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.

The encapsulin from Thermotoga maritima is a flavoprotein with a symmetry matched ferritin-like cargo protein.

Bacterial nanocompartments, also known as encapsulins, are an emerging class of protein-based 'organelles' found in bacteria and archaea. Encapsulins are virus-like icosahedral particles comprising a ~ 25-50 nm shell surrounding a specific cargo enzyme. Compartmentalization is thought to create a unique chemical environment to facilitate catalysis and isolate toxic intermediates. Many questions regarding nanocompartment structure-function remain unanswered, including how shell symmetry dictates cargo loading and to what extent the shell facilitates enzymatic activity. Here, we explore these questions using the model Thermotoga maritima nanocompartment known to encapsulate a redox-active ferritin-like protein. Biochemical analysis revealed the encapsulin shell to possess a flavin binding site located at the interface between capsomere subunits, suggesting the shell may play a direct and active role in the function of the encapsulated cargo. Furthermore, we used cryo-EM to show that cargo proteins use a form of symmetry-matching to facilitate encapsulation and define stoichiometry. In the case of the Thermotoga maritima encapsulin, the decameric cargo protein with fivefold symmetry preferentially binds to the pentameric-axis of the icosahedral shell. Taken together, these observations suggest the shell is not simply a passive barrier-it also plays a significant role in the structure and function of the cargo enzyme.

Ultrafast dynamics of fully reduced flavin in catalytic structures of thymidylate synthase ThyX.

Thymidylate is a vital DNA precursor synthesized by thymidylate synthases. ThyX is a flavin-dependent thymidylate synthase found in several human pathogens and absent in humans, which makes it a potential target for antimicrobial drugs. This enzyme methylates the 2'-deoxyuridine 5'-monophosphate (dUMP) to 2'-deoxythymidine 5'-monophosphate (dTMP) using a reduced flavin adenine dinucleotide (FADH-) as prosthetic group and (6R)-N5,N10-methylene-5,6,7,8-tetrahydrofolate (CH2THF) as a methylene donor. Recently, it was shown that ThyX-catalyzed reaction is a complex process wherein FADH- promotes both methylene transfer and reduction of the transferred methylene into a methyl group. Here, we studied the dynamic and photophysics of FADH- bound to ThyX, in several substrate-binding states (no substrate, in the presence of dUMP or folate or both) by femtosecond transient absorption spectroscopy. This methodology provides valuable information about the ground-state configuration of the isoalloxazine moiety of FADH- and the rigidity of its local environment, through spectra shape and excited-state lifetime parameters. In the absence of substrate, the environment of FADH- in ThyX is only mildly more constrained than that of free FADH- in solution. The addition of dUMP however narrows the distribution of ground-state configurations and increases the constraints on the butterfly bending motion in the excited state. Folate binding results in the selection of new ground-state configurations, presumably located at a greater distance from the conical intersection where excited-state decay occurs. When both substrates are present, the ground-state configuration appears on the contrary rather limited to a geometry close to the conical intersection, which explains the relatively fast excited-state decay (100 ps on the average), even if the environment of the isoalloxazine is densely packed. Hence, although the environment of the flavin is dramatically constrained, FADH- retains a dynamic necessary to shuttle carbon from folate to dUMP. Our study demonstrates the high sensitivity of FADH- photophysics to the constraints exerted by its immediate surroundings.

Post-translational flavinylation is associated with diverse extracytosolic redox functionalities throughout bacterial life.

Disparate redox activities that take place beyond the bounds of the prokaryotic cell cytosol must connect to membrane or cytosolic electron pools. Proteins post-translationally flavinylated by the enzyme ApbE mediate electron transfer in several characterized extracytosolic redox systems but the breadth of functions of this modification remains unknown. Here, we present a comprehensive bioinformatic analysis of 31,910 prokaryotic genomes that provides evidence of extracytosolic ApbEs within ~50% of bacteria and the involvement of flavinylation in numerous uncharacterized biochemical processes. By mining flavinylation-associated gene clusters, we identify five protein classes responsible for transmembrane electron transfer and two domains of unknown function (DUF2271 and DUF3570) that are flavinylated by ApbE. We observe flavinylation/iron transporter gene colocalization patterns that implicate functions in iron reduction and assimilation. We find associations with characterized and uncharacterized respiratory oxidoreductases that highlight roles of flavinylation in respiratory electron transport chains. Finally, we identify interspecies gene cluster variability consistent with flavinylation/cytochrome functional redundancies and discover a class of 'multi-flavinylated proteins' that may resemble multi-heme cytochromes in facilitating longer distance electron transfer. These findings provide mechanistic insight into an important facet of bacterial physiology and establish flavinylation as a functionally diverse mediator of extracytosolic electron transfer.

