Flavin Imbalance as an Important Player in Diabetic Retinopathy.

The retina and RPE together constitute the most metabolically active ecosystem in the body, harboring high levels of flavins. Although diabetic patients have been reported to suffer from riboflavin deficiency and use of flavins as nutritional interventions to combat diabetic insult on other tissues have been investigated, such attempts have never been tested for the retina to avoid diabetic retinopathy. Furthermore, the role of flavins in pathophysiology of the retina and RPE has mostly been overlooked. Herein, we review the impact of flavins on various clinical manifestations of diabetic retinopathy and discuss possible ways to address them.

Physiology, Biochemistry, and Applications of F420- and Fo-Dependent Redox Reactions.

5-Deazaflavin cofactors enhance the metabolic flexibility of microorganisms by catalyzing a wide range of challenging enzymatic redox reactions. While structurally similar to riboflavin, 5-deazaflavins have distinctive and biologically useful electrochemical and photochemical properties as a result of the substitution of N-5 of the isoalloxazine ring for a carbon. 8-Hydroxy-5-deazaflavin (Fo) appears to be used for a single function: as a light-harvesting chromophore for DNA photolyases across the three domains of life. In contrast, its oligoglutamyl derivative F420 is a taxonomically restricted but functionally versatile cofactor that facilitates many low-potential two-electron redox reactions. It serves as an essential catabolic cofactor in methanogenic, sulfate-reducing, and likely methanotrophic archaea. It also transforms a wide range of exogenous substrates and endogenous metabolites in aerobic actinobacteria, for example mycobacteria and streptomycetes. In this review, we discuss the physiological roles of F420 in microorganisms and the biochemistry of the various oxidoreductases that mediate these roles. Particular focus is placed on the central roles of F420 in methanogenic archaea in processes such as substrate oxidation, C1 pathways, respiration, and oxygen detoxification. We also describe how two F420-dependent oxidoreductase superfamilies mediate many environmentally and medically important reactions in bacteria, including biosynthesis of tetracycline and pyrrolobenzodiazepine antibiotics by streptomycetes, activation of the prodrugs pretomanid and delamanid by Mycobacterium tuberculosis, and degradation of environmental contaminants such as picrate, aflatoxin, and malachite green. The biosynthesis pathways of Fo and F420 are also detailed. We conclude by considering opportunities to exploit deazaflavin-dependent processes in tuberculosis treatment, methane mitigation, bioremediation, and industrial biocatalysis.

Diflavin oxidoreductases activate the bioreductive prodrug PR-104A under hypoxia.

The clinical agent PR-104 is converted systemically to PR-104A, a nitrogen mustard prodrug designed to target tumor hypoxia. Reductive activation of PR-104A is initiated by one-electron oxidoreductases in a process reversed by oxygen. The identity of these oxidoreductases is unknown, with the exception of cytochrome P450 reductase (POR). To identify other hypoxia-selective PR-104A reductases, nine candidate oxidoreductases were expressed in HCT116 cells. Increased PR-104A-cytotoxicity was observed in cells expressing methionine synthase reductase (MTRR), novel diflavin oxidoreductase 1 (NDOR1), and inducible nitric-oxide synthase (NOS2A), in addition to POR. Plasmid-based expression of these diflavin oxidoreductases also enhanced bioreductive metabolism of PR-104A in an anoxia-specific manner. Diflavin oxidoreductase-dependent PR-104A metabolism was suppressed >90% by pan-flavoenzyme inhibition with diphenyliodonium chloride. Antibodies were used to quantify endogenous POR, MTRR, NDOR1, and NOS2A expression in 23 human tumor cell lines; however, only POR protein was detectable and its expression correlated with anoxic PR-104A reduction (r(2) = 0.712). An anti-POR monoclonal antibody was used to probe expression using human tissue microarrays; 13 of 19 cancer types expressed detectable POR with 21% of cores (185 of 874) staining positive; this heterogeneity suggests that POR is a useful biomarker for PR-104A activation. Immunostaining for carbonic anhydrase 9 (CAIX), reportedly an endogenous marker of hypoxia, revealed only moderate coexpression (9.6%) of both CAIX and POR across a subset of five cancer types.

Flavin-dependent quinone reductases.

Quinones are abundant cyclic organic compounds present in the environment as well as in pro- and eukaryotic cells. Several species have been shown to possess enzymes that afford the two-electron reduction to the hydroquinone form in an attempt to avoid the generation of one-electron reduced semiquinone known to cause oxidative stress. These enzymes utilize a flavin cofactor, either FMN or FAD, to transfer a hydride from an electron donor, such as NAD(P)H, to a quinone substrate. This family of flavin-dependent quinone reductases shares a flavodoxin-like structure and reaction mechanism pointing towards a common evolutionary origin. Recent studies of their physiological functions in eukaryotes suggest a role beyond detoxication of quinones and involvement in the oxygen stress response. Accordingly, mammalian quinone reductases emerge as central molecular switches that control the lifespan of transcription factors, such as p53, and hence participate in the development of apoptosis and cell transformation.

