118A SsuE variant converted the typically flavin-free enzyme to a flavin-bound form. Mass spectrometric analysis of the extracted flavin gave a mass of 457.11 similar to that of the FMN cofactor, suggesting the Y118A SsuE variant retained flavin specificity. The Y118A SsuE FMN cofactor was reduced with approximately 1 equiv of NADPH in anaerobic titration experiments, and the flavin remained bound following reduction. Although reactivity of the reduced flavin with oxygen was slow in NADPH oxidase assays, the variant supported electron transfer to ferricyanide. In addition, there was no measurable sulfite product in coupled assays with the Y118A SsuE variant and SsuD, further demonstrating that flavin transfer was no longer supported. The results from these studies suggest that the π-helix enables SsuE to effectively utilize flavin as a substrate in the two-component monooxygenase system and provides a foundation for further studies aimed at evaluating the functional properties of the π-helix in SsuE and related two-component flavin reductase enzymes.

Flavins are known to enhance extracellular electron transfer (EET) in Shewanella oneidensis MR-1 bacteria, which reduce electron acceptors through outer-membrane (OM) cytochromes c. Free-shuttle and bound-redox cofactor mechanisms were proposed to explain this enhancement, but recent electrochemical reports favor a flavin-bound model, proposing two one-electron reductions of flavin, namely, oxidized (Ox) to semiquinone (Sq) and semiquinone to hydroquinone (Hq), at anodic and cathodic conditions, respectively. In this work, to provide a mechanistic understanding of riboflavin (RF) binding at the multiheme OM cytochrome OmcA, we explored binding configurations at hemes 2, 5, 7, and 10. Subsequently, on the basis of molecular dynamics (MD) simulations, binding free energies and redox potential shifts upon RF binding for the Ox/Sq and Sq/Hq reductions were analyzed. Our results demonstrated an upshift in the Ox/Sq and a downshift in the Sq/Hq redox potentials, consistent with a bound RF-OmcA model. Furthermore, binding free energy MD simulations indicated an RF binding preference at heme 7. MD simulations of the OmcA-MtrC complex interfacing at hemes 5 revealed a small interprotein redox potential difference with an electron transfer rate of 10(7)-10(8)/s.

Many microorganisms use flavin-dependent thymidylate synthase (FDTS) to synthesize the essential nucleotide 2'-deoxythymidine 5'-monophosphate (dTMP) from 2'-deoxyuridine 5'-monophosphate (dUMP), 5,10-methylenetetrahydrofolate (CH2THF), and NADPH. FDTSs have a structure that is unrelated to the thymidylate synthase used by humans and a very different mechanism. Here we report nuclear magnetic resonance evidence that FDTS ionizes N3 of dUMP using an active-site arginine. The ionized form of dUMP is largely responsible for the changes in the flavin absorbance spectrum of FDTS upon dUMP binding. dUMP analogues also suggest that the phosphate of dUMP acts as the base that removes the proton from C5 of the dUMP-methylene intermediate in the FDTS-catalyzed reaction. These findings establish additional differences between the mechanisms of FDTS and human thymidylate synthase.

Tyrosyl-DNA phosphodiesterase 2 repairs irreversible topoisomerase II-mediated cleavage complexes generated by anticancer topoisomerase-targeted drugs and processes replication intermediates for picornaviruses (VPg unlinkase) and hepatitis B virus. There is currently no TDP2 inhibitor in clinical development. Here, we report a series of deazaflavin derivatives that selectively inhibit the human TDP2 enzyme in a competitive manner both with recombinant and native TDP2. We show that mouse, fish, and C. elegans TDP2 enzymes are highly resistant to the drugs and that key protein residues are responsible for drug resistance. Among them, human residues L313 and T296 confer high resistance when mutated to their mouse counterparts. Moreover, deazaflavin derivatives show potent synergy in combination with the topoisomerase II inhibitor etoposide in human prostate cancer DU145 cells and TDP2-dependent synergy in TK6 human lymphoblast and avian DT40 cells. Deazaflavin derivatives represent the first suitable platform for the development of potent and selective TDP2 inhibitors.

