A combination of NADHP and hispidin is not essential for bioluminescence in luminous fungal living gills of Mycena chlorophos

The chemical mechanisms underlying visible bioluminescence in the fungus Mycena chlorophos are not clear. A combination of dihydronicotinamide adenine dinucleotide phosphate (NADPH) and hispidin, which has been reported to increase the intensity of in vitro luminescence in crude cold‐water extracts prepared from the bioluminescent fruiting bodies of M. chlorophos, exhibited potential bioluminescence activation in the early bioluminescence stages, in which the bioluminescence was ultra‐weak, for living gills and luminescence activation for non‐bioluminescent gills, which was collapsed by freezing and subsequent thawing, at all bioluminescence stages. These abilities were not evident in considerably bioluminescent gills. These abilities were blocked by trans‐4‐hydroxycinnamic acid and trans‐3,4‐dihydroxycinnamic acid, which were identified as in vivo bioluminescence‐activating components. Original bioluminescence and bioluminescence produced from the addition of trans‐4‐hydroxycinnamic acid and trans‐3,4‐dihydroxycinnamic acid in living gills were almost completely inhibited by 10 mM NaN₃, whereas the luminescence produced form the combination of NADPH and hispidin in thawed non‐bioluminescent and living gills at the early weak bioluminescence stages was not inhibited by 10 mM NaN₃. Thus, the combination of NADPH and hispidin plays different roles in luminescence systems compared with essential bioluminescence systems, and the combination of NADPH and hispidin was not essential for visible bioluminescence in living gills.     

Second bioluminescence‐activating component in the luminous fungus Mycena chlorophos

Mycena chlorophos is an oxygen‐dependent bioluminescent fungus. The mechanisms underlying its light emission are unknown. A component that increased the bioluminescence intensity of the immature living gills of M. chlorophos was isolated from mature M. chlorophos gills and chemically characterized. The bioluminescence‐activating component was found to be trans‐3,4‐dihydroxycinnamic acid and its bioluminescence activation was highly structure‐specific. ¹³C‐ and ¹⁸O‐labelling studies using the immature living gills showed that trans‐3,4‐dihydroxycinnamic acid was synthesized from trans‐4‐hydroxycinnamic acid in the gills by hydroxylation with molecular oxygen as well as by the general metabolism, and trans‐3,4‐dihydroxycinnamic acid did not produce hispidin (detection‐limit concentration: 10 pmol/1 g wet gill). Addition of 0.01 mM hispidin to the immature living gills generated no bioluminescence activation. These results suggested that the prompt bioluminescence activation resulting from addition of trans‐3,4‐dihydroxycinnamic acid could not be attributed to the generation of hispidin. Copyright © 2016 John Wiley & Sons, Ltd.     

Lampteromyces bioluminescence—1. Identification of riboflavin as the light emitter in the mushroom L. japonicus

The bioluminescence of the luminous mushroom, Lampteromyces japonicus, was studied by using the mushroom gills and also the luminous mycelia, the latter being cultured from the isolated spores and grown in a potato sucrose medium. The luminescence intensity of the mushroom gills and the cultured mycelia was measured in an aqueous suspension under various conditions. The original intensity was enhanced by exposing the luminous cells to oxygen for several hours or to acids or bases for a short period. This enhancement enabled measurement of their bioluminescence spectra which were identical to the fluorescence spectrum of riboflavin, having a maximum at 524 nm. The green fluorescent substance was extracted with cold water from the mushroom and it was identified as riboflavin by spectroscopic and chromatographic analyses. Riboflavin was concluded to be the light emitter of this mushroom.     