In bacteria, certain chemical reactions required for life do not take place directly inside the cells. For instance, 'redox' reactions essential to gather minerals, repair proteins and obtain energy are localised in the membranes and space that surround a bacterium. These chemical reactions involve electrons being transferred from one molecule to another in a cascade that connects the exterior of a cell to its internal space. The enzyme ApbE allows proteins to perform electron transfer by equipping them with ring-like compounds called flavins, through a process known as flavinylation. Yet, the prevelance of flavinylation in bacteria and the scope of redox reactions it facilitates has remained unclear. To investigate this question, Méheust, Huang et al. analysed over 30,000 bacterial genomes, finding genes essential for ApbE flavinylation in about half of all bacterial species across the tree of life. The role of ApbE-flavinylated proteins was then deciphered using a 'guilt by association' approach. In bacteria, genes that perform similar roles are often close to each other in the genome, which helps to infer the function of a protein coded by a specific gene. This approach revealed that flavinylation is involved in processes that allow bacteria to acquire iron and to use various energy sources. A number of interesting proteins were also identified, including a group that carry multiple flavins, and could therefore, in theory, transfer electrons over long distances. This discovery could be relevant to bioelectronic applications, which are already considering another class of bacterial electron-carrying molecules as candidates to form minuscule electric wires.

Efficient flavinylation of glycosomal fumarate reductase by its own ApbE domain in Trypanosoma brucei.

A subset of flavoproteins has a covalently attached flavin prosthetic group enzymatically attached via phosphoester bonding. In prokaryotes, this is catalysed by alternative pyrimidine biosynthesis E (ApbE) flavin transferases. ApbE-like domains are present in few eukaryotic taxa, for example the N-terminal domain of fumarate reductase (FRD) of Trypanosoma, a parasitic protist known as a tropical pathogen causing African sleeping sickness. We use the versatile reverse genetic tools available for Trypanosoma to investigate the flavinylation of glycosomal FRD (FRDg) in vivo in the physiological and organellar context. Using direct in-gel fluorescence detection of covalently attached flavin as proxy for activity, we show that the ApbE-like domain of FRDg has flavin transferase activity in vivo. The ApbE domain is preceded by a consensus flavinylation target motif at the extreme N terminus of FRDg, and serine 9 in this motif is essential as flavin acceptor. The preferred mode of flavinylation in the glycosome was addressed by stoichiometric expression and comparison of native and catalytically inactive ApbE domains. In addition to the trans-flavinylation activity, the ApbE domain catalyses the intramolecular cis-flavinylation with at least fivefold higher efficiency. We discuss how the higher efficiency due to unusual fusion of the ApbE domain to its substrate protein FRD may provide a selective advantage by faster FRD biogenesis during rapid metabolic adaptation of trypanosomes. The first 37 amino acids of FRDg, including the consensus motif, are sufficient as flavinylation target upon fusion to other proteins. We propose FRDg(1-37) as 4-kDa heat-stable, detergent-resistant fluorescent protein tag and suggest its use as a new tool to study glycosomal protein import.

Characterization of a broadly specific cadaverine N-hydroxylase involved in desferrioxamine B biosynthesis in Streptomyces sviceus.

