Misregulation of bromotyrosine compromises fertility in male Drosophila.

Biological regulation often depends on reversible reactions such as phosphorylation, acylation, methylation, and glycosylation, but rarely halogenation. A notable exception is the iodination and deiodination of thyroid hormones. Here, we report detection of bromotyrosine and its subsequent debromination during Drosophila spermatogenesis. Bromotyrosine is not evident when Drosophila express a native flavin-dependent dehalogenase that is homologous to the enzyme responsible for iodide salvage from iodotyrosine in mammals. Deletion or suppression of the dehalogenase-encoding condet (cdt) gene in Drosophila allows bromotyrosine to accumulate with no detectable chloro- or iodotyrosine. The presence of bromotyrosine in the cdt mutant males disrupts sperm individualization and results in decreased fertility. Transgenic expression of the cdt gene in late-staged germ cells rescues this defect and enhances tolerance of male flies to bromotyrosine. These results are consistent with reversible halogenation affecting Drosophila spermatogenesis in a process that had previously eluded metabolomic, proteomic, and genomic analyses.

Hypothiocyanous acid reductase is critical for host colonization and infection by Streptococcus pneumoniae.

The major human pathogen Streptococcus pneumoniae encounters the immune-derived oxidant hypothiocyanous acid (HOSCN) at sites of colonization and infection. We recently identified the pneumococcal hypothiocyanous acid reductase (Har), a member of the flavoprotein disulfide reductase enzyme family, and showed that it contributes to the HOSCN tolerance of S. pneumoniae in vitro. Here, we demonstrate in mouse models of pneumococcal infection that Har is critical for colonization and invasion. In a colonization model, bacterial load was attenuated dramatically in the nasopharynx when har was deleted in S. pneumoniae. The Δhar strain was also less virulent compared to wild type in an invasion model as reflected by a significant reduction in bacteria in the lungs and no dissemination to the blood and brain. Kinetic measurements with recombinant Har demonstrated that this enzyme reduced HOSCN with near diffusion-limited catalytic efficiency, using either NADH (kcat/KM = 1.2 × 108 M-1s-1) or NADPH (kcat/KM = 2.5 × 107 M-1s-1) as electron donors. We determined the X-ray crystal structure of Har in complex with the FAD cofactor to 1.50 Å resolution, highlighting the active site architecture characteristic for this class of enzymes. Collectively, our results demonstrate that pneumococcal Har is a highly efficient HOSCN reductase, enabling survival against oxidative host immune defenses. In addition, we provide structural insights that may aid the design of Har inhibitors.

The rapid-reaction kinetics of an electron-bifurcating flavoprotein, the crotonyl-CoA-dependent NADH:ferredoxin oxidoreductase EtfAB:bcd.

We have investigated the kinetic behavior of the electron-bifurcating crotonyl-CoA-dependent NADH: ferredoxin oxidoreductase EtfAB:bcd from Megasphaera elsdenii. The overall behavior of the complex in both the reductive and the oxidative half-reactions is consistent with that previously determined for the individual EtfAB and bcd components. This includes an uncrossing of the half-potentials of the bifurcating flavin of the EtfAB component in the course of ferredoxin-reducing catalysis, ionization of the bcd flavin semiquinone and the appearance of a charge transfer complex upon binding of the high potential acceptor crotonyl-CoA. The observed rapid-reaction rates of ferredoxin reduction are independent of [NADH], [crotonyl-CoA], and [ferredoxin], with an observed rate of ∼0.2 s-1, consistent with the observed steady-state kinetics. In enzyme-monitored turnover experiments, an approach to steady-state where the complex's flavins become reduced but no ferredoxin is generated is followed by a steady-state phase characterized by extensive ferredoxin reduction but little change in overall levels of flavin reduction. The approach to the steady-state phase can be eliminated by prior reduction of the complex, in which case there is no lag in the onset of ferredoxin reduction; this is consistent with the et FAD needing to be reduced to the level of the (anionic) semiquinone for bifurcation and concomitant ferredoxin reduction to occur. Single-turnover experiments support this conclusion, with the accumulation of the anionic semiquinone of the et FAD apparently required to prime the system for subsequent bifurcation and ferredoxin reduction.