The spread of the absorbance of the stable FADH(•) radical (300-700 nm) allows CPD photolyase to highly efficiently form FADH(-), making it functional for DNA repair. In this study, FTIR spectroscopy detected a strong hydrogen bond, from FAD N5-H to the carbonyl group of the Asn378 side chain, that is modulated by the redox state of FAD. The observed characteristic frequency shifts were reproduced in quantum-mechanical models of the flavin binding site, which were then employed to elucidate redox tuning governed by Asn378. We demonstrate that enhanced hydrogen bonding of the Asn378 side chain with the FADH(•) radical increases thermodynamic stabilization of the radical state, and further ensures kinetic stabilization and accumulation of the fully reduced FADH(-) state.

miniSOG is the first flavin-binding protein that has been developed with the specific aim of serving as a genetically-encodable light-induced source of singlet oxygen (1O2). We have determined its 1.17 Å resolution structure, which has allowed us to investigate its mechanism of photosensitization using an integrated approach combining spectroscopic and structural methods. Our results provide a structural framework to explain the ability of miniSOG to produce 1O2 as a competition between oxygen- and protein quenching of its triplet state. In addition, a third excited-state decay pathway has been identified that is pivotal for the performance of miniSOG as 1O2 photosensitizer, namely the photo-induced transformation of flavin mononucleotide (FMN) into lumichrome, which increases the accessibility of oxygen to the flavin FMN chromophore and makes protein quenching less favourable. The combination of the two effects explains the increase in the 1O2 quantum yield by one order of magnitude upon exposure to blue light. Besides, we have identified several surface electron-rich residues that are progressively photo-oxidized, further contributing to facilitate the production of 1O2. Our results help reconcile the apparent poor level of 1O2 generation by miniSOG and its excellent performance in correlative light and electron microscopy experiments., AR acknowledges funding from the French Agence Nationale de la Recherche (project SOxygen, ANR-11-JSV5-0009 and project CrystalBall, ANR-14-CE06-0005-02), from the Spanish Ministerio de Economía y Competitividad (CTQ2016-78454-C2-1-R, MAT2015-66605-P and SEV-2016-0686) and the Fundació la Marató de TV3 (grant No. 20133133). This work used the platforms of the Grenoble Instruct-ERIC Center (ISBG: UMS 3518 CNRS-CEA-UGA-EMBL) with support from FRISBI (ANR-10-INBS-05-02) and GRAL (ANR-10-LABX-49-01) within the Grenoble Partnership for Structural Biology (PSB).

A single mutation within a flavoprotein is capable of switching the catalytic activity of a dehalogenase into a nitroreductase. This change in function correlates with a destabilization of the one-electron-reduced flavin semiquinone that is differentially expressed in the nitro-FMN reductase superfamily during redox cycling. The diversity of function within such a superfamily therefore has the potential to arise from rapid evolution, and its members should provide a convenient basis for developing new catalysts with an altered specificity of choice.

The electrochemical reduction mechanisms of 2 synthesized flavins (Flox) were examined in detail in deoxygenated solutions of DMSO containing varying amounts of water, utilizing variable scan rate cyclic voltammetry (ν = 0.1-20 V s(-1)), controlled-potential bulk electrolysis, and UV-vis spectroscopy. Flavin 1, which contains a hydrogen atom at N(3), is capable of donating its proton to other reduced flavin species. After 1e(-) reduction, the initially formed Fl(•-) receives a proton from another Flox to form FlH(•) (and concomitantly produce the deprotonated flavin, Fl(-)), although the equilibrium constant for this process favors the back reaction. Any FlH(•) formed at the electrode surface immediately undergoes another 1e(-) reduction to form FlH(-), which reacts with Fl(-) to form 2 molecules of Fl(•-). Further 1e(-) reduction of Fl(•-) at more negative potentials produces the dianion, Fl(2-), which can also be protonated by another Flox to form FlH(-) and Fl(-). Flavin 2, which is methylated at N(3) (and therefore has no acidic proton), undergoes a simple chemically reversible 1e(-) reduction process in DMSO provided the water content is low (100 mM). Further 1e(-) reduction of Fl(•-) (from flavin 2) at more negative potentials leads to the dianion, Fl(2-), which is protonated by trace water in solution to form FlH(-), similar to the mechanism of flavin 1 at high scan rates. Addition of sufficient amounts of water to nonaqueous solvents results in protonation of the anion radical species, Fl(•-), for both flavins, causing an increase in the amount of FlH(-) in solution. This behavior contrasts with what is observed for quinones, which are also reduced in two 1e(-) steps in aprotic organic solvents to form the radical anions and dianions, but are able to exist in hydrogen-bonded forms (with trace or added water) without undergoing protonation.