Evidence of a light-sensing role for folate in Arabidopsis cryptochrome blue-light receptors.

Arabidopsis cryptochromes cry1 and cry2 are blue-light signalling molecules with significant structural similarity to photolyases--a class of blue-light-sensing DNA repair enzymes. Like photolyases, purified plant cryptochromes have been shown to bind both flavin and pterin chromophores. The flavin functions as a light sensor and undergoes reduction in response to blue light that initiates the signalling cascade. However, the role of the pterin in plant cryptochromes has until now been unknown. Here, we show that the action spectrum for light-dependent degradation of cry2 has a significant peak of activity at 380 nm, consistent with absorption by a pterin cofactor. We further show that cry1 protein expressed in living insect cells responds with greater sensitivity to 380 nm light than to 450 nm, consistent with a light-harvesting antenna pigment that transfers excitation energy to the oxidized flavin of cry1. The pterin biosynthesis inhibitor DHAP selectively reduces cryptochrome responsivity at 380 nm but not 450 nm blue light in these cell cultures, indicating that the antenna pigment is a folate cofactor similar to that of photolyases.

Regulation of normal cell cycle progression by flavin-containing oxidases.

Mechanisms underlying the role of reactive oxygen species (ROS) generated by flavin-containing oxidases in regulating cell cycle progression were examined in human and rodent fibroblasts. Incubation of confluent cell cultures with nontoxic/nonclastogenic concentrations of the flavoprotein inhibitor, diphenyleneiodonium (DPI), reduced nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase activity and basal ROS levels, but increased proteolysis of cyclin D1, p21(Waf1) and phospho-p38(MAPK). When these cells were allowed to proliferate by subculture in DPI-free medium, an extensive G(1) delay was observed with concomitant activation of p53/p21(Waf1) signaling and reduced phosphorylation of mitogen-activated kinases. Compensation for decreased oxidant generation by simultaneous exposure to DPI and nontoxic doses of the ROS generators, gamma-radiation or t-butyl-hydroperoxide, attenuated the G(1) delay. Whereas the DPI-induced G(1) checkpoint was completely dependent on PHOX91, ATM and WAF1, it was only partially dependent on P53. Interestingly, G(1) to S progression was not affected when another flavin-containing enzyme, nitric oxide synthase, was inhibited nor was it associated with changes in mitochondrial membrane potential. Proliferating cells treated with DPI also experienced a significant but attenuated delay in G(2). We propose that ATM performs a critical function in mediating normal cellular proliferation that is regulated by nonphagocytic NAD(P)H oxidase enzymes activity, which may serve as a novel target for arresting cancer cells in G(1).

The rhizosphere signal molecule lumichrome alters seedling development in both legumes and cereals.

The stimulatory role of lumichrome, a rhizosphere metabolite, was assessed on the growth of legume and cereal seedlings. At a very low nanomolar concentration (5 nm), lumichrome elicited growth promotion in cowpea, soybean, sorghum, millet and maize, but not in common bean, Bambara groundnut and Sudan grass. In soybean and cowpea only, 5 nm lumichrome caused early initiation of trifoliate leaf development, expansion in unifoliate and trifoliate leaves, increased stem elongation and, as a result, an increase in shoot and plant total biomass relative to control. Lumichrome (5 nm) also increased leaf area in maize and sorghum, and thus raised shoot and total biomass but there was no effect on the leaf area of the other cereals. Root growth was also stimulated in sorghum and millet by the supply of 5 nm lumichrome. By contrast, the application of a higher dose of lumichrome (50 nm) depressed development of unifoliate leaves in soybean, the second trifoliate leaf in cowpea, and shoot biomass in soybean. The 50 nm concentration also consistently decreased root development in cowpea and millet, but had no effect on the other species. These data show that lumichrome is a rhizosphere signal molecule that affects seedling development in both monocots and dicots.

Xylem transport and shoot accumulation of lumichrome, a newly recognized rhizobial signal, alters root respiration, stomatal conductance, leaf transpiration and photosynthetic rates in legumes and cereals.

