ABSTRACT
The hydroxylated and diacetylated cyclo-l-Trp-l-Leu derivative (-)-protubonine B was isolated from a culture of Aspergillus ustus 3.3904. Genome mining led to the identification of a putative biosynthetic gene cluster coding for a bimodular nonribosomal peptide synthetase, a flavin-dependent monooxygenase, and two acetyltransferases. Heterologous expression of the pbo cluster in Aspergillus nidulans showed that it is responsible for the formation of the isolated metabolite. Gene deletion experiments and structural elucidation of the isolated intermediates confirmed the biosynthetic steps. In vitro experiments with the recombinant protein proved that the flavin-dependent oxygenase is responsible for stereospecific hydroxylation at the indole ring accompanied by pyrrolidine ring formation.
Subject(s)
Aspergillus nidulans , Oxygenases , Oxygenases/genetics , Hydroxylation , Aspergillus nidulans/genetics , Flavins/genetics , Multigene FamilyABSTRACT
Enzymatic halogenation captures scientific interest considering its feasibility in modifying compounds for chemical diversity. Currently, majority of flavin-dependent halogenases (F-Hals) were reported from bacterial origin, and as far as we know, none from lichenized fungi. Fungi are well-known producers of halogenated compounds, so using available transcriptomic dataset of Dirinaria sp., we mined for putative gene encoding for F-Hal. Phylogenetic-based classification of the F-Hal family suggested a non-tryptophan F-Hals, similar to other fungal F-Hals, which mainly act on aromatic compounds. However, after the putative halogenase gene from Dirinaria sp., dnhal was codon-optimized, cloned, and expressed in Pichia pastoris, the ~63 kDa purified enzyme showed biocatalytic activity towards tryptophan and an aromatic compound methyl haematommate, which gave the tell-tale isotopic pattern of a chlorinated product at m/z 239.0565 and 241.0552; and m/z 243.0074 and 245.0025, respectively. This study is the start of understanding the complexities of lichenized fungal F-hals and its ability to halogenate tryptophan and other aromatic. compounds which can be used as green alternatives for biocatalysis of halogenated compounds.
Subject(s)
Lichens , Oxidoreductases , Oxidoreductases/metabolism , Lichens/metabolism , Tryptophan/metabolism , Phylogeny , Halogenation , Organic Chemicals , Flavins/genetics , Flavins/metabolismABSTRACT
Contezolid (MRX-I), a safer antibiotic of the oxazolidinone class, is a promising new antibiotic with potent activity against Mycobacterium tuberculosis (MTB) both in vitro and in vivo. To identify resistance mechanisms of contezolid in MTB, we isolated several in vitro spontaneous contezolid-resistant MTB mutants, which exhibited 16-fold increases in the MIC of contezolid compared with the parent strain but were still unexpectedly susceptible to linezolid. Whole-genome sequencing revealed that most of the contezolid-resistant mutants bore mutations in the mce3R gene, which encodes a transcriptional repressor. The mutations in mce3R led to markedly increased expression of a monooxygenase encoding gene Rv1936. We then characterized Rv1936 as a putative flavin-dependent monooxygenase that catalyzes the degradation of contezolid into its inactive 2,3-dihydropyridin-4-one (DHPO) ring-opened metabolites, thereby conferring drug resistance. While contezolid is an attractive drug candidate with potent antimycobacterial activity and low toxicity, the occurrence of mutations in Mce3R should be considered when designing combination therapy using contezolid for treating tuberculosis.