Physcomitrium patens flavodiiron proteins form heterotetrametric complexes.

Flavodiiron proteins (FLVs) catalyze the reduction of oxygen to water by using electrons from Photosystem I (PSI). In several photosynthetic organisms such as cyanobacteria, green algae, mosses and gymnosperms, FLV-dependent electron flow protects PSI from over-reduction and consequent damage especially under fluctuating light conditions. In this work we investigated biochemical and structural properties of FLVA and FLVB from the model moss Physcomitrium patens. The two proteins, expressed and purified from Escherichia coli, bind both iron and flavin cofactors and show NAD(P)H oxidase activity as well as oxygen reductase capacities. Moreover, the co-expression of both FLVA and FLVB, coupled to a tandem affinity purification procedure with two different affinity tags, enabled the isolation of the stable and catalytically active FLVA/B hetero tetrameric protein complex with cooperative nature. The multimeric organization was shown to be stabilized by inter-subunit disulfide bonds. This investigation provides valuable new information on the biochemical properties of FLVs, with new insights into their in vivo activity.

Singlet oxygen-mediated photochemical cross-linking of an engineered fluorescent flavoprotein iLOV.

Genetically encoded photoactive proteins are integral tools in modern biochemical and molecular biological research. Within this tool box, truncated variants of the phototropin two light-oxygen-voltage flavoprotein have been developed to photochemically generate singlet oxygen (1O2) in vitro and in vivo, yet the effect of 1O2 on these genetically encoded photosensitizers remains underexplored. In this study, we demonstrate that the "improved" light-oxygen-voltage flavoprotein is capable of photochemical 1O2 generation. Once generated, 1O2 induces protein oligomerization via covalent cross-linking. The molecular targets of protein oligomerization by cross-linking are not endogenous tryptophans or tyrosines, but rather primarily histidines. Substitution of surface-exposed histidines for serine or glycine residues effectively eliminates protein cross-linking. When used in biochemical applications, such protein-protein cross-links may interfere with native biological responses to 1O2, which can be ameliorated by substitution of the surface exposed histidines of improved" light-oxygen-voltage or other 1O2-generating flavoproteins.

Rapid reaction studies on the chemistry of flavin oxidation in urocanate reductase.

Urocanate reductase (UrdA) is a bacterial flavin-dependent enzyme that reduces urocanate to imidazole propionate, enabling bacteria to use urocanate as an alternative respiratory electron acceptor. Elevated serum levels of imidazole propionate are associated with the development of type 2 diabetes, and, since UrdA is only present in humans in gut bacteria, this enzyme has emerged as a significant factor linking the health of the gut microbiome and insulin resistance. Here, we investigated the chemistry of flavin oxidation by urocanate in the isolated FAD domain of UrdA (UrdA') using anaerobic stopped-flow experiments. This analysis unveiled the presence of a charge-transfer complex between reduced FAD and urocanate that forms within the dead time of the stopped-flow instrument (∼1 ms), with flavin oxidation subsequently occurring with a rate constant of ∼60 s-1. The pH dependence of the reaction and analysis of an Arg411Ala mutant of UrdA' are consistent with Arg411 playing a crucial role in catalysis by serving as the active site acid that protonates urocanate during hydride transfer from reduced FAD. Mutational analysis of urocanate-binding residues suggests that the twisted conformation of urocanate imposed by the active site of UrdA' facilitates urocanate reduction. Overall, this study provides valuable insight into the mechanism of urocanate reduction by UrdA.

Coupling and regulation mechanisms of the flavin-dependent halogenase PyrH observed by infrared difference spectroscopy.