Understanding how flavin-dependent enzymes activate oxygen for their oxidation and oxygenation reactions is one of the most challenging issues in flavoenzymology. Density functional calculations and transient kinetics were performed to investigate the mechanism of oxygen activation in the oxygenase component (C2) of p-hydroxyphenylacetate 3-hydroxylase (HPAH). We found that the protonation of dioxygen by His396 via a proton-coupled electron transfer mechanism is the key step in the formation of the triplet diradical complex of flavin semiquinone and (•)OOH. This complex undergoes intersystem crossing to form the open-shell singlet diradical complex before it forms the closed-shell singlet C4a-hydroperoxyflavin intermediate (C4aOOH). Notably, density functional calculations indicated that the formation of C4aOOH is nearly barrierless, possibly facilitated by the active site arrangement in which His396 positions the proximal oxygen of the (•)OOH in an optimum position to directly attack the C4a atom of the isoalloxazine ring. The nearly barrierless formation of C4aOOH agrees well with the experimental results; based on transient kinetics and Eyring plot analyses, the enthalpy of activation for the formation of C4aOOH is only 1.4 kcal/mol and the formation of C4aOOH by C2 is fast (∼10(6) M(-1) s(-1) at 4 °C). The calculations identified Ser171 as the key residue that stabilizes C4aOOH by accepting a hydrogen bond from the H(N5) of the isoalloxazine ring. Both Ser171 and Trp112 facilitate H2O2 elimination by donating hydrogen bonds to the proximal oxygen of the OOH moiety during the proton transfer. According to our combined theoretical and experimental studies, the existence of a positively charged general acid at the position optimized for facilitating the proton-coupled electron transfer has emerged as an important catalytic feature for the oxygen activation process in flavin-dependent enzymes.

Flavins regulate the rate and direction of extracellular electron transfer (EET) in Shewanella oneidensis. However, low concentration of endogenously secreted flavins by the wild-type S. oneidensis MR-1 limits its EET efficiency in bioelectrochemical systems (BES). Herein, a synthetic flavin biosynthesis pathway from Bacillus subtilis was heterologously expressed in S. oneidensis MR-1, resulting in ∼25.7 times' increase in secreted flavin concentration. This synthetic flavin module enabled enhanced bidirectional EET rate of MR-1, in which its maximum power output in microbial fuel cells increased ∼13.2 times (from 16.4 to 233.0 mW/m(2)), and the inward current increased ∼15.5 times (from 15.5 to 255.3 μA/cm(2)).

The dibenzothiophene catabolic pathway converts dibenzothiophene to 2-hydroxybiphenyl and sulfite. The third step of the pathway, involving the conversion of dibenzothiophene sulfone to 2-(2-hydroxyphenyl)-benzenesulfinic acid, is catalyzed by a unique flavoenzyme DszA. Mechanistic studies on this reaction suggest that the C2 hydroperoxide of dibenzothiophene sulfone reacts with flavin to form a flavin-N5-oxide. The intermediacy of the flavin-N5-oxide was confirmed by LC-MS analysis, a co-elution experiment with chemically synthesized FMN-N5-oxide and (18)O2 labeling studies.

Thymidylate is a critical DNA nucleotide that has to be synthesized in cells de novo by all organisms. Flavin-dependent thymidylate synthase (FDTS) catalyzes the final step in this de novo production of thymidylate in many human pathogens, but it is absent from humans. The FDTS reaction proceeds via a chemical route that is different from its human enzyme analogue, making FDTS a potential antimicrobial target. The chemical mechanism of FDTS is still not understood, and the two most recently proposed mechanisms involve reaction intermediates that are unusual in pyrimidine biosynthesis and biology in general. These mechanisms differ in the relative timing of the reaction of the flavin with the substrate. The consequence of this difference is significant: the intermediates are cationic in one case and neutral in the other, an important consideration in the construction of mechanism-based enzyme inhibitors. Here we test these mechanisms via chemical trapping of reaction intermediates, stopped-flow, and substrate hydrogen isotope exchange techniques. Our findings suggest that an initial activation of the pyrimidine substrate by reduced flavin is required for catalysis, and a revised mechanism is proposed on the basis of previous and new data. These findings and the newly proposed mechanism add an important piece to the puzzle of the mechanism of FDTS and suggest a new class of intermediates that, in the future, may serve as targets for mechanism-based design of FDTS-specific inhibitors.