* Root respiration, stomatal conductance, leaf transpiration and photosynthetic rates were measured in phytotron and field-grown plants following the application of 5 or 10 nM lumichrome, 10 nM ABA (abscisic acid) and 10 ml of 0.2 OD600 infective rhizobial cells. * Providing soybean and cowpea roots with their respective homologous rhizobia and/or purified lumichrome increased the concentration of this molecule in xylem sap and leaf extracts. Relative to control, rhizobial inoculation and lumichrome application significantly increased root respiration in maize, decreased it in lupin, but had no effect on the other test species. * Applying either lumichrome (10 nM), infective rhizobial cells or ABA to roots of plants for 44 h in growth chambers altered leaf stomatal conductance and transpiration in cowpea, lupin, soybean, Bambara groundnut and maize, but not in pea or sorghum. Where stomatal conductance was increased by lumichrome application or rhizobial inoculation, it resulted in increased leaf transpiration relative to control plants. Treating roots of field plants of cowpea with this metabolite up to 63 d after planting showed decreased stomatal conductance, which affected CO2 intake and reduction by Rubisco. * The effect of rhizobial inoculation closely mirrored that of lumichrome application to roots, indicating that rhizobial effects on these physiological activities were most likely due to lumichrome released into the rhizosphere.

Deflavination and reconstitution of flavoproteins.

Flavoproteins are ubiquitous redox proteins that are involved in many biological processes. In the majority of flavoproteins, the flavin cofactor is tightly but noncovalently bound. Reversible dissociation of flavoproteins into apoprotein and flavin prosthetic group yields valuable insights in flavoprotein folding, function and mechanism. Replacement of the natural cofactor with artificial flavins has proved to be especially useful for the determination of the solvent accessibility, polarity, reaction stereochemistry and dynamic behaviour of flavoprotein active sites. In this review we summarize the advances made in the field of flavoprotein deflavination and reconstitution. Several sophisticated chromatographic procedures to either deflavinate or reconstitute the flavoprotein on a large scale are discussed. In a subset of flavoproteins, the flavin cofactor is covalently attached to the polypeptide chain. Studies from riboflavin-deficient expression systems and site-directed mutagenesis suggest that the flavinylation reaction is a post-translational, rather than a cotranslational, process. These genetic approaches have also provided insight into the mechanism of covalent flavinylation and the rationale for this atypical protein modification.

The chemical and biological versatility of riboflavin.

Since their discovery and chemical characterization in the 1930s, flavins have been recognized as being capable of both one- and two-electron transfer processes, and as playing a pivotal role in coupling the two-electron oxidation of most organic substrates to the one-electron transfers of the respiratory chain. In addition, they are now known as versatile compounds that can function as electrophiles and nucleophiles, with covalent intermediates of flavin and substrate frequently being involved in catalysis. Flavins are thought to contribute to oxidative stress through their ability to produce superoxide, but at the same time flavins are frequently involved in the reduction of hydroperoxides, products of oxygen-derived radical reactions. Flavoproteins play an important role in soil detoxification processes via the hydroxylation of many aromatic compounds, and a simple flavoprotein in liver microsomes catalyses many reactions similar to those carried out by cytochrome P450 enzymes. Flavins are involved in the production of light in bioluminescent bacteria, and are intimately connected with light-initiated reactions such as plant phototropism and nucleic acid repair processes. Recent reports also link them to programmed cell death. The chemical versatility of flavoproteins is clearly controlled by specific interactions with the proteins with which they are bound. One of the main thrusts of current research is to try to define the nature of these interactions, and to understand in chemical terms the various steps involved in catalysis by flavoprotein enzymes.

Lumichrome. A larval metamorphosis-inducing substance in the ascidian Hhalocynthia roretzi.

It has long been known that metamorphosis of ascidian larvae is induced by exposure to adult tunic extract or larval-conditioned seawater. However, such a natural 'inducer' has not been identified, probably due to its very low concentration in organisms. Here we have succeeded in isolating the same metamorphosis-inducing substance from the larvae, the larval-conditioned seawater, and the adult tunic of the ascidian Halocynthia roretzi. Structural analysis revealed that this substance was identical to lumichrome. Lumichrome was active toward H. roretzi larvae, but inactive toward another ascidian larvae, suggesting that lumichrome is species-specific. Riboflavin (vitamin B2), from which lumichrome might be derived from, was found to be inactive in induction of larval metamorphosis. In addition, it was demonstrated that lumichrome is localized predominantly in the basal region of the adhesive organ and the posterior part of the larval trunk. Thus, we propose that lumichrome functions as a natural inducer for larval metamorphosis in H. roretzi. This is the first natural metamorphosis-inducing substance to be identified in ascidians.

[Marine natural products influencing larval settlement and metamorphosis of marine sessile organisms].