An essential role for UshA in processing of extracellular flavin electron shuttles by Shewanella oneidensis

The facultative anaerobe Shewanella oneidensis can reduce a number of insoluble extracellular metals. Direct adsorption of cells to the metal surface is not necessary, and it has been shown that S. oneidensis releases low concentrations flavins, including riboflavin and flavin mononucleotide (FMN), into the surrounding medium to act as extracellular electron shuttles. However, the mechanism of flavin release by Shewanella remains unknown. We have conducted a transposon mutagenesis screen to identify mutants deficient in extracellular flavin accumulation. Mutations in ushA, encoding a predicted 5′‐nucleotidase, resulted in accumulation of flavin adenine dinucleotide (FAD) in culture supernatants, with a corresponding decrease in FMN and riboflavin. Cellular extracts of S. oneidensis convert FAD to FMN, whereas extracts of ushA mutants do not, and fractionation experiments show that UshA activity is periplasmic. We hypothesize that S. oneidensis secretes FAD into the periplasmic space, where it is hydrolysed by UshA to FMN and adenosine monophosphate (AMP). FMN diffuses through outer membrane porins where it accelerates extracellular electron transfer, and AMP is dephosphorylated by UshA and reassimilated by the cell. We predict that transport of FAD into the periplasm also satisfies the cofactor requirement of the unusual periplasmic fumarate reductase found in Shewanella.     

Identification and characterization of the missing phosphatase on the riboflavin biosynthesis pathway in Arabidopsis thaliana

Despite the importance of riboflavin as the direct precursor of the cofactors flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), the physiologically relevant catalyst dephosphorylating the riboflavin biosynthesis pathway intermediate 5‐amino‐6‐ribitylamino‐2,4(1H,3H) pyrimidinedione 5′‐phosphate (ARPP) has not been characterized from any organism. By using as the query sequence a previously identified plastidial FMN hydrolase AtcpFHy1 (At1g79790), belonging to the haloacid dehalogenase (HAD) superfamily, seven candidates for the missing ARPP phosphatase were found, cloned, recombinantly expressed, and purified. Activity screening showed that the enzymes encoded by AtcpFHy1, At4g11570, and At4g25840 catalyze dephosphorylation of ARPP. AtcpFHy1 was renamed AtcpFHy/PyrP1, At4g11570 and At4g25840 were named AtPyrP2 and AtGpp1/PyrP3, respectively. Subcellular localization in planta indicated that AtPyrP2 was localized in plastids and AtGpp1/PyrP3 in mitochondria. Biochemical characterization of AtcpFHy/PyrP1 and AtPyrP2 showed that they have similar K_m values for the substrate ARPP, with AtcpFHy/PyrP1 having higher catalytic efficiency. Screening of 21 phosphorylated substrates showed that AtPyrP2 is specific for ARPP. Molecular weights of AtcpFHy/PyrP1 and AtPyrP2 were estimated at 46 and 72 kDa, suggesting dimers. pH and temperature optima for AtcpFHy/PyrP1 and AtPyrP2 were ~7.0–8.5 and 40–50°C. T‐DNA knockout of AtcpFHy/PyrP1 did not affect the flavin profile of the transgenic plants, whereas silencing of AtPyrP2 decreased accumulation of riboflavin, FMN, and FAD. Our results strongly support AtPyrP2 as the missing phosphatase on the riboflavin biosynthesis pathway in Arabidopsis thaliana. The identification of this enzyme closes a long‐standing gap in understanding of the riboflavin biosynthesis in plants.     

Adenosine diphosphate moiety does not participate in structural changes for the signaling state in the sensor of blue-light using FAD domain of AppA

A sensor of blue light using FAD (BLUF) protein is a flavin adenine dinucleotide (FAD) based new class blue-light sensory flavoprotein. The BLUF domain of AppA was reconstituted in vitro from apoprotein and flavin adenine dinucleotide, flavin adenine mononucleotide or riboflavin. The light-induced FTIR spectra of the domain reconstituted from various flavins and the 13C-labeled apoprotein showed that identical light-induced structural changes occur in both the flavin chromophore and protein for the signaling state in all of the reconstituted holoproteins. The results showed that an adenosine 5'-dinucleotide moiety is not required for signaling-state formation in a BLUF domain.     

