Production of Riboflavin by Arthrobacter globiformis

S ummary . The yellow pigment excreted by a strain of Arthrobacter globiformis during exponential growth was identified as riboflav in. The amount produced was approximately 5 μ/mg of cells formed (dry weight) in all media tested, rich as well as poor. This value was also found in media containing 1% of Tween So, even though the concentration of internal flavins then was nearly one-half the normal value. Over production of riboflavin in most media was 40-50 fold. The specific growth rate of the riboflavin producer was sevral per cent less than that of a normal strain of the same species.     

Abiotic Photophosphorylation Model Based on Abiogenic Flavin and Pteridine Pigments

A model for abiotic photophosphorylation of adenosine diphosphate by orthophosphate with the formation of adenosine triphosphate was studied. The model was based on the photochemical activity of the abiogenic conjugates of pigments with the polymeric material formed after thermolysis of amino acid mixtures. The pigments formed showed different fluorescence parameters depending on the composition of the mixture of amino acid precursors. Thermolysis of the mixture of glutamic acid, glycine, and lysine (8:3:1) resulted in a predominant formation of a pigment fraction which had the fluorescence maximum at 525 nm and the excitation band maxima at 260, 375, and 450 nm and was identified as flavin. When glycine in the initial mixture was replaced with alanine, a product formed whose fluorescence parameters were typical to pteridines (excitation maximum at 350 nm, emission maximum at 440 nm). When irradiated with the quasi-monochromatic light (over the range 325–525 nm), microspheres in which flavin pigments were prevailing showed a maximum photophosphorylating activity at 375 and 450 nm, and pteridine-containing chromoproteinoid microspheres were most active at 350 nm. The positions and the relative height of maxima in the action spectra correlate with those in the excitation spectra of the pigments, which point to the involvement of abiogenic flavins and pteridines in photophosphorylation.     

Reduction of exogenous flavins and mobilization of iron from ferritin by isolated mitochondria

The release mechanism for ferritin iron and the nature of the compound(s) which donate iron to the mitochondria are two important problems of intracellular iron metabolism which still await their solution. We have previously shown that isolated mitochondria reduce exogenously added flavins in a ubiquinol-flavin oxidoreductase reaction at the C-side of the inner membrane and that the resulting dihydroflavins function as reductants in mitochondrial mobilization of iron from ferritin (Ulvik, R. J., and Romslo, I. (1981). Biochim. Biophys. Acta 635, 457-469). In the present study it is shown that the rate at which iron is removed from ferritin depends on the capability of the flavins to penetrate (1) the mitochondrial outer membrane and (2) the intersubunit channels of the ferritin protein shell. Intact mitochondria reduce flavins at rates which decrease in the following order: riboflavin > FAD > FMN. The ferritin iron mobilization rates decrease in the order of riboflavin > FMN > FAD. The results are further support for the operation of a flavin-dependent mitochondrial ferrireductase, and strengthen the suggested role for ferritin as a donor of iron to the mitochondria.     

Regulation of 6-hydroxy-2,4,5-triaminopyramidine synthesis by riboflavin and iron in riboflavin-deficient mutants of Pichia guilliermondii yeast

The effect of riboflavin and iron on 6-hydroxy-2,4,5-triaminopyrimidine synthesis rate was investigated in the cultures of the yeast Pichia guilliermondii (rib₂ mutants) with the blocked second reaction of flavinogenesis.It was shown that riboflavin inhibited the 6-hydroxy-2,4,5-triaminopyrimidine synthesis rate in iron-rich and iron-deficient cells of mutants with low riboflavin requirements. Cycloheximide did not prevent the stimulation of 6-hydroxy-2,4,5-triaminopyrimidine synthesis caused by riboflavin starvation.7-methyl-8-trifluoromethyl-10-(1′- -ribityl)isoalloxazine strongly inhibited the 6-hydroxy-2,4,5-triaminopyrimidine synthesis, while 7-mithyl-8-trifluoromethyl-10-(β-hydroxyethyl) izoalloxazine and galactoflavin exerted only a slight effect on this process.The 6-hydroxy-2,4,5-triaminopyrimidine synthesis rate in iron-deficient cells was significantly higher than in iron-rich cells. The 2,2′-dipyridyl treatment of iron-rich cells caused the stimulation of 6-hydroxy-2,4,5-triaminopyrimidine synthesis and cycloheximide abolished this effect.The results suggest that the activity of the first enzyme of flavinogenesis (guanylic cyclohydrolase) is under the control of feedback inhibition by flavins and the biosynthesis of this enzyme is regulated by iron.     