Here, we investigate the effect of urea in the unfolding dynamics of flavin adenine dinucleotide (FAD), an important enzymatic cofactor, through steady state, time-resolved fluorescence spectroscopic and molecular dynamics (MD) simulation studies. Steady state results indicate the possibility of urea induced unfolding of FAD, inferred from increasing emission intensity of FAD with urea. The TCSPC and up-conversion results suggest that the stack-unstack dynamics of FAD severely gets affected in the presence of urea and leads to an increase in the unstack conformation population from 15% in pure water to 40% in 12 M urea. Molecular dynamics simulation was employed to understand the nature of the interaction between FAD and urea at the molecular level. Results depict that urea molecules replace many of the water molecules around adenine and isoalloxazine rings of FAD. However, the major driving force for the stability of this unstack conformations arises from the favorable stacking interaction of a significant fraction of the urea molecules with adenine and isoalloxazine rings of FAD, which overcomes the intramolecular stacking interaction between themselves observed in pure water.

Augmenter of liver regeneration is a member of the ERV family of small flavin-dependent sulfhydryl oxidases that contain a redox-active CxxC disulfide bond in redox communication with the isoalloxazine ring of bound FAD. These enzymes catalyze the oxidation of thiol substrates with the reduction of molecular oxygen to hydrogen peroxide. This work studies the catalytic mechanism of the short, cytokine form of augmenter of liver regeneration (sfALR) using model thiol substrates of the enzyme. The redox potential of the proximal disulfide in sfALR was found to be approximately 57 mV more reducing than the flavin chromophore, in agreement with titration experiments. Rapid reaction studies show that dithiothreitol (DTT) generates a transient mixed disulfide intermediate with sfALR signaled by a weak charge-transfer interaction between the thiolate of C145 and the oxidized flavin. The subsequent transfer of reducing equivalents to the flavin ring is relatively slow, with a limiting apparent rate constant of 12.4 s(-1). However, reoxidation of the reduced flavin by molecular oxygen is even slower (2.3 s(-1) at air saturation) and thus largely limits turnover at 5 mM DTT. The nature of the charge-transfer complexes observed with DTT was explored using a range of simple monothiols to mimic the initial nucleophilic attack on the proximal disulfide. While β-mercaptoethanol is a very poor substrate of sfALR (∼0.3 min(-1) at 100 mM thiol), it rapidly generates a mixed disulfide intermediate allowing the thiolate of C145 to form a strong charge-transfer complex with the flavin. Unlike the other monothiols tested, glutathione is unable to form charge-transfer complexes and is an undetectable substrate of the oxidase. These data are rationalized on the basis of the stringent steric requirements for thiol-disulfide exchange reactions. The inability of the relatively bulky glutathione to attain the in-line geometry required for efficient disulfide exchange in sfALR may be physiologically important in preventing the oxidase from catalyzing the potentially harmful oxidation of intracellular glutathione.

Examination of electron transfer and proton transfer reactions of lumiflavin and proton transfer reactions of the lumiflavin radical anion by Fourier transform ion cyclotron resonance mass spectrometry is described. From the equilibrium constant determined for electron transfer between 1,4-naphthoquinone and lumiflavin the electron affinity of lumiflavin is deduced to be 1.86 ± 0.1 eV. Measurements of the rate constants and efficiencies for proton transfer reactions indicate that the proton affinity of the lumiflavin radical anion is between that of difluoroacetate (331.0 kcal/mol) and p-formyl-phenoxide (333.0 kcal/mol). Combining the electron affinity of lumiflavin with the proton affinity of the lumiflavin radical anion gives a lumiflavin hydrogen atom affinity of 59.7 ± 2.2 kcal/mol. The ΔG298 deduced from these results for adding an H atom to gas phase lumiflavin, 52.1 ± 2.2 kcal/mol, is in good agreement with ΔG298 for adding an H atom to aqueous lumiflavin from electrochemical measurements in the literature, 51.0 kcal/mol, and that from M06-L density functional calculations in the literature, 51.2 kcal/mol, suggesting little, if any, solvent effect on the H atom addition. The proton affinity of lumiflavin deduced from the equilibrium constant for the proton transfer reaction between lumiflavin and 2-picoline is 227.3 ± 2.0 kcal mol(-1). Density functional theory calculations on isomers of protonated lumiflavin provide a basis for assigning the most probable site of protonation as position 1 on the isoalloxazine ring and for estimating the ionization potentials of lumiflavin neutral radicals.