Photoirradiation products of flavin derivatives,and the effects of photooxidation on guanine

Photoirradiation in the presence of riboflavin led to guanine oxidation and the formation of imidazolone. Meanwhile, riboflavin itself was degraded by ultraviolet light A (UV-A) and visible light (VIS) radiation, and the end product was lumichrome. VIS radiation in the presence of riboflavin oxidized guanine similarly to UV-A radiation. Although UV-A radiation with lumichrome oxidized guanine, VIS radiation with lumichrome did not. Thus, UV-A radiation with riboflavin can oxidize guanine even if riboflavin is degraded to lumichrome. In contrast, following VIS radiation degradation of riboflavin to lumichrome, VIS radiation with riboflavin is hardly capable of oxidizing guanine. The consequences of riboflavin degradation and guanine photooxidation can be extended to flavin mononucleotide and flavin adenine dinucleotide. In addition, we report advanced synthesis; carboxymethylflavin was obtained by oxidation of formylmethylflavin with chlorite and hydrogen peroxide; lumichrome was obtained by heating of formylmethylflavin in 50% AcOH; lumiflavin was obtained by incubation of formylmethylflavin in 2 M NaOH, followed by isolation by step-by-step concentration.     

Bioluminescence characteristics of the fruiting body of Mycena chlorophos

Bioluminescent fungi are widely distributed on land and most belong to the class Basidomycetes. Light of about 530 nm wavelength maximum is emitted continuously. The molecular basis for the light‐emitting process remains unclear. We investigated the characteristics of the bioluminescence using cultivated fruiting bodies of M. chlorophos. Only fresh fruiting bodies exhibited long‐lasting light emission; rapid decay of light emission was observed with frozen and freeze‐dried samples. Freeze‐dried samples can be stored at room temperature under dry conditions and may be useful for the isolation of luciferin. The light emission of the fresh fruiting bodies was maintained in various buffers at varying pH; it could be stopped with pH 4 acetate buffer and could be recovered at pH 6. The isolation of luciferin from the fresh fruiting bodies might be possible by the control of buffer pH. The effect of temperature on the light emission of fruiting bodies indicated that bioluminescence in M. chlorophos may involve enzymatic reaction(s). The solubilization of bioluminescent components from the fruiting bodies could not be achieved with various surfactants. Copyright © 2011 John Wiley & Sons, Ltd.     

Regulation of flavin biosynthesis in the methylotrophic yeast Hansenula polymorpha

Under various conditions of growth of the methylotrophic yeast Hansenula polymorpha, a tight correlation was observed between the levels of flavin adenine dinucleotide (FAD)-containing alcohol oxidase, and the levels of intracellularly bound FAD and flavin biosynthetic enzymes. Adaptation of the organism to changes in the physiological requirement for FAD was by adjustment of the levels of the enzymes catalyzing the last three steps in flavin biosynthesis, riboflavin synthetase, riboflavin kinase and flavin mononucleotide adenylyltransferase. The regulation of the synthesis of the latter enzymes in relation to that of alcohol oxidase synthesis was studied in experiments involving addition of glucose to cells of H. polymorpha growing on methanol in batch cultures or in carbon-limited continuous cultures. This resulted not only in selective inactivation of alcohol oxidase and release of FAD, as previously reported, but invariably also in repression/inactivation of the flavin biosynthetic enzymes. In further experiments involving addition of FAD to the same type of cultures it became clear that inactivation of the latter enzymes was not caused directly by glucose, but rather by free FAD that accumulated intracellularly. In these experiments no repression or inactivation of alcohol oxidase occurred and it is therefore concluded that the synthesis of this enzyme and the flavin biosynthetic enzymes is under separate control, the former by glucose (and possibly methanol) and the latter by intracellular levels of free FAD.Abbreviations FAD Flavin adenine dinucleotide - FMN riboflavin-5-phosphate; flavin mononucleotide - Rf riboflavin     

Riboflavin metabolism in the hypothyroid human adult

It had been shown that thyroxine regulates the conversion of riboflavin to riboflavin mononucleotide and flavin adenine dinucleotide (FAD) in laboratory animals. In the hypothyroid rat, the flavin adenine dinucleotide level of the liver decreases to levels observed in riboflavin deficiency. We have shown that in six hypothyroid human adults, the activity of erythrocyte glutathione reductase, an accessible FAD-containing enzyme, is decreased to levels observed during riboflavin deficiency. Thyroxine therapy resulted in normal levels of this enzyme while the subjects were on a controlled dietary regimen. This demonstrates that thyroid hormone regulates the enzymatic conversion of riboflavin to its active coenzyme forms in the human adult.     