Studies on the flavins in rat liver mitochondrial outer membranes

The incorporation of radioactivity derived from [2-¹⁴C] riboflavin into the flavins of rat liver mitochondrial outer membranes was studied. These membranes were found to contain about 0.6 nmol of non-covalently bound flavins per mg protein; the majority is in the form of FAD (73%) and FMN (24%). The membranes also contain about 1.5 nmol per mg of covalently bound flavins.After labeling, radioactive flavins appeared in the non-covalently bound flavins for about 4 h. Most of this radioactivity was in FAD (77%). Neither the rate nor extent of this labelling was affected by cycloheximide (1 mg/kg) administered 30 min prior to the radioactive riboflavin. With the covalently bound flavins, radioactivity was incorporated into the coenzymes for at least 18 h, but the rate of incorporation was much slower. After cycloheximide, radioactive flavins continued to appear in covalently bound flavins for about 2 h, but then stopped. Labeling of both types of flavins after [¹⁴C] riboflavin was considerably slower than the incorporation of [³H] leucine into outer membrane proteins. These results suggest that with flavoproteins from the mitochondrial outer membranes, the incorporation of flavins occurs after synthesis of the various apoenyzmes is complete.     

Riboflavin Biosynthesis by Candida robusta

During the study on the oxidative sugar metabolism of yeasts, it was found that six strains of Candida robusta, isolated from fruits, produced large amounts of yellow pigment in shaking culture and this pigment was identified as riboflavin. Riboflavin production by C. robusta has never been reported.

Some notable characteristics of C. robusta in riboflavin production were found. As nitrogen sources, ammonium salts and urea were favorable, but nitrate and organic nitrogen sources such as glycine, asparagine and peptone were not utilized for riboflavin production. Riboflavin was not produced in still culture; a highly aerobic condition, as may be obtained by shaking culture, wa, essential. The addition of excess CaCO₃ was also necessary. Acetic acid, added as the Ca salt in its production as a sole carbon source, was more effective than sugars and optimum concentration of this acid was 7%. Riboflavin were obtained in yields as high as 32 to 34 mg % from the acetate medium after 8 days.     

Identification of protein-bound riboflavin in rat hepatocyte plasma membrane as a source of autofluorescence

The presence of flavin compound(s) giving a yellowish-green autofluorescence in rat hepatocyte plasma membrane has recently been reported (Nokubo, M. et al. (1988) Biochim. Biophys. Acta 939, 441-448). The fluorophore can quantitatively be extracted with water at 80 degrees C from isolated plasma membranes. Gel filtration of the extract eluted with water showed two peaks, the fluorescence of which closely resembled that of riboflavin. The major peak comigrated with proteins and the minor one displayed a position identical to authentic riboflavin. When the components of the major peak were rechromatographed after acetic acid treatment and eluted with 20 mM of acetic acid, the fluorescent compound separated from the proteins and eluted at the same position as riboflavin. In paper chromatography and HPLC, the behavior of the fluorescent compound (separated by acid treatment from the proteins) was identical to that of riboflavin. SDS gel filtration of subcellular fractions of rat liver revealed that riboflavin was the dominant flavin, whereas FAD and FMN were not detectable in the plasma membrane. Microsomes and mitochondria contain predominantly FAD and FMN, and only minor quantities of riboflavin. The presence of riboflavin in the plasma membrane is a novel finding, the functional significance of which is still unclear; however, a hypothesis can be forwarded on the basis of the ability of flavins to generate superoxide anion radicals during their autoxidation.     

Mechanism of specific inhibition of phototropism by phenylacetic Acid in corn seedling

Using geotropism as a control for phototropism, compounds similar to phenylacetic acid that photoreact with flavins and/or have auxin-like activity were examined for their ability to specifically inhibit phototropism in corn seedlings using geotropism as a control. Results using indole-3-acetic acid, napthalene-1-acetic acid, naphthalene-2-acetic acid, phenylacetic acid, and β-phenylpyruvic acid suggest that such compounds will specifically inhibit phototropism primarily because of their photoreactivity with flavins and not their auxin activity. For example, strong auxins, indole-3-acetic acid and naphthalene-1-acetic acid, affected both tropic responses at all concentrations tested whereas weak auxins, phenylacetic acid and naphthalene-2-acetic acid, exhibited specific inhibition. In addition, the in vivo concentration of phenylacetic acid required to induce specificity was well below that required to stimulate coleoptile growth. Estimates of the percentage of photoreceptor pigment inactivated by phenylacetic acid (>10%) suggest that phenylacetic acid could be used to photoaffinity label the flavoprotein involved in corn seedling phototropism.     