N-hydroxylating flavin-dependent monooxygenases (FMOs) are involved in the biosynthesis of hydroxamate siderophores, playing a key role in microbial virulence. Herein, we report the first structural and kinetic characterization of a novel alkyl diamine N-hydroxylase DesB from Streptomyces sviceus (SsDesB). This enzyme catalyzes the first committed step in the biosynthesis of desferrioxamine B, a clinical drug used to treat iron overload disorders. X-ray crystal structures of the SsDesB holoenzyme with FAD and the ternary complex with bound NADP+ were solved at 2.86 Å and 2.37 Å resolution, respectively, providing a structural view of the active site environment. SsDesB crystallized as a tetramer and the structure of the individual protomers closely resembles the structures of homologous N-hydroxylating FMOs from Erwinia amylovora (DfoA), Pseudomonas aeruginosa (PvdA), and Aspergillus fumigatus (SidA). Using NADPH oxidation, oxygen consumption, and product formation assays, kinetic parameters were determined for various substrates with SsDesB. SsDesB exhibited typical saturation kinetics with substrate inhibition at high concentrations of NAD(P)H as well as cadaverine. The apparent kcat values for NADPH in steady-state NADPH oxidation and oxygen consumption assays were 0.28 ± 0.01 s-1 and 0.24 ± 0.01 s-1, respectively. However, in product formation assays used to measure the rate of N-hydroxylation, the apparent kcat for NADPH (0.034 ± 0.008 s-1) was almost 10-fold lower under saturating FAD and cadaverine concentrations, reflecting an uncoupled reaction, and the apparent NADPH KM was 33 ± 24 µM. Under saturating FAD and NADPH concentrations, the apparent kcat and KM for cadaverine in Csaky assays were 0.048 ± 0.004 s-1 and 19 ± 9 µM, respectively. SsDesB also N-hydroxylated putrescine, spermidine, and L-lysine substrates but not alkyl (di)amines that were branched or had fewer than four methylene units in an alkyl chain. These data demonstrate that SsDesB has wider substrate scope compared to other well-studied ornithine and lysine N-hydroxylases, making it an amenable biocatalyst for the production of desferrioxamine B, derivatives, and other N-substituted products.

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.

Flavin-mediated reductive iron mobilization from frog M and Mycobacterial ferritins: impact of their size, charge and reactivities with NADH/O2.

In vitro, reductive mobilization of ferritin iron using suitable electron transfer mediators has emerged as a possible mechanism to mimic the iron release process, in vivo. Nature uses flavins as electron relay molecules for important biological oxidation and oxygenation reactions. Therefore, the current work utilizes three flavin analogues: riboflavin (RF), flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), which differ in size and charge but have similar redox potentials, to relay electron from nicotinamide adenine dinucleotide (NADH) to ferritin mineral core. Of these, the smallest/neutral analogue, RF, released more iron (~ three fold) in comparison to the larger and negatively charged FMN and FAD. Although iron mobilization got marred during the initial stages under aerobic conditions, but increased with a greater slope at the later stages of the reaction kinetics, which gets inhibited by superoxide dismutase, consistent with the generation of O2∙- in situ. The initial step, i.e., interaction of flavins with NADH played critical role in the iron release process. Overall, the flavin-mediated reductive iron mobilization from ferritins occurred via two competitive pathways, involving the reduced form of flavins either alone (anaerobic condition) or in combination with O2∙- intermediate (aerobic condition). Moreover, faster iron release was observed for ferritins from Mycobacterium tuberculosis than from bullfrog, indicating the importance of protein nanocage and the advantages they provide to the respective organisms. Therefore, these structure-reactivity studies of flavins with NADH/O2 holds significance in ferritin iron release, bioenergetics, O2-based cellular toxicity and may be potentially exploited in the treatment of methemoglobinemia. Smaller sized/neutral flavin analogue, riboflavin (RF) exhibits faster reactivity towards both NADH and O2 generating more amount of O2∙- and releases higher amount of iron from different ferritins, compared to its larger sized/negatively charged derivatives such as FMN and FAD.

Functional diversity of prokaryotic HdrA(BC) modules: Role in flavin-based electron bifurcation processes and beyond.

In methanogenic archaea, the archetypical complex of heterodisulfide reductase (HdrABC) and hydrogenase (MvhAGD) couples the endergonic reduction of CO2 by H2 to the exergonic reduction of the CoB-S-S-CoM heterodisulfide by H2 via flavin-based electron bifurcation. Presently known enzymes containing HdrA(BC)-like components play key roles in methanogenesis, acetogenesis, respiratory sulfate reduction, lithotrophic reduced sulfur compound oxidation, aromatic compound degradation, fermentations, and probably many further processes. This functional diversity is achieved by a modular architecture of HdrA(BC) enzymes, where a big variety of electron input/output modules may be connected either directly or via adaptor modules to the HdrA(BC) components. Many, but not all HdrA(BC) complexes are proposed to catalyse a flavin-based electron bifurcation/confurcation. Despite the availability of HdrA(BC) crystal structures, fundamental questions of electron transfer and energy coupling processes remain. Here, we address the common properties and functional diversity of HdrA(BC) core modules integrated into electron-transfer machineries of outstanding complexity.