Flavin-dependent halogenases are central enzymes in the production of halogenated secondary metabolites in various organisms and they constitute highly promising biocatalysts for regioselective halogenation. The mechanism of these monooxygenases includes formation of hypohalous acid from a reaction of fully reduced flavin with oxygen and halide. The hypohalous acid then diffuses via a tunnel to the substrate-binding site for halogenation of tryptophan and other substrates. Oxidized flavin needs to be reduced for regeneration of the enzyme, which can be performed in vitro by a photoreduction with blue light. Here, we employed this photoreduction to study characteristic structural changes associated with the transition from oxidized to fully reduced flavin in PyrH from Streptomyces rugosporus as a model for tryptophan-5-halogenases. The effect of the presence of bromide and chloride or the absence of any halides on the UV-vis spectrum of the enzyme demonstrated a halide-dependent structure of the flavin-binding pocket. Light-induced FTIR difference spectroscopy was applied and the signals assigned by selective isotope labeling of the protein moiety. The identified structural changes in α-helix and ß-sheet elements were strongly dependent on the presence of bromide, chloride, the substrate tryptophan, and the product 5-chloro-tryptophan, respectively. We identified a clear allosteric coupling in solution at ambient conditions between cofactor-binding site and substrate-binding site that is active in both directions, despite their separation by a tunnel. We suggest that this coupling constitutes a fine-tuned mechanism for the promotion of the enzymatic reaction of flavin-dependent halogenases in dependence of halide and substrate availability.

Biosynthetic Strategies of Berberine Bridge Enzyme-like Flavoprotein Oxidases toward Structural Diversification in Natural Product Biosynthesis.

Berberine bridge enzyme-like oxidases are often involved in natural product biosynthesis and are seen as essential enzymes for the generation of intricate pharmacophores. These oxidases have the ability to transfer a hydride atom to the FAD cofactor, which enables complex substrate modifications and rearrangements including (intramolecular) cyclizations, carbon-carbon bond formations, and nucleophilic additions. Despite the diverse range of activities, the mechanistic details of these reactions often remain incompletely understood. In this Review, we delve into the complexity that BBE-like oxidases from bacteria, fungal, and plant origins exhibit by providing an overview of the shared catalytic features and emphasizing the different reactivities. We propose four generalized modes of action by which BBE-like oxidases enable the synthesis of natural products, ranging from the classic alcohol oxidation reactions to less common amine and amide oxidation reactions. Exploring the mechanisms utilized by nature to produce its vast array of natural products is a subject of considerable interest and can lead to the discovery of unique biochemical activities.

Rapid-reaction kinetics of the bifurcating NAD+-dependent NADPH:ferredoxin oxidoreductase NfnI from Pyrococcus furiosus.

We have investigated the kinetics of NAD+-dependent NADPH:ferredoxin oxidoreductase (NfnI), a bifurcating transhydrogenase that takes two electron pairs from NADPH to reduce two ferredoxins and one NAD+ through successive bifurcation events. NADPH reduction takes place at the bifurcating FAD of NfnI's large subunit, with high-potential electrons transferred to the [2Fe-2S] cluster and S-FADH of the small subunit, ultimately on to NAD+; low-potential electrons are transferred to two [4Fe-4S] clusters of the large subunit and on to ferredoxin. Reduction of NfnI by NADPH goes to completion only at higher pH, with a limiting kred of 36 ± 1.6 s-1 and apparent KdNADPH of 5 ± 1.2 µM. Reduction of one of the [4Fe-4S] clusters of NfnI occurs within a second, indicating that in the absence of NAD+, the system can bifurcate and generate low-potential electrons without NAD+. When enzyme is reduced by NADPH in the absence of NAD+ but the presence of ferredoxin, up to three equivalents of ferredoxin become reduced, although the reaction is considerably slower than seen during steady-state turnover. Bifurcation appears to be limited by transfer of the first, high-potential electron into the high-potential pathway. Ferredoxin reduction without NAD+ demonstrates that electron bifurcation is an intrinsic property of the bifurcating FAD and is not dependent on the simultaneous presence of NAD+ and ferredoxin. The tight coupling between NAD+ and ferredoxin reduction observed under multiple-turnover conditions is instead simply due to the need to remove reducing equivalents from the high-potential electron pathway under multiple-turnover conditions.

Noncovalent interactions that tune the reactivities of the flavins in bifurcating electron transferring flavoprotein.