15N nuclear magnetic resonance of flavins.

Ninety-nine percent 15N-enriched flavins were synthesized and their proton decoupled 15N resonances were observed. The enriched compounds were [1,3-15N]riboflavin, [1,3,5-15N]riboflavin, [1,3-15N]riboflavin 5'-phosphate, [1,3,5-15N]riboflavin 5'-phosphate, and [1,3,5-15N] flavin adenine dinucleotide, [1,3,5-15N] lumiflavin, and [1,3,5-15N] lumichrome. By comparison of their spectra and from th- nuclear Overhauser effect data each 15N resonance peak could be assigned to each 15N nucleus. The order of the chemical shifts well corresponds to that of the calculated pi-electron densities. The N-3 nucleus gives the most intense inverted peak and the N-5 nucleus a small noninverted peak. By changing pH from neutral to alkaline, the chemical shift and the intensity of signal were mostly affected in the N-3 resonance of riboflavin 5'-phosphate. The N-5 signal of flavin adenine dinucleotide showed a fairly large downfield shift with the increase of temperature. These observations can be well interpreted by the chemical structure and the proposed conformation of riboflavin 5'-phosphate and flavin adenine dinucleotide.     

Fluorescence titration with apoflavodoxin: a sensitive assay for riboflavin 5'-phosphate and flavin adenine dinucleotide in mixtures.

A method is described for determining riboflavin 5′-phosphate (FMN) and flavin adenine dinucleotide (FAD) in mixtures by fluorimetric titration with the FMN-specific apoprotein of flavodoxin from Peptostreptococcus elsdenii. Accurate determinations can be carried out in the presence of a variety of compounds that decrease the fluorescence yield of FMN; the method may therefore be especially useful in the analysis of crude protein-free extracts of biological materials.     

A key enzyme for flavin synthesis is required for nitric oxide and reactive oxygen species production in disease resistance

Nitric oxide (NO) and reactive oxygen species (ROS) play key roles in plant immunity. However, the regulatory mechanisms of the production of these radicals are not fully understood. Hypersensitive response (HR) cell death requires the simultaneous and balanced production of NO and ROS. In this study we indentified NbRibA encoding a bifunctional enzyme, guanosine triphosphate cyclohydrolase II/3,4‐dihydroxy‐2‐butanone‐4‐phosphate synthase, which participates in the biosynthesis of flavin, by screening genes related to mitogen‐activated protein kinase‐mediated cell death, using virus‐induced gene silencing. Levels of endogenous riboflavin and its derivatives, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), which are important prosthetic groups for several enzymes participating in redox reactions, decreased in NbRibA‐silenced Nicotiana benthamiana. Silencing NbRibA compromised not only HR cell death, but also the NO and ROS production induced by INF1 elicitin and a constitutively active form of NbMEK2 (NbMEK2^DD), and also induced high susceptibility to oomycete Phytophthora infestans and ascomycete Colletotrichum orbiculare. Compromised radical production and HR cell death induced by INF1 in NbRibA‐silenced leaves were rescued by adding riboflavin, FMN or FAD. These results indicate that flavin biosynthesis participates in regulating NO and ROS production, and HR cell death.     

Dyakia bioluminescence—1. Bioluminescence and fluorescence spectra of the land snail,D. striata

The luminescent land snail Dyakia striata displayed a bioluminescence spectrum with a maximum wavelength of 515 nm. A green fluorescent substance extracted from the photogenic organ of an adult snail had a similar wavelength maximum but its fluorescence spectrum differed from that of flavin chromophore substances involved in light emission in some other luminescent organisms.     