Riboflavin analogs and inhibitors of riboflavin biosynthesis

Flavins are active components of many enzymes. In most cases, riboflavin (vitamin B₂) as a coenzyme represents the catalytic part of the holoenzyme. Riboflavin is an amphiphatic molecule and allows a large variety of different interactions with the enzyme itself and also with the substrate. A great number of active riboflavin analogs can readily be synthesized by chemical methods and, thus, a large number of possible inhibitors for many different enzyme targets is conceivable. As mammalian and especially human biochemistry depends on flavins as well, the target of the inhibiting flavin analog has to be carefully selected to avoid unwanted effects. In addition to flavoproteins, enzymes, which are involved in the biosynthesis of flavins, are possible targets for anti-infectives. Only a few flavin analogs or inhibitors of flavin biosynthesis have been subjected to detailed studies to evaluate their biological activity. Nevertheless, flavin analogs certainly have the potential to serve as basic structures for the development of novel anti-infectives and it is possible that, in the future, the urgent need for new molecules to fight multiresistant microorganisms will be met.     

Reduction of substituted flavins by .CO2- and cyclic disulphide anions

5-Deazariboflavin was observed to undergo one electron-equivalent reduction on reaction with .CO2-, DS2-. or a mixture of .CO2- and eaq-. This is attributed to formation of .FlH, followed by dimerization: 2 5-DRFlH.----(5-DRFlH)2 a process not previously confirmed. In contrast, 3-deazariboflavin, 1-deazariboflavin 7-chlororiboflavin, 2-thioriboflavin, lumiflavin, flavin adenine dinucleotide and riboflavin all underwent two electron-equivalent reductions to FlH2 or FlH-species via the disproportionation: 2.FlH----FlH2+Fl Spectra of the reduced species are reported for 3-deazariboflavin, 1-deazarboflavin and 2-thioriboflavin. The anion radicals of dithiothreitol (DS2-.) and lipoamide (LS2-.) did not effect reduction beyond the two electron equivalence point. However, .CO2- was seen to effect further reactions with the dihydroforms of the higher redox potential flavins 7-chlororiboflavin and 2-thioriboflavin at pH 3 to 10, but for the other flavins only at or below pH 2. Except for the sulphur-containing 2-thioriboflavin, the protonated DS2.H and LS2.H radicals were unable to reduce any of the flavins at pH less than or equal to 3. This cause a 'falloff' in the yield of the reduced flavin which is in accord with the reaction rate constants and radical pK values. Despite their differences in absorbance, the reactions of DS2.H and LS2.H towards flavins were similar in all aspects examined. Several new rate constants are reported.     

Specific inhibition of phototropism in corn seedlings

Geotropism was used as a control for the specificity of potential inhibitors of phototropism by the coleoptiles of corn (Zea mays) seedlings. The compounds tested fall into three categories showing: (a) no inhibition of either phototropism or geotropism (KCl); (b) nonspecific inhibition of both phototropism and geotropism (KCN); and (c) specific inhibition of phototropism (KI, NaN₃, and phenylacetic acid). Simultaneous irradiation of coleoptiles with phototropically inert light in addition to the phototropically active blue light also results in an inhibition of phototropism. Since azide, iodide, and phenylacetic acid are known to interact with flavins while a simultaneous irradiation with a phototropically inert light may depopulate the first triplet state of flavins, these data support the hypothesis that the photoreceptor pigment for phototropism in corn is a flavin.     

A study of flavin-protein and flavoprotein-ligand interactions. Binding aspects and spectral properties of D-amino acid oxidase and riboflavin binding protein.

To study flavin-protein and flavoprotein-ligand interaction, the absorption, CD and MCD spectra of riboflavin, FAD, roseoflavin, the complexes of riboflavin and roseoflavin with riboflavin binding protein(RBP),D-amino acid oxidase(D-AO) and its complexes with ligands were observed in the spectral region of 310-600 nm and the binding properties of D-AO with di-substituted benzoate derivatives and of RBP with roseoflavin were also measured. The dimer of D-amino acid oxidase has a higher affinity for di-substituted benzoate derivatives than the monomer. The change in the absorption of FAD in D-AO caused by the binding of the first ligand to the dimer, which can bind two ligands, was similar to that caused by the binding of the second ligand. Roseoflavin could bind to RBP in a 1 : 1 ratio and the dissociation constant was 3.8 x 10(-8)M. The protein fluorescence of RBP was quenched by about 86% due to complex formation with roseoflavin. The MCD spectra showed similar patterns for all molecular complexes of riboflavin and FAD, with two negative extrema of ellipticity which probably correspond to the Faraday B-term, but the Faraday A-term could not be observed, suggesting that there was no degeneracy in the excited state of flavins. It is also suggested, based on a comparison of the absorption, CD and MCD spectra, that the vibronic structure of flavin was modified differently by each flavin-protein or flavoprotein-ligand interaction. Comparison of the absorption, CD and MCD spectra(310-600 nm) for roseoflavin and the roseoflavin-RBP complex revealed that there were five spectral components around 320, 340, 400, 500, and 550 nm in roseoflavin.     