Role of reduced flavin in dehalogenation reactions.

Halogenated organic compounds are extensively used in the cosmetic, pharmaceutical, and chemical industries. Several naturally occurring halogen-containing natural products are also produced, mainly by marine organisms. These compounds accumulate in the environment due to their chemical stability and lack of biological pathways for their degradation. However, a few enzymes have been identified that perform dehalogenation reactions in specific biological pathways and others have been identified to have secondary activities toward halogenated compounds. Various mechanisms for dehalogenation of I, Cl, Br, and F containing compounds have been elucidated. These have been grouped into reductive, oxidative, and hydrolytic mechanisms. Flavin-dependent enzymes have been shown to catalyze oxidative dehalogenation reactions utilizing the C4a-hydroperoxyflavin intermediate. In addition, flavoenzymes perform reductive dehalogenation, forming transient flavin semiquinones. Recently, flavin-dependent enzymes have also been shown to perform dehalogenation reactions where the reduced form of the flavin produces a covalent intermediate. Here, recent studies on the reactions of flavoenzymes in dehalogenation reactions, with a focus on covalent catalytic dehalogenation mechanisms, are described.

Electron flow between the worlds of Marcus and Warburg.

Living organisms are characterized by the ability to process energy (all release heat). Redox reactions play a central role in biology, from energy transduction (photosynthesis, respiratory chains) to highly selective catalyzed transformations of complex molecules. Distance and scale are important: electrons transfer on a 1 nm scale, hydrogen nuclei transfer between molecules on a 0.1 nm scale, and extended catalytic processes (cascades) operate most efficiently when the different enzymes are under nanoconfinement (10 nm-100 nm scale). Dynamic electrochemistry experiments (defined broadly within the term "protein film electrochemistry," PFE) reveal details that are usually hidden in conventional kinetic experiments. In PFE, the enzyme is attached to an electrode, often in an innovative way, and electron-transfer reactions, individual or within steady-state catalytic flow, can be analyzed in terms of precise potentials, proton coupling, cooperativity, driving-force dependence of rates, and reversibility (a mark of efficiency). The electrochemical experiments reveal subtle factors that would have played an essential role in molecular evolution. This article describes how PFE is used to visualize and analyze different aspects of biological redox chemistry, from long-range directional electron transfer to electron/hydride (NADPH) interconversion by a flavoenzyme and finally to NADPH recycling in a nanoconfined enzyme cascade.

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.

Hydrogen movements in the oxidative half-reaction of kynurenine 3-monooxygenase from Pseudomonas fluorescens reveal the mechanism of hydroxylation.

Kynurenine 3-monoxygenase (KMO) catalyzes the conversion of l-kynurenine (L-Kyn) to 3-hydroxykynurenine (3-OHKyn) in the pathway for tryptophan catabolism. We have investigated the effects of pH and deuterium substitution on the oxidative half-reaction of KMO from P. fluorescens (PfKMO). The three phases observed during the oxidative half reaction are formation of the hydroperoxyflavin, hydroxylation and product release. The measured rate constants for these phases proved largely unchanging with pH, suggesting that the KMO active site is insulated from exchange with solvent during catalysis. A solvent inventory study indicated that a solvent isotope effect of 2-3 is observed for the hydroxylation phase and that two or more protons are in flight during this step. An inverse isotope effect of 0.84 ± 0.01 on the rate constant for the hydroxylation step with ring perdeutero-L-Kyn as a substrate indicates a shift from sp2 to sp3 hybridization in the transition state leading to the formation of a non-aromatic intermediate. The pH dependence of transient state data collected for the substrate analog meta-nitrobenzoylalanine indicate that groups proximal to the hydroperoxyflavin are titrated in the range pH 5-8.5 and can be described by a pKa of 8.8. That higher pH values do not slow the rate of hydroxylation precludes that the pKa measured pertains to the proton of the hydroperoxflavin. Together, these observations indicate that the C4a-hydroperoxyflavin has a pKa ≫ 8.5, that a non-aromatic species is the immediate product of hydroxylation and that at least two solvent derived protons are in-flight during oxygen insertion to the substrate aromatic ring. A unifying mechanistic proposal for these observations is proposed.