Subject(s)
Mycobacterium tuberculosis , Oxazolidinones , Linezolid , Anti-Bacterial Agents , Mutation , Mixed Function Oxygenases/metabolism , Flavins/genetics , Flavins/metabolism , Antitubercular Agents/pharmacology , Antitubercular Agents/metabolism , Microbial Sensitivity TestsABSTRACT
Proton-coupled electron transfer (PCET) is key to the activation of the blue light using flavin (BLUF) domain photoreceptors. Here, to elucidate the photocycle of the central FMN-Gln-Tyr motif in the BLUF domain of OaPAC, we eliminated the intrinsic interfering W90 in the mutant design. We integrated the stretched exponential function into the target analysis to account for the dynamic heterogeneity arising from the active-site solvation relaxation and the flexible H-bonding network as shown in the molecular dynamics simulation results, facilitating a simplified expression of the kinetics model. We find that, in both the functional wild-type (WT) and the nonfunctional Q48E and Q48A, forward PCET happens in the range of 105 ps to 344 ps, with a kinetic isotope effect (KIE) measured to be â¼1.8 to 2.4, suggesting that the nature of the forward PCET is concerted. Remarkably, only WT proceeds with an ultrafast reverse PCET process (31 ps, KIE = 4.0), characterized by an inverted kinetics of the intermediate FMNHË. Our results reveal that the reverse PCET is driven by proton transfer via an intervening imidic Gln.
Subject(s)
Electron Transport , Flavins , Light , Flavins/genetics , Flavins/metabolism , Molecular Dynamics Simulation , ProtonsABSTRACT
Heme has been shown to have a crucial role in the signal transduction mechanism of the facultative photoheterotrophic bacterium Rhodobacter sphaeroides. It interacts with the transcriptional regulatory complex AppA/PpsR, in which AppA and PpsR function as the antirepressor and repressor, respectively, of photosynthesis gene expression. The mechanism, however, of this interaction remains incompletely understood. In this study, we combined electron paramagnetic resonance (EPR) spectroscopy and Förster resonance energy transfer (FRET) to demonstrate the ligation of heme in PpsR with a proposed cysteine residue. We show that heme binding in AppA affects the fluorescent properties of the dark-adapted state of the protein, suggesting a less constrained flavin environment compared with the absence of heme and the light-adapted state. We performed ultrafast transient absorption measurements in order to reveal potential differences in the dynamic processes in the full-length AppA and its heme-binding domain alone. Comparison of the CO-binding dynamics demonstrates a more open heme pocket in the holo-protein, qualitatively similar to what has been observed in the CO sensor RcoM-2, and suggests a communication path between the blue-light-using flavin (BLUF) and sensing containing heme instead of cobalamin (SCHIC) domains of AppA. We have also examined quantitatively the affinity of PpsR to bind to individual DNA fragments of the puc promoter using fluorescence anisotropy assays. We conclude that oligomerization of PpsR is initially triggered by binding of one of the two DNA fragments and observe a â¼10-fold increase in the dissociation constant Kd for DNA binding upon heme binding to PpsR. Our study provides significant new insight at the molecular level on the regulatory role of heme that modulates the complex transcriptional regulation in R. sphaeroides and supports the two levels of heme signaling, via its binding to AppA and PpsR and via the sensing of gases like oxygen.
Subject(s)
Gene Expression Regulation, Bacterial , Rhodobacter sphaeroides , Bacterial Proteins/metabolism , Dinucleoside Phosphates , Flavins/genetics , Flavins/metabolism , Flavoproteins , Heme/metabolism , Repressor Proteins/metabolism , Rhodobacter sphaeroides/genetics , Rhodobacter sphaeroides/metabolismABSTRACT
Thiol-containing nucleophiles such as cysteine react spontaneously with the citric acid cycle intermediate fumarate to form S-(2-succino)-adducts. In Bacillus subtilis, a salvaging pathway encoded by the yxe operon has recently been identified for the detoxification and exploitation of these compounds as sulfur sources. This route involves acetylation of S-(2-succino)cysteine to N-acetyl-2-succinocysteine, which is presumably converted to oxaloacetate and N-acetylcysteine, before a final deacetylation step affords cysteine. The critical oxidative cleavage of the C-S bond of N-acetyl-S-(2-succino)cysteine was proposed to depend on the predicted flavoprotein monooxygenase YxeK. Here, we characterize YxeK and verify its role in S-(2-succino)-adduct detoxification and sulfur metabolism. Detailed biochemical and mechanistic investigation of YxeK including 18 O-isotope-labeling experiments, homology modeling, substrate specificity tests, site-directed mutagenesis, and (pre-)steady-state kinetics provides insight into the enzyme's mechanism of action, which may involve a noncanonical flavin-N5-peroxide species for C-S bond oxygenolysis.