Bifurcating electron transferring flavoproteins (Bf-ETFs) tune chemically identical flavins to two contrasting roles. To understand how, we used hybrid quantum mechanical molecular mechanical calculations to characterize noncovalent interactions applied to each flavin by the protein. Our computations replicated the differences between the reactivities of the flavins: the electron transferring flavin (ETflavin) was calculated to stabilize anionic semiquinone (ASQ) as needed to execute its single-electron transfers, whereas the Bf flavin (Bfflavin) was found to disfavor the ASQ state more than does free flavin and to be less susceptible to reduction. The stability of ETflavin ASQ was attributed in part to H-bond donation to the flavin O2 from a nearby His side chain, via comparison of models employing different tautomers of His. This H-bond between O2 and the ET site was uniquely strong in the ASQ state, whereas reduction of ETflavin to the anionic hydroquinone (AHQ) was associated with side chain reorientation, backbone displacement, and reorganization of its H-bond network including a Tyr from the other domain and subunit of the ETF. The Bf site was less responsive overall, but formation of the Bfflavin AHQ allowed a nearby Arg side chain to adopt an alternative rotamer that can H-bond to the Bfflavin O4. This would stabilize the anionic Bfflavin and rationalize effects of mutation at this position. Thus, our computations provide insights on states and conformations that have not been possible to characterize experimentally, offering explanations for observed residue conservation and raising possibilities that can now be tested.

Vanillyl alcohol oxidase from Diplodia corticola: Residues Ala420 and Glu466 allow for efficient catalysis of syringyl derivatives.

Vanillyl alcohol oxidases (VAOs) belong to the 4-phenol oxidases family and are found predominantly in lignin-degrading ascomycetes. Systematical investigation of the enzyme family at the sequence level resulted in discovery and characterization of the second recombinantly produced VAO member, DcVAO, from Diplodia corticola. Remarkably high activities for 2,6-substituted substrates like 4-allyl-2,6-dimethoxy-phenol (3.5 ± 0.02 U mg-1) or 4-(hydroxymethyl)-2,6-dimethoxyphenol (6.3 ± 0.5 U mg-1) were observed, which could be attributed to a Phe to Ala exchange in the catalytic center. In order to rationalize this rare substrate preference among VAOs, we resurrected and characterized three ancestral enzymes and performed mutagenesis analyses. The results indicate that a Cys/Glu exchange was required to retain activity for É£-hydroxylations and shifted the acceptance towards benzyl ethers (up to 4.0 ± 0.1 U mg-1). Our findings contribute to the understanding of the functionality of VAO enzyme group, and with DcVAO, we add a new enzyme to the repertoire of ether cleaving biocatalysts.

5-Formyltetrahydrofolate promotes conformational remodeling in a methylenetetrahydrofolate reductase active site and inhibits its activity.

The flavoprotein methylenetetrahydrofolate reductase (MTHFR) catalyzes the reduction of N5, N10-methylenetetrahydrofolate (CH2-H4folate) to N5-methyltetrahydrofolate (CH3-H4folate), committing a methyl group from the folate cycle to the methionine one. This committed step is the sum of multiple ping-pong electron transfers involving multiple substrates, intermediates, and products all sharing the same active site. Insight into folate substrate binding is needed to better understand this multifunctional active site. Here, we performed activity assays with Thermus thermophilus MTHFR (tMTHFR), which showed pH-dependent inhibition by the substrate analog, N5-formyltetrahydrofolate (CHO-H4folate). Our crystal structure of a tMTHFR•CHO-H4folate complex revealed a unique folate-binding mode; tMTHFR subtly rearranges its active site to form a distinct folate-binding environment. Formation of a novel binding pocket for the CHO-H4folate p-aminobenzoic acid moiety directly affects how bent the folate ligand is and its accommodation in the active site. Comparative analysis of the available active (FAD- and folate-bound) MTHFR complex structures reveals that CHO-H4folate is accommodated in the active site in a conformation that would not support hydride transfer, but rather in a conformation that potentially reports on a different step in the reaction mechanism after this committed step, such as CH2-H4folate ring-opening. This active site remodeling provides insights into the functional relevance of the differential folate-binding modes and their potential roles in the catalytic cycle. The conformational flexibility displayed by tMTHFR demonstrates how a shared active site can use a few amino acid residues in lieu of extra domains to accommodate chemically distinct moieties and functionalities.

Rapid-reaction kinetics of the butyryl-CoA dehydrogenase component of the electron-bifurcating crotonyl-CoA-dependent NADH:ferredoxin oxidoreductase from Megasphaera elsdenii.