Discovery of Novel Pseudomonas putida Flavin-Binding Fluorescent Protein Variants with Significantly Improved Quantum Yield.

Oxygen-independent, flavin-binding fluorescent proteins (FbFPs) are emerging as alternatives to green fluorescent protein (GFP), which has limited applicability in studying anaerobic microorganisms, such as human gastrointestinal bacteria, which grow in oxygen-deficient environments. However, the utility of these FbFPs has been compromised because of their poor fluorescence emission. To overcome this limitation, we have employed a high-throughput library screening strategy and engineered an FbFP derived from Pseudomonas putida (SB2) for enhanced quantum yield. Of the resulting SB2 variants, KOFP-7 exhibited a significantly improved quantum yield (0.61) compared to other reported engineered FbFPs, which was even higher than that of enhanced GFP (EGFP, 0.60), with significantly enhanced tolerance against a strong reducing agent.

Insights into the FMNAT Active Site of FAD Synthase: Aromaticity is Essential for Flavin Binding and Catalysis.

The last step in the biosynthesis of flavin adenine dinucleotide (FAD) is considered a target for the design of antimicrobial drugs because it is carried out by two non-homologous proteins in eukaryotic and prokaryotic organisms. Monofunctional FMN: adenylyltransferases (FMNAT) in Eukarya and FMNAT modules of bifunctional FAD synthases (FADS) in Prokarya belong to different structural families with dissimilar chemistry and binding modes for the substrates. In this study, we analyzed the relevance of the hydrophobic environment of the flavin isoalloxazine in the FMNAT active site of Corynebacterium ammoniagenes FADS (CaFADS) through the mutational analysis of its F62, Y106, and F128 residues. They form the isoalloxazine binding cavity and are highly conserved in the prokaryotic FADS family. The spectroscopic, steady-state kinetics and thermodynamic data presented indicate that distortion of aromaticity at the FMNAT isoalloxazine binding cavity prevents FMN and FAD from correct accommodation in their binding cavity and, as a consequence, decreases the efficiency of the FMNAT activity. Therefore, the side-chains of F62, Y106 and F128 are relevant in the formation of the catalytic competent complex during FMNAT catalysis in CaFADS. The introduced mutations also modulate the activity occurring at the riboflavin kinase (RFK) module of CaFADS, further evidencing the formation of quaternary assemblies during catalysis.

Reductive Evolution and Diversification of C5-Uracil Methylation in the Nucleic Acids of Mollicutes.

The C5-methylation of uracil to form 5-methyluracil (m5U) is a ubiquitous base modification of nucleic acids. Four enzyme families have converged to catalyze this methylation using different chemical solutions. Here, we investigate the evolution of 5-methyluracil synthase families in Mollicutes, a class of bacteria that has undergone extensive genome erosion. Many mollicutes have lost some of the m5U methyltransferases present in their common ancestor. Cases of duplication and subsequent shift of function are also described. For example, most members of the Spiroplasma subgroup use the ancestral tetrahydrofolate-dependent TrmFO enzyme to catalyze the formation of m5U54 in tRNA, while a TrmFO paralog (termed RlmFO) is responsible for m5U1939 formation in 23S rRNA. RlmFO has replaced the S-adenosyl-L-methionine (SAM)-enzyme RlmD that adds the same modification in the ancestor and which is still present in mollicutes from the Hominis subgroup. Another paralog of this family, the TrmFO-like protein, has a yet unidentified function that differs from the TrmFO and RlmFO homologs. Despite having evolved towards minimal genomes, the mollicutes possess a repertoire of m5U-modifying enzymes that is highly dynamic and has undergone horizontal transfer.

Bioinformatic Analysis of the Flavin-Dependent Amine Oxidase Superfamily: Adaptations for Substrate Specificity and Catalytic Diversity.