Subject(s)
Cysteine/analogs & derivatives , Cysteine/genetics , Flavoproteins/genetics , Mixed Function Oxygenases/genetics , Acetylation , Bacillus subtilis/genetics , Bacillus subtilis/metabolism , Cysteine/metabolism , Flavins/genetics , Flavins/metabolism , Flavoproteins/metabolism , Fumarates/metabolism , Kinetics , Models, Chemical , Mutagenesis, Site-Directed , Operon/genetics , Substrate Specificity/genetics , Sulfhydryl Compounds/metabolismABSTRACT
Cyanogenic glycosides form part of a binary plant defense system that, upon catabolism, detonates a toxic hydrogen cyanide bomb. In seed plants, the initial step of cyanogenic glycoside biosynthesis-the conversion of an amino acid to the corresponding aldoxime-is catalyzed by a cytochrome P450 from the CYP79 family. An evolutionary conundrum arises, as no CYP79s have been identified in ferns, despite cyanogenic glycoside occurrence in several fern species. Here, we report that a flavin-dependent monooxygenase (fern oxime synthase; FOS1), catalyzes the first step of cyanogenic glycoside biosynthesis in two fern species (Phlebodium aureum and Pteridium aquilinum), demonstrating convergent evolution of biosynthesis across the plant kingdom. The FOS1 sequence from the two species is near identical (98%), despite diversifying 140 MYA. Recombinant FOS1 was isolated as a catalytic active dimer, and in planta, catalyzes formation of an N-hydroxylated primary amino acid; a class of metabolite not previously observed in plants.
Subject(s)
Ferns/genetics , Flavins/genetics , Glycosides/genetics , Oxygenases/genetics , Amino Acid Sequence/genetics , Catalysis , Cytochrome P-450 Enzyme System/genetics , Ferns/enzymology , Gene Expression Regulation, Plant/genetics , Glycosides/biosynthesis , Kinetics , Oximes , Phylogeny , Plant Proteins/genetics , Sequence AlignmentABSTRACT
The enzymes involved in bacterial bioluminescence are encoded in the lux operon with a conserved gene order of luxCDABEG. Some photobacterial strains carry an additional gene, termed luxF, which produces the LuxF protein, whose function and influence on bacterial bioluminescence is still uncertain. The LuxF protein binds the flavin derivative 6-(3'-(R)-myristyl)-flavin mononucleotide (myrFMN), which is generated as a side product in the luciferase-catalyzed reaction. This study utilized an Escherichia coli (E. coli) based lux operon expression system where the lux operons of Photobacterium leiognathi subsp. mandapamensis 27561 or of Photobacterium leiognathi subsp. leiognathi 25521, namely luxCDAB(F)EG, were cloned into a single expression vector. Exclusion of luxF gene from the lux operon enabled novel insights into the role of LuxF protein in light emission. E. coli cultures harboring and expressing the genes of the lux operon including luxF gene emit more light than without luxF gene. Furthermore, isolation of the tightly bound flavin derivative revealed the presence of at least three different flavin derivatives. Analysis by UV/Vis absorption and NMR spectroscopy as well as mass spectrometry showed that the flavin derivatives bear fatty acids of various chain lengths. This distribution of FMN derivatives is vastly different to what was found in bioluminescent bacteria and indicates that the luciferase is supplied with a range of aldehyde substrates in E. coli.