Localization of the bioluminescence system in the pileus of Mycena chlorophos

Mycena chlorophos, which is primarily distributed in Southeast Asia, is a luminous fungus that emits a bright green light from its pileus for about 2 days at approximately 20°C and high relative humidity. The distribution of bioluminescent tissues in the whole pileus and its sections was heterogeneous. The light intensity in the cap and the upper region of the gill was greater than that in the lower region of the gill. At the microscopic level, the light was predominantly emitted from the membranes of hymenium and basidia cells on the gill. The emission was both cell and region specific. The luminescence system was localized in the cell membrane, and a part of the system was on the cell membrane surface. Copyright © 2015 John Wiley & Sons, Ltd.     

Riboflavin-binding sites associated with flagella of Euglena: A candidate for blue-light photoreceptor?

The hypothesis was tested that reversible riboflavin (RF)-binding sites are part of the photoreceptor in Euglena gracilis. Published evidence shows that the phototactic stimulus — with a flavin-type action spectrum — is perceived at the paraflagellar body (PFB). Flagella with PFBs were isolated from Euglena gracilis by a combined cold and Ca²⁺ shock. Saturable binding of [¹⁴C]RF was demonstrated with such preparations, in the oxidized state as well as under reducing conditions in the presence of dithionite. Affinities for RF were high: K _D (oxidized)=0.08 M, and K _D (reduced)=0.7 M. Flavin mononucleotide and flavin adenine dinucleotide showed lower binding affinities. The in vitro RF binding per unit of protein was enriched approximately tenfold in the flagellar preparations when compared with homogenates of whole cells. The number of (reduced) binding sites per entire flagellum was determined to be in the order of 10⁶. This number is in line with published estimates of chromophores bound in or at the PFB.Abbreviations FAD flavin adenine dinucleotide - FMN flavin mononucleotide - PFB paraflagellar body - RF riboflavin This work was supported by the Deutsche Forscungsgemeinschaft.     

Singlet oxygen generation by UVA light exposure of endogenous photosensitizers

UVA light (320-400 nm) has been shown to produce deleterious biological effects in tissue due to the generation of singlet oxygen by substances like flavins or urocanic acid. Riboflavin, flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), beta-nicotinamide adenine dinucleotide (NAD), and beta-nicotinamide adenine dinucleotide phosphate (NADP), urocanic acid, or cholesterol in solution were excited at 355 nm. Singlet oxygen was directly detected by time-resolved measurement of its luminescence at 1270 nm. NAD, NADP, and cholesterol showed no luminescence signal possibly due to the very low absorption coefficient at 355 nm. Singlet oxygen luminescence of urocanic acid was clearly detected but the signal was too weak to quantify a quantum yield. The quantum yield of singlet oxygen was precisely determined for riboflavin (PhiDelta = 0.54 +/- 0.07), FMN (PhiDelta = 0.51 +/- 0.07), and FAD (PhiDelta = 0.07 +/- 0.02). In aerated solution, riboflavin and FMN generate more singlet oxygen than exogenous photosensitizers such as Photofrin, which are applied in photodynamic therapy to kill cancer cells. With decreasing oxygen concentration, the quantum yield of singlet oxygen generation decreased, which must be considered when assessing the role of singlet oxygen at low oxygen concentrations (inside tissue).     

Identification of a flavin‐containing S‐oxygenating monooxygenase involved in alliin biosynthesis in garlic

