Structural Basis of Hydrogenotrophic Methanogenesis.

Most methanogenic archaea use the rudimentary hydrogenotrophic pathway-from CO2 and H2 to methane-as the terminal step of microbial biomass degradation in anoxic habitats. The barely exergonic process that just conserves sufficient energy for a modest lifestyle involves chemically challenging reactions catalyzed by complex enzyme machineries with unique metal-containing cofactors. The basic strategy of the methanogenic energy metabolism is to covalently bind C1 species to the C1 carriers methanofuran, tetrahydromethanopterin, and coenzyme M at different oxidation states. The four reduction reactions from CO2 to methane involve one molybdopterin-based two-electron reduction, two coenzyme F420-based hydride transfers, and one coenzyme F430-based radical process. For energy conservation, one ion-gradient-forming methyl transfer reaction is sufficient, albeit supported by a sophisticated energy-coupling process termed flavin-based electron bifurcation for driving the endergonic CO2 reduction and fixation. Here, we review the knowledge about the structure-based catalytic mechanism of each enzyme of hydrogenotrophic methanogenesis.

An uncharacteristically low-potential flavin governs the energy landscape of electron bifurcation.

Electron bifurcation, an energy-conserving process utilized extensively throughout all domains of life, represents an elegant means of generating high-energy products from substrates with less reducing potential. The coordinated coupling of exergonic and endergonic reactions has been shown to operate over an electrochemical potential of ∼1.3 V through the activity of a unique flavin cofactor in the enzyme NADH-dependent ferredoxin-NADP+ oxidoreductase I. The inferred energy landscape has features unprecedented in biochemistry and presents novel energetic challenges, the most intriguing being a large thermodynamically uphill step for the first electron transfer of the bifurcation reaction. However, ambiguities in the energy landscape at the bifurcating site deriving from overlapping flavin spectral signatures have impeded a comprehensive understanding of the specific mechanistic contributions afforded by thermodynamic and kinetic factors. Here, we elucidate an uncharacteristically low two-electron potential of the bifurcating flavin, resolving the energetic challenge of the first bifurcation event.

Ultrafast photooxidation of protein-bound anionic flavin radicals.

The photophysical properties of anionic semireduced flavin radicals are largely unknown despite their importance in numerous biochemical reactions. Here, we studied the photoproducts of these intrinsically unstable species in five different flavoprotein oxidases where they can be stabilized, including the well-characterized glucose oxidase. Using ultrafast absorption and fluorescence spectroscopy, we unexpectedly found that photoexcitation systematically results in the oxidation of protein-bound anionic flavin radicals on a time scale of less than ∼100 fs. The thus generated photoproducts decay back in the remarkably narrow 10- to 20-ps time range. Based on molecular dynamics and quantum mechanics computations, positively charged active-site histidine and arginine residues are proposed to be the electron acceptor candidates. Altogether, we established that, in addition to the commonly known and extensively studied photoreduction of oxidized flavins in flavoproteins, the reverse process (i.e., the photooxidation of anionic flavin radicals) can also occur. We propose that this process may constitute an excited-state deactivation pathway for protein-bound anionic flavin radicals in general. This hitherto undocumented photochemical reaction in flavoproteins further extends the family of flavin photocycles.

Polar Interactions between Substrate and Flavin Control Iodotyrosine Deiodinase Function.

Flavin cofactors offer a wide range of chemical mechanisms to support a great diversity in catalytic function. As a corollary, such diversity necessitates careful control within each flavoprotein to limit its function to an appropriate subset of possible reactions and substrates. This task falls to the protein environment surrounding the flavin in most enzymes. For iodotyrosine deiodinase that catalyzes a reductive dehalogenation of halotyrosines, substrates can dictate the chemistry available to the flavin. Their ability to stabilize the necessary one-electron reduced semiquinone form of flavin strictly depends on a direct coordination between the flavin and α-ammonium and carboxylate groups of its substrates. While perturbations to the carboxylate group do not significantly affect binding to the resting oxidized form of the deiodinase, dehalogenation (kcat/Km) is suppressed by over 2000-fold. Lack of the α-ammonium group abolishes detectable binding and dehalogenation. Substitution of the ammonium group with a hydroxyl group does not restore measurable binding but does support dehalogenation with an efficiency greater than those of the carboxylate derivatives. Consistent with these observations, the flavin semiquinone does not accumulate during redox titration in the presence of inert substrate analogues lacking either the α-ammonium or carboxylate groups. As a complement, a nitroreductase activity based on hydride transfer is revealed for the appropriate substrates with perturbations to their zwitterion.