Subject(s)
Escherichia coli/genetics , Flavins/genetics , Photobacterium/genetics , Bacteria , DNA, Bacterial/metabolism , Escherichia coli/metabolism , Fatty Acids/metabolism , Flavins/metabolism , Gene Expression Regulation , Light , Luciferases/genetics , Luminescent Measurements , Operon/genetics , Photochemical Processes , Tandem Mass Spectrometry , Water MicrobiologyABSTRACT
The flavin transferase ApbE plays essential roles in bacterial physiology, covalently incorporating FMN cofactors into numerous respiratory enzymes that use the integrated cofactors as electron carriers. In this work we performed a detailed kinetic and structural characterization of Vibrio cholerae WT ApbE and mutants of the conserved residue His-257, to understand its role in substrate binding and in the catalytic mechanism of this family. Bi-substrate kinetic experiments revealed that ApbE follows a random Bi Bi sequential kinetic mechanism, in which a ternary complex is formed, indicating that both substrates must be bound to the enzyme for the reaction to proceed. Steady-state kinetic analyses show that the turnover rates of His-257 mutants are significantly smaller than those of WT ApbE, and have increased Km values for both substrates, indicating that the His-257 residue plays important roles in catalysis and in enzyme-substrate complex formation. Analyses of the pH dependence of ApbE activity indicate that the pKa of the catalytic residue (pKES1) increases by 2 pH units in the His-257 mutants, suggesting that this residue plays a role in substrate deprotonation. The crystal structures of WT ApbE and an H257G mutant were determined at 1.61 and 1.92 Å resolutions, revealing that His-257 is located in the catalytic site and that the substitution does not produce major conformational changes. We propose a reaction mechanism in which His-257 acts as a general base that deprotonates the acceptor residue, which subsequently performs a nucleophilic attack on FAD for flavin transfer.
Subject(s)
Flavins/metabolism , Transferases/metabolism , Vibrio cholerae/metabolism , Bacterial Proteins/metabolism , Catalysis , Catalytic Domain , Conserved Sequence , Flavin Mononucleotide/metabolism , Flavin-Adenine Dinucleotide/metabolism , Flavins/genetics , Histidine/metabolism , Kinetics , Oxidation-Reduction , Substrate Specificity/genetics , Transferases/genetics , Vibrio cholerae/geneticsABSTRACT
Ferric reductase B (FerB) is a flavin mononucleotide (FMN)-containing NAD(P)H:acceptor oxidoreductase structurally close to the Gluconacetobacter hansenii chromate reductase (ChrR). The crystal structure of ChrR was previously determined with a chloride bound proximal to FMN in the vicinity of Arg101, and the authors suggested that the anionic electron acceptors, chromate and uranyl tricarbonate, bind similarly. Here, we identify the corresponding arginine residue in FerB (Arg95) as being important for the reaction of FerB with superoxide. Four mutants at position 95 were prepared and found kinetically to have impaired capacity for superoxide binding. Stopped-flow data for the flavin cofactor showed that the oxidative step is rate limiting for catalytic turnover. The findings are consistent with a role for FerB as a superoxide scavenging contributor.
Subject(s)
FMN Reductase/chemistry , Flavins/genetics , Protein Conformation , Superoxides/metabolism , Amino Acid Sequence/genetics , Arginine/genetics , Catalytic Domain/genetics , Crystallography, X-Ray , FMN Reductase/genetics , Flavin Mononucleotide/chemistry , Flavin Mononucleotide/genetics , Flavins/metabolism , Kinetics , Oxidation-Reduction , Oxidoreductases/chemistry , Oxidoreductases/genetics , Paracoccus denitrificans/chemistry , Paracoccus denitrificans/enzymologyABSTRACT
miniSOG is the first flavin-binding protein that has been developed with the specific aim of serving as a genetically-encodable light-induced source of singlet oxygen (1O2). We have determined its 1.17 Å resolution structure, which has allowed us to investigate its mechanism of photosensitization using an integrated approach combining spectroscopic and structural methods. Our results provide a structural framework to explain the ability of miniSOG to produce 1O2 as a competition between oxygen- and protein quenching of its triplet state. In addition, a third excited-state decay pathway has been identified that is pivotal for the performance of miniSOG as 1O2 photosensitizer, namely the photo-induced transformation of flavin mononucleotide (FMN) into lumichrome, which increases the accessibility of oxygen to the flavin FMN chromophore and makes protein quenching less favourable. The combination of the two effects explains the increase in the 1O2 quantum yield by one order of magnitude upon exposure to blue light. Besides, we have identified several surface electron-rich residues that are progressively photo-oxidized, further contributing to facilitate the production of 1O2. Our results help reconcile the apparent poor level of 1O2 generation by miniSOG and its excellent performance in correlative light and electron microscopy experiments.