We have investigated the equilibrium properties and rapid-reaction kinetics of the isolated butyryl-CoA dehydrogenase (bcd) component of the electron-bifurcating crotonyl-CoA-dependent NADH:ferredoxin oxidoreductase (EtfAB-bcd) from Megasphaera elsdenii. We find that a neutral FADH• semiquinone accumulates transiently during both reduction with sodium dithionite and with NADH in the presence of catalytic concentrations of EtfAB. In both cases full reduction of bcd to the hydroquinone is eventually observed, but the accumulation of FADH• indicates that a substantial portion of reduction occurs in sequential one-electron processes rather than a single two-electron event. In rapid-reaction experiments following the reaction of reduced bcd with crotonyl-CoA and oxidized bcd with butyryl-CoA, long-wavelength-absorbing intermediates are observed that are assigned to bcdred:crotonyl-CoA and bcdox:butyryl-CoA charge-transfer complexes, demonstrating their kinetic competence in the course of the reaction. In the presence of crotonyl-CoA there is an accumulation of semiquinone that is unequivocally the anionic FAD•- rather than the neutral FADH• seen in the absence of substrate, indicating that binding of substrate/product results in ionization of the bcd semiquinone. In addition to fully characterizing the rapid-reaction kinetics of both the oxidative and reductive half-reactions, our results demonstrate that one-electron processes play an important role in the reduction of bcd in EtfAB-bcd.

The Pseudomonas aeruginosa PAO1 metallo flavoprotein d-2-hydroxyglutarate dehydrogenase requires Zn2+ for substrate orientation and activation.

Pseudomonas aeruginosa PAO1 d-2-hydroxyglutarate (D2HG) dehydrogenase (PaD2HGDH) oxidizes D2HG to 2-ketoglutarate during the vital l-serine biosynthesis and is a potential therapeutic target against P. aeruginosa. PaD2HGDH, which oxidizes d-malate as an alternative substrate, has been demonstrated to be a metallo flavoprotein that requires Zn2+ for activity. However, the role of Zn2+ in the enzyme has not been elucidated, making it difficult to rationalize why nature employs both a redox center and a metal ion for catalysis in PaD2HGDH and other metallo flavoenzymes. In this study, recombinant His-tagged PaD2HGDH was purified to high levels in the presence of Zn2+ or Co2+ to investigate the metal's role in catalysis. We found that the flavin reduction step was reversible and partially rate limiting for the enzyme's turnover at pH 7.4 with either D2HG or d-malate with similar rate constants for both substrates, irrespective of whether Zn2+ or Co2+ was bound to the enzyme. The steady-state pL profiles of the kcat and kcat/Km values with d-malate demonstrate that Zn2+ mediates the activation of water coordinated to the metal. Our data are consistent with a dual role for the metal, which orients the hydroxy acid substrate in the enzyme's active site and rapidly deprotonates the substrate to yield an alkoxide species for hydride transfer to the flavin. Thus, we propose a catalytic mechanism for PaD2HGDH oxidation that establishes Zn2+ as a cofactor required for substrate orientation and activation during enzymatic turnover.

Uncovering Zn2+ as a cofactor of FAD-dependent Pseudomonas aeruginosa PAO1 d-2-hydroxyglutarate dehydrogenase.