Mediating the performance of anaerobic granular sludge by exogenous flavin supplementation: Flavin binding capacity and behavior, interspecies electron transfer, and methane production.

Although flavins are known as effective electron mediators, the binding capacity of exogenous flavins by anaerobic granular sludge (AGS) and their role in interspecies electron transfer (IET) remains unknown. In this study, AGS was mediated by using three exogenous flavins of riboflavin (RF), flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD). Results showed that the total amounts of flavins associated with extracellular polymeric substance (EPS) of AGS increased by 2.03-2.42 and 3.83-4.94 folds, after exposure to 50 and 200 µM of exogenous flavins, respectively. A large portion of FMN and FAD was transformed into RF by AGS. Exogenous flavin mediation also stimulated the production of EPS and cytochrome c (c-Cyts) as well as cytochrome-bound flavins. The increased abundance of these electron mediators led to a reduced electrochemical impedance of EPS and improved extracellular electron transfer capacity. The methane production of AGS after mediation with exogenous RF, FMN, and FAD increased by 19.03-31.71%, 22.86-26.04%, and 28.51-33.44%, respectively. This study sheds new light on the role of exogenous flavins in promoting the IET process of a complex microbial aggregate of AGS.

Acetogenic bacteria utilize light-driven electrons as an energy source for autotrophic growth.

Acetogenic bacteria use cellular redox energy to convert CO2 to acetate using the Wood-Ljungdahl (WL) pathway. Such redox energy can be derived from electrons generated from H2 as well as from inorganic materials, such as photoresponsive semiconductors. We have developed a nanoparticle-microbe hybrid system in which chemically synthesized cadmium sulfide nanoparticles (CdS-NPs) are displayed on the cell surface of the industrial acetogen Clostridium autoethanogenum The hybrid system converts CO2 into acetate without the need for additional energy sources, such as H2, and uses only light-induced electrons from CdS-NPs. To elucidate the underlying mechanism by which C. autoethanogenum uses electrons generated from external energy sources to reduce CO2, we performed transcriptional analysis. Our results indicate that genes encoding the metal ion or flavin-binding proteins were highly up-regulated under CdS-driven autotrophic conditions along with the activation of genes associated with the WL pathway and energy conservation system. Furthermore, the addition of these cofactors increased the CO2 fixation rate under light-exposure conditions. Our results demonstrate the potential to improve the efficiency of artificial photosynthesis systems based on acetogenic bacteria integrated with photoresponsive nanoparticles.

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.

Directed Evolution of Flavin-Dependent Halogenases for Site- and Atroposelective Halogenation of 3-Aryl-4(3H)-Quinazolinones via Kinetic or Dynamic Kinetic Resolution.

In this study, we engineer a variant of the flavin-dependent halogenase RebH that catalyzes site- and atroposelective halogenation of 3-aryl-4(3H)-quinazolinones via kinetic or dynamic kinetic resolution. The required directed evolution uses a combination of random and site-saturation mutagenesis, substrate walking using two probe substrates, and a two-tiered screening approach involving the analysis of variant conversion and then enantioselectivity of improved variants. The resulting variant, 3-T, provides >99:1 e.r. for the (M)-atropisomer of the major brominated product, 25-fold improved conversion, and 91-fold improved site selectivity relative to the parent enzyme on the probe substrate used in the final rounds of evolution. This high activity and selectivity translate well to several additional substrates with varied steric and electronic properties. Computational modeling and docking simulations are used to rationalize the effects of key mutations on substrate binding. Given the range of substrates that have been used for atroposelective synthesis via electrophilic halogenation in the literature, these results suggest that flavin-dependent halogenases (FDHs) could find many additional applications for atroposelective catalysis. More broadly, this study highlights how RebH can be engineered to accept structurally diverse substrates that enable its use for enantioselective catalysis.

Slow Conformational Changes of Blue Light Sensor BLUF Proteins in Milliseconds.