Subject(s)
Arabidopsis Proteins/genetics , Photosensitizing Agents/metabolism , Protein Conformation , Protein Serine-Threonine Kinases/genetics , Singlet Oxygen/metabolism , Arabidopsis Proteins/chemistry , Arabidopsis Proteins/ultrastructure , Biophysical Phenomena , Flavins/chemistry , Flavins/genetics , Light , Microscopy, Electron , Oxidation-Reduction , Oxygen/metabolism , Photosensitivity Disorders , Photosensitizing Agents/chemistry , Protein Binding/genetics , Protein Engineering , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/ultrastructure , Singlet Oxygen/chemistryABSTRACT
Flavin-dependent halogenases can be used as biocatalysts because they regioselectively halogenate their substrates under mild reaction conditions. New halogenases with novel substrate specificities will add to the toolbox of enzymes available to organic chemists. HalX, the product of the xcc-b100_4193 gene, is a putative flavin-dependent halogenase from Xanthomonas campestris. The enzyme was recombinantly expressed and crystallized in order to aid in identifying its hitherto unknown substrate. Native data collected to a resolution of 2.5â Å showed indications of merohedral twinning in a hexagonal lattice. Attempts to solve the phase problem by molecular replacement failed. Here, a detailed analysis of the suspected twinning is presented. It is most likely that the crystals are trigonal (point group 3) and exhibit perfect hemihedral twinning so that they appear to be hexagonal (point group 6). As there are several molecules in the asymmetric unit, noncrystallographic symmetry may complicate twinning analysis and structure determination.
Subject(s)
Flavins/chemistry , Flavins/genetics , Xanthomonas campestris/enzymology , Xanthomonas campestris/genetics , Amino Acid Sequence , Crystallization/methods , Crystallography/methods , HalogenationABSTRACT
MAIN CONCLUSION: Transcriptomic analysis indicates that the bacterial signalling molecule lumichrome enhances plant growth through a combination of enhanced cell division and cell enlargement, and possibly enhances photosynthesis. Lumichrome (7,8 dimethylalloxazine), a novel multitrophic signal molecule produced by Sinorhizobium meliloti bacteria, has previously been shown to elicit growth promotion in different plant species (Phillips et al. in Proc Natl Acad Sci USA 96:12275-12280, https://doi.org/10.1073/pnas.96.22.12275 , 1999). However, the molecular mechanisms that underlie this plant growth promotion remain obscure. Global transcript profiling using RNA-seq suggests that lumichrome enhances growth by inducing genes impacting on turgor driven growth and mitotic cell cycle that ensures the integration of cell division and expansion of developing leaves. The abundance of XTH9 and XPA4 transcripts was attributed to improved mediation of cell-wall loosening to allow turgor-driven cell enlargement. Mitotic CYCD3.3, CYCA1.1, SP1L3, RSW7 and PDF1 transcripts were increased in lumichrome-treated Arabidopsis thaliana plants, suggesting enhanced growth was underpinned by increased cell differentiation and expansion with a consequential increase in biomass. Synergistic ethylene-auxin cross-talk was also observed through reciprocal over-expression of ACO1 and SAUR54, in which ethylene activates the auxin signalling pathway and regulates Arabidopsis growth by both stimulating auxin biosynthesis and modulating the auxin transport machinery to the leaves. Decreased transcription of jasmonate biosynthesis and responsive-related transcripts (LOX2; LOX3; LOX6; JAL34; JR1) might contribute towards suppression of the negative effects of methyl jasmonate (MeJa) such as chlorophyll loss and decreases in RuBisCO and photosynthesis. This work contributes towards a deeper understanding of how lumichrome enhances plant growth and development.