Effects of pH and ionic strength on the binding of egg white riboflavin binding protein with flavins

The effects of pH and ionic strength on the equilibrium constants and rate constants (binding and dissociation rate constants) between riboflavin binding protein (RBP) and flavins (riboflavin, 3-carboxymethylriboflavin [CMRF], and FMN) were studied by fluorometry. The equilibrium constant and the binding rate constant between RBP and riboflavin were pH-independent between pH 6 and 9, and both constants were also independent of the ionic strength, while the constants between RBP and CMRF or FMN were dependent on both pH and ionic strength. The dissociation rate constants between RBP and the flavins used here were not so dependent on pH and ionic strength in the pH region 6 to 9, and the patterns of pH profiles as a whole were similar to each other, although the constants for FMN were about 30-60 times larger than those for CMRF or riboflavin. RBP had lower affinity for FMN than for riboflavin in the neutral pH region, which is based on the small binding rate constant and the large dissociation rate constant for FMN. The former is due to an electrostatic repulsion force between negative net charges of RBP and the phosphate group of FMN, and the latter is due to steric interference by the phosphate group of FMN.     

Pigments associated with the flagellum ofEuglena gracilis

The pigments associated with the flagellum of the phytoflagellateEuglena gracllis were characterized by HPLC. The pigment pattern of the wild-type strain was compared with a set of white mutants which did not display phototaxis and photoaccumulation in response to blue light. Flagella of the wild type contained FMN and FAD. Two mutants which lacked the stigma but retained a small paraxonemal body (PAB) contained less flavins. The whiteEuglena mutant FB, which retained a residual stigma and also a PAB, and the white phytoflagellateAstasia longa, a close relative ofEuglena, had normal amounts of flagellar flavins. Cells and flagella ofEuglena wild type contained an unldentified pterin-like pigment, called Pt₁₆, which was substantially reduced inAstasia and theEuglena mutants. A third pigment, designated P₅₂₈ with major absorption at 528 nm and fluorescence emission at 550 nm was present mainly in flagella. The association of the three pigment types with flagella and their respective alterations in the white strains indicates their possible role in photoreception. Dedicated to Pill-Soon Song on the occasion of his 60th birthday.     

Active transport of riboflavin by the isolated choroid plexus in vitro.

In vitro, the transport of [14C]riboflavin into and from the isolated choroid plexus, the anatomical locus of the blood-cerebrospinal fluid barrier, was studied. With concentrations of [14C]riboflavin of 0.7 microM (or greater) in the incubation medium, the choroid plexus accumulated [14C]riboflavin against a large concentration gradient by a process that did not depend on binding or intracellular metabolism of the [14C]riboflavin. The [14C]riboflavin accumulation process in isolated choroid plexus could be described by Michaelis-Menten transport kinetics (kt = 78 microM and Ymax = 1.65 mmol kg-1 (15 min)-1) and was inhibited by other flavins and probenecid but not by ribose, weak bases, or other B vitamins. The accumulation process was markedly depressed by iodoacetate and low temperatures. With a concentration of 0.08 microM [14C]riboflavin in the incubation medium, 28% of the [14C]riboflavin within the choroid plexus was converted to [14C]FAD or [14C]FMN intracellularly. Unlike the active transport of [14C]riboflavin into choroid plexus, accumulated [14C]riboflavin departed choroid plexus by a process independent of intracellular concentration or temperature. The efflux of [14C]riboflavin from choroid plexus could be described by first oder kinetics with a rate constant of -0.08 min-1.     

The effect of flavin electron shuttles in microbial fuel cells current production

The effect of electron shuttles on electron transfer to microbial fuel cell (MFC) anodes was studied in systems where direct contact with the anode was precluded. MFCs were inoculated with Shewanella cells, and flavins used as the electron shuttling compound. In MFCs with no added electron shuttles, flavin concentrations monitored in the MFCs' bulk liquid increased continuously with FMN as the predominant flavin. The maximum concentrations were 0.6 μM for flavin mononucleotide and 0.2 μM for riboflavin. In MFCs with added flavins, micro-molar concentrations were shown to increase current and power output. The peak current was at least four times higher in MFCs with high concentrations of flavins (4.5–5.5 μM) than in MFCs with low concentrations (0.2–0.6 μM). Although high power outputs (around 150 mW/m²) were achieved in MFCs with high concentrations of flavins, a Clostridium-like bacterium along with other reactor limitations affected overall coulombic efficiencies (CE) obtained, achieving a maximum CE of 13%. Electron shuttle compounds (flavins) permitted bacteria to utilise a remote electron acceptor (anode) that was not accessible to the cells allowing current production until the electron donor (lactate) was consumed.     