The flavin-dependent amine oxidase (FAO) superfamily consists of over 9000 nonredundant sequences represented in all domains of life. Of the thousands of members identified, only 214 have been functionally annotated to date, and 40 unique structures are represented in the Protein Data Bank. The few functionally characterized members share a catalytic mechanism involving the oxidation of an amine substrate through transfer of a hydride to the FAD cofactor, with differences observed in substrate specificities. Previous studies have focused on comparing a subset of superfamily members. Here, we present a comprehensive analysis of the FAO superfamily based on reaction mechanism and substrate recognition. Using a dataset of 9192 sequences, a sequence similarity network, and subsequently, a genome neighborhood network were constructed, organizing the superfamily into eight subgroups that accord with substrate type. Likewise, through phylogenetic analysis, the evolutionary relationship of subgroups was determined, delineating the divergence between enzymes based on organism, substrate, and mechanism. In addition, using sequences and atomic coordinates of 22 structures from the Protein Data Bank to perform sequence and structural alignments, active-site elements were identified, showing divergence from the canonical aromatic-cage residues to accommodate large substrates. These specificity determinants are held in a structural framework comprising a core domain catalyzing the oxidation of amines with an auxiliary domain for substrate recognition. Overall, analysis of the FAO superfamily reveals a modular fold with cofactor and substrate-binding domains allowing for diversity of recognition via insertion/deletions. This flexibility allows facile evolution of new activities, as shown by reinvention of function between subfamilies.

Interconnection of the Antenna Pigment 8-HDF and Flavin Facilitates Red-Light Reception in a Bifunctional Animal-like Cryptochrome.

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 Šdistance 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.

Expression profiles of glucosinolate biosynthetic genes in turnip (Brassica rapa var. rapa) at different developmental stages and effect of transformed flavin-containing monooxygenase genes on hairy root glucosinolate content.

BACKGROUND: Glucosinolates (GSLs) are secondary metabolites, mainly existing in Brassica vegetables. Their breakdown products have health benefits and contribute to the distinctive taste of these vegetables. Because of their high value, there is a lot of interest in developing breeding strategies to increase the content of beneficial GSLs in Brassica species. GSLs are synthesized from certain amino acids and their biological roles depend largely on the structure of their side chains. Flavin-containing monooxygenase (FMOGS-OX ) genes are involved in the synthesis of these side chains. To better understand GSL biosynthesis, we sequenced the transcriptomes of turnip (Brassica rapa var. rapa) tubers at four developmental stages (S1-S4) and determined their GSL content. RESULTS: The total GSL content was high at the early stage (S1) of tuber development and increased up to S3, then decreased at S4. We detected 61 differentially expressed genes, including five FMOGS-OX genes, that were related for GSL biosynthesis among the four developmental stages. Most of these genes were highly expressed at stages S1 to S3, but their expression was much lower at S4. We estimated the effect of the five FMOGS-OX genes on GSL content by overexpressing them in turnip hairy roots and found that the amount of aliphatic GSLs increased significantly in the transgenic plants. CONCLUSION: The transcriptome data and characterization of genes involved in GSL biosynthesis, particularly the FMOGS-OX genes, will be valuable for improving the yield of beneficial GSLs in turnip and other Brassica crops. © 2019 Society of Chemical Industry.

Mutational analysis of the flavinylation and binding motifs in two protein targets of the flavin transferase ApbE.

Many flavoproteins belonging to three domain types contain an FMN residue linked through a phosphoester bond to a threonine or serine residue found in a conserved seven-residue motif. The flavinylation reaction is catalyzed by a specific enzyme, ApbE, which uses FAD as a substrate. To determine the structural requirements of the flavinylation reaction, we examined the effects of single substitutions in the flavinylation motif of Klebsiella pneumoniae cytoplasmic fumarate reductase on its modification by its own ApbE in recombinant Escherichia coli cells. The replacement of the flavin acceptor threonine with alanine completely abolished the modification reaction, whereas the replacements of conserved aspartate and serine had only minor effects. Effects of other substitutions, including replacing the acceptor threonine with serine, (a 10-55% decrease in the flavinylation degree) pinpointed important glycine and alanine residues and suggested an excessive capacity of the ApbE-based flavinylation system in vivo. Consistent with this deduction, drastic replacements of conserved leucine and threonine residues in the binding pocket that accommodates FMN residue still allowed appreciable flavinylation of the NqrC subunit of Vibrio harveyi Na+-translocating NADH:quinone oxidoreductase, despite a profound weakening of the isoalloxazine ring binding and an increase in its exposure to solvent.