Subject(s)
Arabidopsis/drug effects , Arabidopsis/growth & development , Flavins/pharmacology , Plant Growth Regulators/metabolism , Signal Transduction/drug effects , Sinorhizobium meliloti/genetics , Acetates/metabolism , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Bacterial Proteins/pharmacology , Biomass , Cell Division/drug effects , Cell Enlargement/drug effects , Cell Wall/drug effects , Chlorophyll/metabolism , Cyclopentanes/metabolism , Ethylenes/metabolism , Flavins/genetics , Flavins/metabolism , Gene Expression Profiling , Indoleacetic Acids/metabolism , Oxylipins/metabolism , Plant Leaves/drug effects , Plant Leaves/genetics , Plant Leaves/growth & developmentABSTRACT
The UbiX-UbiD system consists of the flavin prenyltransferase UbiX that produces prenylated FMN that serves as the cofactor for the (de)carboxylase UbiD. Recent developments have provided structural insights into the mechanism of both enzymes, detailing unusual chemistry in each case. The proposed reversible 1,3-dipolar cycloaddition between the cofactor and substrate serves as a model to explain many of the key UbiD family features. However, considerable variation exists in the many branches of the UbiD family tree.
Subject(s)
Carboxy-Lyases , Dimethylallyltranstransferase , Escherichia coli Proteins , Escherichia coli , Flavins , Flavoproteins , Prenylation/physiology , Carboxy-Lyases/chemistry , Carboxy-Lyases/genetics , Carboxy-Lyases/metabolism , Dimethylallyltranstransferase/chemistry , Dimethylallyltranstransferase/genetics , Dimethylallyltranstransferase/metabolism , Escherichia coli/chemistry , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Flavins/biosynthesis , Flavins/chemistry , Flavins/genetics , Flavoproteins/chemistry , Flavoproteins/genetics , Flavoproteins/metabolismABSTRACT
RNAs molecules fulfill key roles in the expression and regulation of the genetic information stored within the DNA chromosomes. In addition to the four canonical bases, U, C, A and G, RNAs harbor various chemically modified derivatives which are generated post-transcriptionally by specific enzymes acting directly at the polymer level. More than one hundred naturally occurring modified nucleosides have been identified to date, the largest number of which is found in tRNAs and rRNA. This remarkable biochemical process produces widely diversified RNAs further expanding the functional repertoires of these nucleic acids. Interestingly, several RNA-modifying enzymes use a flavin bioorganic molecule as a coenzyme in RNA modification pathways. Some of these reactions are simple while others are extremely complex using challenging chemistry orchestrated by large flavoenzymatic systems. In this review, we summarize recent knowledges on the flavin-dependent RNA-modifying enzymes and discuss the relevance of their activity within a cellular context.