Blue light sensor using flavin (BLUF) proteins consist of flavin-binding BLUF domains and functional domains. Upon blue light excitation, the hydrogen bond network around the flavin chromophore changes, and the absorption spectrum in the visible region exhibits a red shift. Ultimately, the light information received in the BLUF domain is transmitted to the functional region. It has been believed that this red shift is complete within nanoseconds. In this study, slow reaction kinetics were discovered in milliseconds (τ1- and τ2-phase) for all the BLUF proteins examined (AppA, OaPAC, BlrP1, YcgF, PapB, SyPixD, and TePixD). Despite extensive reports on BLUF, this is the first clear observation of the BLUF protein absorption change with the duration in the millisecond time region. From the measurements of some domain-deleted mutants of OaPAC and two chimeric mutants of PixD proteins, it was found that the slower dynamics (τ2-phase) are strongly affected by the size and nature of the C-terminal region adjacent to the BLUF domain. Hence, this millisecond reaction is a significant indicator of conformational changes in the C-terminal region, which is essential for the biological functions. On the other hand, the τ1-phase commonly exists in all BLUF proteins, including any mutants. The origin of the slow dynamics was studied using site-specific mutants. These results clearly show the importance of Trp in the BLUF domain. Based on this, a reaction scheme for the BLUF reaction is proposed.

Flavin-Dependent Monooxygenase-Mediated 1,2-Oxazine Construction via Meisenheimer Rearrangement in the Biosynthesis of Paeciloxazine.

The [1,2]-Meisenheimer rearrangement is well known as the [1,2]-migration of an O-substituted hydroxylamine from a tertiary amine N-oxide, and it is frequently employed in organic synthesis to enforce adjacent carbon oxidation or install a 1,2-oxazine core, which is a prevalent structural feature and pharmacophore of many bioactive natural products. Although the [1,2]-Meisenheimer rearrangement was proposed to occur in the biosynthesis of a number of 1,2-oxazine-containing natural products, it has never been proved biosynthetically. Here, we identified the biosynthetic gene cluster of an insecticidal natural product, paeciloxazine (1), from Penicillium janthinellum and characterized a flavin-dependent monooxygenase, PaxA, as the first example that mediates the formation of a 1,2-oxazine moiety via Meisenheimer rearrangement. In vitro biochemical assays, site-directed mutations, docking and molecular dynamics simulations, and density functional theory calculations support the mechanism that PaxA first catalyzes N-oxidation to form an N-oxide intermediate, which undergoes [1,2]-Meisenheimer rearrangement with the assistance of an amino acid with proton transfer property. This study expands the repertoire of rearrangement reactions during the biosynthesis of natural products and provides a new strategy for discovering natural products with N-O tethers by genome mining.

A π-Stacking Based Fluorescent Probe for Labeling of Flavin Analogues in Live Cells through Unusual FRET Process.

The flavin adenine dinucleotide (FAD) is an indispensable coenzyme in live cells. It acts as a catalyst in many redox responsive metabolic reactions, including oxidative phosphorylation in mitochondria. The real-time monitoring of flavin is important to understand the disorder in the metabolic process, redox system, etc. Thus, we have developed a fluorescent probe CPy-1 that noncovalently binds with flavin to exhibit the FRET process. 1H- NMR and docking study indicated that there is a strong hydrophobic interaction between flavins and CPy-1. Also, a π-π stacking between isoalloxazine ring in flavin and quinoline and coumarin moieties of CPy-1 favors self-assembly. The nontoxic probe CPy-1 could distinguish cancer cells from normal cells based on expressions of endogenous FAD.

Engineered Bacterial Flavin-Dependent Monooxygenases for the Regiospecific Hydroxylation of Polycyclic Phenols.

4-Hydroxyphenylacetate 3-hydroxylase (4HPA3H), a flavin-dependent monooxygenase from E. coli that catalyzes the hydroxylation of monophenols to catechols, was modified by rational redesign to convert also more bulky substrates, especially phenolic natural products like phenylpropanoids, flavones or coumarins. Selected amino acid positions in the binding pocket of 4HPA3H were exchanged with residues from the homologous protein from Pseudomonas aeruginosa, yielding variants with improved conversion of spacious substrates such as the flavonoid naringenin or the alkaloid mimetic 2-hydroxycarbazole. Reactions were followed by an adapted Fe(III)-catechol chromogenic assay selective for the products. Especially substitution of the residue Y301 facilitated modulation of substrate specificity: introduction of nonaromatic but hydrophobic (iso)leucine resulted in the preference of the substrate ferulic acid (having a guaiacyl (guajacyl) moiety, part of the vanilloid motif) over unsubstituted monophenols. The in vivo (whole-cell biocatalysts) and in vitro (three-enzyme cascade) transformations of substrates by 4HPA3H and its optimized variants was strictly regiospecific and proceeded without generation of byproducts.