Effect of oxygen on the per‐cell extracellular electron transfer rate of Shewanella oneidensis MR‐1 explored in bioelectrochemical systems

Extracellular electron transfer (EET) is a mechanism that enables microbes to respire solid‐phase electron acceptors. These EET reactions most often occur in the absence of oxygen, since oxygen can act as a competitive electron acceptor for many facultative microbes. However, for Shewanella oneidensis MR‐1, oxygen may increase biomass development, which could result in an overall increase in EET activity. Here, we studied the effect of oxygen on S. oneidensis MR‐1 EET rates using bioelectrochemical systems (BESs). We utilized optically accessible BESs to monitor real‐time biomass growth, and studied the per‐cell EET rate as a function of oxygen and riboflavin concentrations in BESs of different design and operational conditions. Our results show that oxygen exposure promotes biomass development on the electrode, but significantly impairs per‐cell EET rates even though current production does not always decrease with oxygen exposure. Additionally, our results indicated that oxygen can affect the role of riboflavin in EET. Under anaerobic conditions, both current density and per‐cell EET rate increase with the riboflavin concentration. However, as the dissolved oxygen (DO) value increased to 0.42 mg/L, riboflavin showed very limited enhancement on per‐cell EET rate and current generation. Since it is known that oxygen can promote flavins secretion in S. oneidensis, the role of riboflavin may change under anaerobic and aerobic conditions. Biotechnol. Bioeng. 2017;114: 96–105. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

Lipid and lipoprotein oxidation: basic mechanisms and unresolved questions in vivo

Abstract

The kinetics and mechanistic aspects of the riboflavin-photosensitised oxidation of the topically administrable ophthalmic drugs Timolol (Tim) and Pindolol (Pin) were investigated in water–MeOH (9:1, v/v) solution employing light of wavelength > 400 nm. riboflavin, belonging to the vitamin B₂ complex, is a known human endogenous photosensitiser. The irradiation of riboflavin in the presence of ophthalmic drugs triggers a complex picture of competitive reactions which produces the photodegradation of both the drugs and the pigment itself. The mechanism was elucidated employing stationary photolysis, polarographic detection of dissolved oxygen, stationary and time-resolved fluorescence spectroscopy, and laser flash photolysis. Ophthalmic drugs quench riboflavin-excited singlet and triplet states. From the quenching of excited triplet riboflavin, the semireduced form of the pigment is generated, through an electron transfer process from the drug, with the subsequent production of superoxide anion radical (O₂^?–) by reaction with dissolved molecular oxygen. Through the interaction of dissolved oxygen with excited triplet riboflavin, the species singlet oxygen (O₂(¹Δ_g)) is also generated to a lesser extent. Both O₂^?– and O₂(¹Δ_g) induce photodegradation of ophthalmic drugs, Tim being ~3-fold more easily photooxidisable than Pin, as estimated by oxygen consumption experiments.     

Chemical and enzymatic properties of riboflavin analogues.

The chemical and enzymatic properties of 26 analogues of riboflavin are presented. These analogues include both endo- and exocyclically substituted isoalloxazines with redox potentials from -370 to -128 mV. Physical and chemical data such as the electronic absorption spectra, pKas, and redox potentials of the analogues are presented and are discussed with respect to preferred tautomeric and resonance forms. Like riboflavin, most of the analogues are shown to be catalytic oxidants of dihydro-5-deazaflavins. Analogue binding to egg white binding apoprotein has been quantitated and serves to determine the origins of binding site specificity for this protein. Nearly all of the analogues that possess D-ribityl groups are found to be processed to the FAD level by the flavokinase/FAD synthetase system of Brevibacterium ammoniagenes. Most extensively studied are the reactivities of the analogues with the NAD(P)H:flavin oxidoreductase of Beneckea harveyi. Many of the analogues are substrates in this enzymatic redox reaction, and a linear free energy-rate relation (log Vmax vs. E0' of the analogue) is seen that parallels similar relationships in the nonenzymatic oxidation of dihydro-5-deazaflavins. This suggests a common mechanism for the reactions of such diverse flavins as riboflavin, 5-deazariboflavin, and 1-deazariboflavin.