Subject(s)
Flavins/metabolism , RNA Processing, Post-Transcriptional/physiology , RNA, Ribosomal/metabolism , RNA, Transfer/metabolism , DNA/genetics , DNA/metabolism , Flavins/genetics , RNA, Ribosomal/genetics , RNA, Transfer/geneticsABSTRACT
Heat shock is known to accelerate mitochondrial ROS production in Saccharomyces cerevisiae cells. But how yeast mitochondria produce ROS under heat-shock condition is not completely clear. Previously, it was shown that ROS production in heat-stressed fermenting yeast cells was accompanied by mitochondrial membrane potential (MMP) increase. In the current investigation the relationship between ROS production and MMP was studied in respiring yeast cells in stationary phase, using diphenyleneiodonium chloride (DPI), an inhibitor of flavin-containing proteins, as well as the mutants deleted for NDE1, NDE2 and NDI1 genes, encoding flavin-containing external and internal NADH dehydrogenases. It was shown that heat shock induced a transient burst in mitochondrial ROS production, which was paralleled by MMP rise. ROS production and MMP was significantly suppressed by DPI addition and deletion of NDE1. The effect of DPI on ROS production and MMP rise was specific for respiring cells. The results obtained suggest that the functioning of mitochondrial flavin-binding enzymes, Nde1p for instance, is required for the hyperpolarization of inner mitochondrial membrane and ROS production in respiring S. cerevisiae cells under heat-shock conditions.
Subject(s)
Electron Transport Complex I/genetics , Membrane Potential, Mitochondrial/genetics , Mitochondrial Membranes/metabolism , Saccharomyces cerevisiae Proteins/genetics , Cell Respiration/genetics , Electron Transport Complex I/antagonists & inhibitors , Flavins/genetics , Flavins/metabolism , Heat-Shock Response , Mitochondria/genetics , Onium Compounds/pharmacology , Reactive Oxygen Species/metabolism , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/antagonists & inhibitorsABSTRACT
BACKGROUND: Riboflavin is the precursor of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), essential cofactors for many metabolic enzymes that catalyze a variety of biochemical reactions. Previously we showed that free flavin (riboflavin, FMN, and FAD) concentrations were decreased in leaves of transgenic Arabidopsis plants expressing a turtle riboflavin-binding protein (RfBP). Here, we report that flavin downregulation by RfBP induces the early flowering phenotype and enhances expression of floral promoting photoperiod genes. RESULTS: Early flowering was a serendipitous phenomenon and was prudently characterized as a constant phenotype of RfBP-expressing transgenic Arabidopsis plants in both long days and short days. The phenotype was eliminated when leaf free flavins were brought back to the steady-state levels either by the RfBP gene silencing and consequently nullified production of the RfBP protein, or by external riboflavin feeding treatment. RfBP-induced early flowering was correlated with enhanced expression of floral promoting photoperiod genes and the florigen gene FT in leaves but not related to genes assigned to vernalization, autonomous, and gibberellin pathways, which provide flowering regulation mechanisms alternative to the photoperiod. RfBP-induced early flowering was further correlated with increased expression of the FD gene encoding bZIP transcription factor FD essential for flowering time control and the floral meristem identity gene AP1 in the shoot apex. By contrast, the expression of FT and photoperiod genes in leaves and the expression of FD and AP1 in the shoot apex were no longer enhanced when the RfBP gene was silenced, RfBP protein production canceled, and flavin concentrations were elevated to the steady-state levels inside plant leaves. CONCLUSIONS: Token together, our results provide circumstantial evidence that downregulation of leaf flavin content by RfBP induces early flowering and coincident enhancements of genes that promote flowering through the photoperiod pathway.
Subject(s)
Arabidopsis/genetics , Flavins/genetics , Flavins/metabolism , Flowers/genetics , Gene Expression Regulation, Plant , Photoperiod , Animals , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Flavin Mononucleotide/metabolism , Flavin-Adenine Dinucleotide/metabolism , Flowers/metabolism , Membrane Transport Proteins/genetics , Membrane Transport Proteins/metabolism , Plant Leaves , Plant Shoots/genetics , Plant Shoots/metabolism , Plant Shoots/physiology , Plants, Genetically Modified/genetics , Plants, Genetically Modified/metabolism , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
The marine alkaloid chlorizidine A contains chlorinated pyrroloisoindolone and pyrrolizine rings that are rare chemical features in bacterial natural products. Herein, we report the biosynthetic logic of their construction in Streptomyces sp. CNH-287 based on the identification of the chlorizidine A biosynthetic gene cluster. Using whole pathway heterologous expression and genetic manipulations, we show that chlorizidine A is assembled by a polyketide synthase that uniquely incorporates a fatty acid synthase-derived dichloropyrrolyl extender unit into the pyrroloisoindolone enzymatic product. We further provide the first biochemical characterization of a flavoenzyme associated with the oxidative formation of chlorizidine A's distinctive pyrrolizine ring. This work illuminates new enzymatic assembly line processes leading to rare nitrogen-containing rings in nature.