Pseudomonas aeruginosa couples the oxidation of d-2-hydroxyglutarate (D2HG) to l-serine biosynthesis for survival, using d-2-hydroxyglutarate dehydrogenase from P. aeruginosa (PaD2HGDH). Knockout of PaD2HGDH impedes P. aeruginosa growth, making PaD2HGDH a potential target for therapeutics. Previous studies showed that the enzyme's activity increased with Zn2+, Co2+, or Mn2+ but did not establish the enzyme's metal composition and whether the metal is an activator or a required cofactor for the enzyme, which we addressed in this study. Comparable to the human enzyme, PaD2HGDH showed only 15% flavin reduction with D2HG or d-malate. Upon purifying PaD2HGDH with 1 mM Zn2+, the Zn2+:protein stoichiometry was 2:1, yielding an enzyme with ∼40 s-1kcat for d-malate. Treatment with 1 mM EDTA decreased the Zn2+:protein ratio to 1:1 without changing the kinetic parameters with d-malate. We observed complete enzyme inactivation for the metalloapoenzyme with 100 mM EDTA treatment, suggesting that Zn2+ is essential for PaD2HGDH activity. The presence of Zn2+ increased the flavin N3 atom pKa value to 11.9, decreased the flavin ε450 at pH 7.4 from 13.5 to 11.8 mM-1 cm-1, and yielded a charged transfer complex with a broad absorbance band >550 nm, consistent with a Zn2+-hydrate species altering the electronic properties of the enzyme-bound FAD. The exogenous addition of Zn2+, Co2+, Cd2+, Mn2+, or Ni2+ to the metalloapoenzyme reactivated the enzyme in a sigmoidal pattern, consistent with an induced fit rapid-rearrangement mechanism. Collectively, our data demonstrate that PaD2HGDH is a Zn2+-dependent metallo flavoprotein, which requires Zn2+ as an essential cofactor for enzyme activity.

Synthesis of Pharmaceutically Relevant Arylamines Enabled by a Nitroreductase from Bacillus tequilensis.

Arylamines are essential building blocks for the manufacture of valuable pharmaceuticals, pigments and dyes. However, their current industrial production involves the use of chemocatalytic procedures with a significant environmental impact. As a result, flavin-dependent nitroreductases (NRs) have received increasing attention as sustainable catalysts for more ecofriendly synthesis of arylamines. In this study, we assessed a novel NR from Bacillus tequilensis, named BtNR, for the synthesis of pharmaceutically relevant arylamines, including valuable synthons used in the manufacture of blockbuster drugs such as vismodegib, sonidegib, linezolid and sildenafil. After optimizing the enzymatic reaction conditions, high conversion of nitroaromatics to arylamines (up to 97 %) and good product yields (up to 56 %) were achieved. Our results indicate that BtNR has a broad substrate scope, including bulky nitro benzenes, nitro pyrazoles and nitro pyridines. Hence, BtNR is an interesting biocatalyst for the synthesis of pharmaceutically relevant amine-functionalized aromatics, providing an attractive alternative to traditional chemical synthesis methodologies.

A multifunctional flavoprotein monooxygenase HspB for hydroxylation and C-C cleavage of 6-hydroxy-3-succinoyl-pyridine.

Flavoprotein monooxygenases catalyze reactions, including hydroxylation and epoxidation, involved in the catabolism, detoxification, and biosynthesis of natural substrates and industrial contaminants. Among them, the 6-hydroxy-3-succinoyl-pyridine (HSP) monooxygenase (HspB) from Pseudomonas putida S16 facilitates the hydroxylation and C-C bond cleavage of the pyridine ring in nicotine. However, the mechanism for biodegradation remains elusive. Here, we refined the crystal structure of HspB and elucidated the detailed mechanism behind the oxidative hydroxylation and C-C cleavage processes. Leveraging structural information about domains for binding the cofactor flavin adenine dinucleotide (FAD) and HSP substrate, we used molecular dynamics simulations and quantum/molecular mechanics calculations to demonstrate that the transfer of an oxygen atom from the reactive FAD peroxide species (C4a-hydroperoxyflavin) to the C3 atom in the HSP substrate constitutes a rate-limiting step, with a calculated reaction barrier of about 20 kcal/mol. Subsequently, the hydrogen atom was rebounded to the FAD cofactor, forming C4a-hydroxyflavin. The residue Cys218 then catalyzed the subsequent hydrolytic process of C-C cleavage. Our findings contribute to a deeper understanding of the versatile functions of flavoproteins in the natural transformation of pyridine and HspB in nicotine degradation.IMPORTANCEPseudomonas putida S16 plays a pivotal role in degrading nicotine, a toxic pyridine derivative that poses significant environmental challenges. This study highlights a key enzyme, HspB (6-hydroxy-3-succinoyl-pyridine monooxygenase), in breaking down nicotine through the pyrrolidine pathway. Utilizing dioxygen and a flavin adenine dinucleotide cofactor, HspB hydroxylates and cleaves the substrate's side chain. Structural analysis of the refined HspB crystal structure, combined with state-of-the-art computations, reveals its distinctive mechanism. The crucial function of Cys218 was never discovered in its homologous enzymes. Our findings not only deepen our understanding of bacterial nicotine degradation but also open avenues for applications in both environmental cleanup and pharmaceutical development.