Dynamic association of flavin cofactors to regulate flavoprotein function.

Flavoproteins are key players in numerous redox pathways in cells. Flavin cofactors FMN and FAD confer the required chemical reactivity to flavoenzymes. In most cases, the interaction between the proteins and the flavins is noncovalent, yet stronger in comparison to other redox-active cofactors, such as NADH and NADPH. The association is considered static, but this view has started to change with the recent discovery of the dynamic association of flavins and flavoenzymes. Six cases from different organisms and various metabolic pathways are discussed here. The available mechanistic details span the range from rudimentary, as in the case of the ER-resident oxidoreductase Ero1, to comprehensive, as for the bacterial respiratory complex I. The same holds true in regard to the assumed functional role of the dynamic association presented here. More work is needed to clarify the structural and functional determinants of the known examples. Identification of new cases will help to appreciate the generality of the new principle of intracellular flavoenzyme regulation.

Aminoperoxide adducts expand the catalytic repertoire of flavin monooxygenases.

One of the hallmark reactions catalyzed by flavin-dependent enzymes is the incorporation of an oxygen atom derived from dioxygen into organic substrates. For many decades, these flavin monooxygenases were assumed to use exclusively the flavin-C4a-(hydro)peroxide as their oxygen-transferring intermediate. We demonstrate that flavoenzymes may instead employ a flavin-N5-peroxide as a soft α-nucleophile for catalysis, which enables chemistry not accessible to canonical monooxygenases. This includes, for example, the redox-neutral cleavage of carbon-hetero bonds or the dehalogenation of inert environmental pollutants via atypical oxygenations. We furthermore identify a shared structural motif for dioxygen activation and N5-functionalization, suggesting a conserved pathway that may be operative in numerous characterized and uncharacterized flavoenzymes from diverse organisms. Our findings show that overlooked flavin-N5-oxygen adducts are more widespread and may facilitate versatile chemistry, thus upending the notion that flavin monooxygenases exclusively function as nature's equivalents to organic peroxides in synthetic chemistry.

Comparing ultrafast excited state quenching of flavin 1,N6-ethenoadenine dinucleotide and flavin adenine dinucleotide by optical spectroscopy and DFT calculations.

Flavins are photoenzymatic cofactors often exploiting the absorption of light to energize photoinduced redox chemistry in a variety of contexts. Both flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) are used for this function. The study of these photoenzymes has been facilitated using flavin analogs. Most of these analogs involve modification of the flavin ring, and there is recent evidence that adenine (Ade)-modified FAD can affect enzyme turnover, but so far this has only been shown for enzymes where the adenine and flavin rings are close to each other in a stacked conformation. FAD is also stacked in aqueous solution, and its photodynamics are quite different from unstacked FAD or FMN. Oxidized photoexcited FAD decays rapidly, presumably through PET with Ade as donor and Fl* as acceptor. Definitive identification of the spectral signatures of Ade∙+ and Fl∙- radicals is elusive. Here we use the FAD analog Flavin 1,N6-Ethenoadenine Dinucleotide (εFAD) to study how different photochemical outcomes depend on the identity of the Ade moiety in stacked FAD and its analog εFAD. We have used UV-Vis transient absorption spectroscopy complemented by TD-DFT calculations to investigate the excited state evolution of the flavins. In FAD*, no radicals were observed, suggesting that FAD* does not undergo PET. εFAD* kinetics showed a broad absorption band that suggests a charge transfer state exists upon photoexcitation with evidence for radical pair formation. Surprisingly, significant triplet flavin was produced from εFAD* We hypothesize that the dipolar (ε)Ade moieties differentially modulate the singlet-triplet energy gap, resulting in different intersystem crossing rates. The additional electron density on the etheno group of εFAD supplies better orbital overlap with the flavin S1 state, accelerating charge transfer in that molecule.

One-megadalton metalloenzyme complex in Geobacter metallireducens involved in benzene ring reduction beyond the biological redox window.