Subject(s)
Flavins/metabolism , Indole Alkaloids/metabolism , Oxidoreductases/metabolism , Pyrroles/metabolism , Streptomyces/enzymology , Biosynthetic Pathways , Flavins/genetics , Indole Alkaloids/chemistry , Multigene Family , Oxidoreductases/genetics , Polyketide Synthases/genetics , Polyketide Synthases/metabolism , Pyrroles/chemistry , Streptomyces/genetics , Streptomyces/metabolismABSTRACT
SidA (siderophore A) is a flavin-dependent N-hydroxylating monooxygenase that is essential for virulence in Aspergillus fumigatus. SidA catalyzes the NADPH- and oxygen-dependent formation of N(5)-hydroxyornithine. In this reaction, NADPH reduces the flavin, and the resulting NADP(+) is the last product to be released. The presence of NADP(+) is essential for activity, as it is required for stabilization of the C4a-hydroperoxyflavin, which is the hydroxylating species. As part of our efforts to determine the molecular details of the role of NADP(H) in catalysis, we targeted Ser-257 for site-directed mutagenesis and performed extensive characterization of the S257A enzyme. Using a combination of steady-state and stopped-flow kinetic experiments, substrate analogs, and primary kinetic isotope effects, we show that the interaction between Ser-257 and NADP(H) is essential for stabilization of the C4a-hydroperoxyflavin. Molecular dynamics simulation results suggest that Ser-257 functions as a pivot point, allowing the nicotinamide of NADP(+) to slide into position for stabilization of the C4a-hydroperoxyflavin.
Subject(s)
Aspergillus fumigatus/enzymology , Flavins/chemistry , Fungal Proteins/chemistry , Mixed Function Oxygenases/chemistry , NADP/chemistry , Serine/chemistry , Amino Acid Substitution , Aspergillus fumigatus/genetics , Catalysis , Flavins/genetics , Flavins/metabolism , Fungal Proteins/genetics , Fungal Proteins/metabolism , Mixed Function Oxygenases/genetics , Mixed Function Oxygenases/metabolism , Mutagenesis, Site-Directed , Mutation, Missense , NADP/genetics , NADP/metabolism , Serine/genetics , Serine/metabolismABSTRACT
Hydroxynitrile lyase from Arabidopsis thaliana (AtHNL) was fused to different fluorescent reporter proteins. Whereas all fusion constructs retained enzymatic activity and fluorescence in vivo and in vitro, significant differences in activity and pH stability were observed. In particular, flavin-based fluorescent reporter (FbFP) fusions showed almost 2 orders of magnitude-increased half-lives in the weakly acidic pH range compared to findings for the wild-type enzyme. Analysis of the quaternary structure of the respective FbFP-AtHNL fusion proteins suggested that this increased stability is apparently caused by oligomerization mediated via the FbFP tag. Moreover, the increased stability of the fusion proteins enabled the efficient synthesis of (R)-mandelonitrile in an aqueous-organic two-phase system at a pH of <5. Remarkably, (R)-mandelonitrile synthesis is not possible using wild-type AtHNL under the same conditions due to the inherent instability of this enzyme below pH 5. The fusion strategy presented here reveals a surprising means for the stabilization of enzymes and stresses the importance of a thorough in vitro characterization of in vivo-employed fluorescent fusion proteins.