Electron-transferring flavoprotein and its dehydrogenase contributed to growth development and virulence in Beauveria bassiana.

Electron-transferring flavoprotein (Etf) and its dehydrogenase (Etfdh) are integral components of the electron transport chain in mitochondria. In this study, we characterize two putative etf genes (Bbetfa and Bbetfb) and their dehydrogenase gene Bbetfdh in the entomopathogenic fungus Beauveria bassiana. Individual deletion of these genes caused a significant reduction in vegetative growth, conidiation, and delayed conidial germination. Lack of these genes also led to abnormal metabolism of fatty acid and increasing lipid body accumulation. Furthermore, the virulence of Bbetfs and Bbetfdh deletion mutants was severely impaired due to decreasing infection structure formation. Additionally, all deletion strains showed reduced ATP synthesis compared to the wild-type strain. Taken together, Bbetfa and Bbetfb, along with Bbetfdh, play principal roles in fungal vegetative growth, conidiation, conidial germination, and pathogenicity of B. bassiana due to their essential functions in fatty acid metabolism.

Electron-Transferring Flavoprotein and Its Dehydrogenase Required for Fungal Pathogenicity in Arthrobotrys oligospora.

Electron transfer flavoprotein (ETF) plays an important function in fatty acid beta oxidation and the amino acid metabolic pathway. It can provide pathogenicity to some opportunistic fungi via modulating cellular metabolite composition. Arthrobotrys oligospora is a typical invasion fungus to nematodes. Its ETF characterization is still unknown. Here, we showed that the mutations of A. oligospora ETF (Aoetfα and Aoetfß) and its dehydrogenase (Aoetfdh) led to severe defects in mitochondrial integrity and blocked fatty acid metabolism. The pathogenicity-associated trap structures were completely suppressed when exposed to nematode-derived ascarosides and nutrition signals, including ammonia and urea. Compared to the wild-type strain, the nematode predatory activity was significantly reduced and delayed. But surprisingly, the rich nutrition could restore the massive trap and robust predatory activity in the mutant Aoetfß beyond all induction cues. Moreover, the deletion of Aoetfß has led to the accumulation of butyrate-like smell, which has a strong attraction to Caenorhabditis elegans nematodes. Ultimately, ETF and its dehydrogenase play a crucial role in nematode-trapping fungi, highlighting mitochondrial metabolite fluctuations that are connected to pathogenesis and further regulating the interactions between fungi and nematodes.

A conserved sequence motif in the Escherichia coli soluble FAD-containing pyridine nucleotide transhydrogenase is important for reaction efficiency.

Soluble pyridine nucleotide transhydrogenases (STHs) are flavoenzymes involved in the redox homeostasis of the essential cofactors NAD(H) and NADP(H). They catalyze the reversible transfer of reducing equivalents between the two nicotinamide cofactors. The soluble transhydrogenase from Escherichia coli (SthA) has found wide use in both in vivo and in vitro applications to steer reducing equivalents toward NADPH-requiring reactions. However, mechanistic insight into SthA function is still lacking. In this work, we present a biochemical characterization of SthA, focusing for the first time on the reactivity of the flavoenzyme with molecular oxygen. We report on oxidase activity of SthA that takes place both during transhydrogenation and in the absence of an oxidized nicotinamide cofactor as an electron acceptor. We find that this reaction produces the reactive oxygen species hydrogen peroxide and superoxide anion. Furthermore, we explore the evolutionary significance of the well-conserved CXXXXT motif that distinguishes STHs from the related family of flavoprotein disulfide reductases in which a CXXXXC motif is conserved. Our mutational analysis revealed the cysteine and threonine combination in SthA leads to better coupling efficiency of transhydrogenation and reduced reactive oxygen species release compared to enzyme variants with mutated motifs. These results expand our mechanistic understanding of SthA by highlighting reactivity with molecular oxygen and the importance of the evolutionarily conserved sequence motif.