Reversible biological electron transfer usually occurs between redox couples at standard redox potentials ranging from +0.8 to -0.5 V. Dearomatizing benzoyl-CoA reductases (BCRs), key enzymes of the globally relevant microbial degradation of aromatic compounds at anoxic sites, catalyze a biological Birch reduction beyond the negative limit of this redox window. The structurally characterized BamBC subunits of class II BCRs accomplish benzene ring reduction at an active-site tungsten cofactor; however, the mechanism and components involved in the energetic coupling of endergonic benzene ring reduction have remained hypothetical. We present a 1-MDa, membrane-associated, Bam[(BC)2DEFGHI]2 complex from the anaerobic bacterium Geobacter metallireducens harboring 4 tungsten, 4 zinc, 2 selenocysteines, 6 FAD, and >50 FeS cofactors. The results suggest that class II BCRs catalyze electron transfer to the aromatic ring, yielding a cyclic 1,5-dienoyl-CoA via two flavin-based electron bifurcation events. This work expands our knowledge of energetic couplings in biology by high-molecular-mass electron bifurcating machineries.

Properties and Mechanisms of Flavin-Dependent Monooxygenases and Their Applications in Natural Product Synthesis.

Natural products are usually highly complicated organic molecules with special scaffolds, and they are an important resource in medicine. Natural products with complicated structures are produced by enzymes, and this is still a challenging research field, its mechanisms requiring detailed methods for elucidation. Flavin adenine dinucleotide (FAD)-dependent monooxygenases (FMOs) catalyze many oxidation reactions with chemo-, regio-, and stereo-selectivity, and they are involved in the synthesis of many natural products. In this review, we introduce the mechanisms for different FMOs, with the classical FAD (C4a)-hydroperoxide as the major oxidant. We also summarize the difference between FMOs and cytochrome P450 (CYP450) monooxygenases emphasizing the advantages of FMOs and their specificity for substrates. Finally, we present examples of FMO-catalyzed synthesis of natural products. Based on these explanations, this review will expand our knowledge of FMOs as powerful enzymes, as well as implementation of the FMOs as effective tools for biosynthesis.

Synthesis and Structure-Activity Relationship Studies of Pyrido [1,2-e]Purine-2,4(1H,3H)-Dione Derivatives Targeting Flavin-Dependent Thymidylate Synthase in Mycobacterium tuberculosis.

In 2002, a new class of thymidylate synthase (TS) involved in the de novo synthesis of dTMP named Flavin-Dependent Thymidylate Synthase (FDTS) encoded by the thyX gene was discovered; FDTS is present only in 30% of prokaryote pathogens and not in human pathogens, which makes it an attractive target for the development of new antibacterial agents, especially against multi-resistant pathogens. We report herein the synthesis and structure-activity relationship of a novel series of hitherto unknown pyrido[1,2-e]purine-2,4(1H,3H)-dione analogues. Several synthetics efforts were done to optimize regioselective N1-alkylation through organopalladium cross-coupling. Modelling of potential hits were performed to generate a model of interaction into the active pocket of FDTS to understand and guide further synthetic modification. All those compounds were evaluated on an in-house in vitro NADPH oxidase assays screening as well as against Mycobacterium tuberculosis ThyX. The highest inhibition was obtained for compound 23a with 84.3% at 200 µM without significant cytotoxicity (CC50 > 100 µM) on PBM cells.

Characterization of a Nitro-Forming Enzyme Involved in Fosfazinomycin Biosynthesis.

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 some Streptomyces sp. 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 higher kcat/KM value for NADPH compared to NADH and has a narrow optimal pH range (7.5-8.0). Contrary to other NMOs where the kred is rate-limiting, FzmM exhibits a very fast kred (50 ± 0.01 s-1 at 4 °C) with NADPH. NADPH binds at a KD value of ∼400 µM, and hydride transfer occurs with pro-R stereochemistry. 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 the kcat. This reaction is enhanced in the presence of Asp with a slightly faster kox than the kcat, 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.

Quaternary Charge-Transfer Complex Enables Photoenzymatic Intermolecular Hydroalkylation of Olefins.

Intermolecular C-C bond-forming reactions are underdeveloped transformations in the field of biocatalysis. Here we report a photoenzymatic intermolecular hydroalkylation of olefins catalyzed by flavin-dependent 'ene'-reductases. Radical initiation occurs via photoexcitation of a rare high-order enzyme-templated charge-transfer complex that forms between an alkene, α-chloroamide, and flavin hydroquinone. This unique mechanism ensures that radical formation only occurs when both substrates are present within the protein active site. This active site can control the radical terminating hydrogen atom transfer, enabling the synthesis of enantioenriched γ-stereogenic amides. This work highlights the potential for photoenzymatic catalysis to enable new biocatalytic transformations via previously unknown electron transfer mechanisms.