S‐Alk(en)yl‐l ‐cysteine sulfoxides are cysteine‐derived secondary metabolites highly accumulated in the genus Allium. Despite pharmaceutical importance, the enzymes that contribute to the biosynthesis of S‐alk‐(en)yl‐l ‐cysteine sulfoxides in Allium plants remain largely unknown. Here, we report the identification of a flavin‐containing monooxygenase, AsFMO1, in garlic (Allium sativum), which is responsible for the S‐oxygenation reaction in the biosynthesis of S‐allyl‐l ‐cysteine sulfoxide (alliin). Recombinant AsFMO1 protein catalyzed the stereoselective S‐oxygenation of S‐allyl‐l ‐cysteine to nearly exclusively yield (R_CS_S)‐S‐allylcysteine sulfoxide, which has identical stereochemistry to the major natural form of alliin in garlic. The S‐oxygenation reaction catalyzed by AsFMO1 was dependent on the presence of nicotinamide adenine dinucleotide phosphate (NADPH) and flavin adenine dinucleotide (FAD), consistent with other known flavin‐containing monooxygenases. AsFMO1 preferred S‐allyl‐l ‐cysteine to γ‐glutamyl‐S‐allyl‐l ‐cysteine as the S‐oxygenation substrate, suggesting that in garlic, the S‐oxygenation of alliin biosynthetic intermediates primarily occurs after deglutamylation. The transient expression of green fluorescent protein (GFP) fusion proteins indicated that AsFMO1 is localized in the cytosol. AsFMO1 mRNA was accumulated in storage leaves of pre‐emergent nearly sprouting bulbs, and in various tissues of sprouted bulbs with green foliage leaves. Taken together, our results suggest that AsFMO1 functions as an S‐allyl‐l ‐cysteine S‐oxygenase, and contributes to the production of alliin both through the conversion of stored γ‐glutamyl‐S‐allyl‐l ‐cysteine to alliin in storage leaves during sprouting and through the de novo biosynthesis of alliin in green foliage leaves.     

The Flavin Content of Clovers Relative to Symbiosis with a Riboflavin-requiring Mutant of Rhizobium trifoli

A riboflavin-requiring auxotroph of Rhizobium trifolii (T1/D-his(r)-15) formed ineffective root nodules on red clover and on two cultivars of subterranean clover, but produced almost fully effective nodules on several other cultivars of subterranean clover. Fluorescence and bioassay measurements of the flavin content of the roots and shoots of these cultivars revealed no differences between cultivars which could be correlated with the differences in symbiotic response. The concentration of flavin in nodules formed by the auxotroph (in the absence of riboflavin), by the effective parent strain (T1), or by a partly effective mutant (penicillin-resistant) of T1 was roughly proportional to the effectiveness of the nodules. Effective nodules contained 20 times as much flavin, and ineffective nodules 3 to 4 times as much flavin as non-nodulated root tissue. Approximately 20 to 30% of the flavins in both root and nodule tissue was flavin adenine dinucleotide and 70 to 80% was riboflavin + flavin mononucleotide. Most of the flavin adenine dinucleotide in macerated nodules was associated with host cell fragments, and none was detected in a cell-free fraction. Bacteroids accounted for approximately 20% of flavins in effective nodules and also contained more riboflavin + flavin mononucleotide than cultured rhizobial cells. The total flavin content of noninoculated roots increased from about 1.2 nmoles to 1.7 nmoles flavin/g of tissue after 3 days' exposure to 80 mum riboflavin. Exposure of only the upper or lower portion of preinoculated roots indicated negligible translocation, as effective nodulation occurred only on parts of the root in direct contact with riboflavin. Plants grown in a medium containing combined nitrogen (100 or 300 mum nitrogen added as (NH(4))(2)SO(4)), but no added riboflavin showed an increased root flavin content (about 2.1 nmoles flavin/g tissue) and a partly effective response when inoculated with the mutant. Nitrogen also promoted some upward translocation of exogenous riboflavin in the roots.     

Modulation of the Purine Pathway for Riboflavin Production in Flavinogenic Recombinant Strain of the Yeast Candida famata

Riboflavin (vitamin B₂) is an indispensable nutrient for humans and animals, since it is the precursor of the essential coenzymes flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), involved in variety of metabolic reactions. Riboflavin is produced on commercial scale and is used for feed and food fortification purposes, and in medicine. Until recently, the mutant strains of the flavinogenic yeast Candida famata were used in industry for riboflavin production. Guanosine triphosphate is the immediate precursor of riboflavin synthesis. Therefore, the activation of metabolic flux toward purine nucleotide biosynthesis is a promising approach to improve riboflavin production. The phosphoribosyl pyrophosphate synthetase and phosphoribosyl pyrophosphate amidotransferase are the rate limiting enzymes in purine biosynthesis. Corresponding genes PRS3 and ADE4 from yeast Debaryomyces hansenii are modified to avoid feedback inhibition and cooverexpressed on the background of a previously constructed riboflavin overproducing strain of C. famata. Constructed strain accumulates twofold more riboflavin when compared to the parental strain.