Most marine sessile organisms have a planktonic larval phase in their life cycles, and then larvae settle and metamorphose into their adult forms. The selection of settlement sites is a critical event for these organisms because settlement on unsuitable places affects their survivorship severely. Ascidians live gregariously, and conspecific chemical cues are thought to play an important role in gregarious settlement of larvae. The extracts of conspecific adults or larvae have been claimed to contain "natural metamorphosis inducers." Little is known, however, about their chemical properties. To discover natural signal substances for larval metamorphosis in ascidians, we surveyed the metamorphosis-inducing activity of the medium conditioned by ascidian larvae, and succeeded in isolating a metamorphosis-inducing substance from the conditioned medium of Halocycthia roretzi larvae and found that it was identical to lumichrome. We have also isolated more than 40 active metabolites, which may mimic lumichrome, from marine sponges. On the contrary, marine sessile organisms cause serious problems by settling on fishing nets, hulls of ships, and cooling systems of power plants. Organotin compounds have been widely used for the control of these organisms, but they are known to be toxic to marine biota. Therefore, nontoxic antifouling substances are urgently needed. Marine sessile organisms possess chemical defense systems using their secondary metabolites, which might be potential by nontoxic antifouling agents. We have attempted to obtain antibarnacle substances from marine sponges and isolated 26 antifoulants.

NADPH oxidase-like activity in rainbow trout Oncorhynchus mykiss (Walbaum) macrophages.

Evidence for the existence of an NADPH oxidase-like enzyme in rainbow trout macrophages is given. Reduced-minus-oxidised difference spectroscopy revealed the presence of a cytochrome b with three absorbance peaks, at 430, 533, and 558 nm. The low midpoint potential of the latter peak suggests this cytochrome is the same as the terminal component of NADPH oxidase (i.e., cytochrome b-245). Subcellular fractionation of macrophages revealed two peaks of cytochrome b activity, in accord with the concept of a plasma membrane localisation of cytochrome b activity in addition to a mitochondrial localisation. Finally, that the rainbow trout oxidase is a multicomponent enzyme was suggested by inhibitor studies, where specific inhibitors of the flavin and cytochrome b-245 components of NADPH oxidase induced significant reduction in superoxide anion production.

Flavin-dependent alcohol oxidase from yeast. Studies on the catalytic mechanism and inactivation during turnover.

The kinetic course of the reaction of methanol and deutero-methanol with FAD-dependent alcohol oxidase was investigated under single-turnover conditions [kred approximately equal to 15000 min-1 (1H3COH) and approximately equal to 4300 min-1 (2H3COH)] and multiple-turnover conditions [TNmax approximately equal to 6000 min-1 (1H3COH) and approximately equal to 3100 min-1 (2H3COH)]. A kinetic scheme for the overall catalytic mechanism is proposed, which is characterized by (1) formation of a Michaelis complex between enzyme and substrate, (2) the reductive step involving partly rate-limiting scission of the substrate C-H bond, (3) reaction of the complex of reduced enzyme and aldehyde with dioxygen, and (4) a significant contribution of the dissociation rate of product from its complex with reoxidized enzyme to the overall rate. Prolonged turnover of various alcohols, including methanol, results in progressive inactivation of the enzyme by two processes. In the absence of catalase the inactivation rate increases with time due to accumulation of hydrogen peroxide, which is a potent inactivator (Kd approximately equal to 1.6 mM; kinact approximately equal to 0.55 min-1). In the presence of catalase inactivation during turnover is much slower, the process showing pseudo-first-order kinetics (Kinact approximately equal to 0.6 mM; kinact approximately equal to 0.005 min-1 with methanol). The ratio kcat/kinact varies with different alcohols but is always greater than 10(5). Propargyl alcohol and methylenecyclopropyl alcohol cannot be considered as suicide substrates, as compared to analogous substrates of other flavin oxidases.

Contribution of N-oxygenation to the metabolism of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) by various liver preparations.

Liver microsomes from uninduced mice and rats catalyze NADPH- and oxygen-dependent N-oxygenation of the neurotoxin MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine). The N-oxide is the principal product and accounts for 95-96% of the total MPTP metabolized by microsomes. Demethylation of MPTP is detectable but the rate of nor-MPTP formation was never more than 4-6% of the rate of N-oxygenation. Studies on the biochemical mechanisms for N-oxygenation of MPTP suggest that this reaction is catalyzed exclusively by the flavin-containing monooxygenase. This conclusion is based on the effects of selective cytochrome P-450 inhibitors, positive effectors, and alternate substrates for the flavin-containing monooxygenase as well as on studies with the purified hog liver enzyme. MPTP is an excellent substrate for this monooxygenase with a Km of 30-33 microM. Limited studies with human liver whole homogenates suggest that N-oxygenation is also a major route for the metabolism of MPTP in man and the rate of N-oxide formation is approximately equal to the rate of mitochondrial monoamine oxidase-dependent MPDP+ (1-methyl-4-phenyl-2,3-dihydropyridinium species) production.

Metabolic oxidation of carcinogenic arylamines by rat, dog, and human hepatic microsomes and by purified flavin-containing and cytochrome P-450 monooxygenases.

Hepatic N-oxidation and aryl ring oxidation are generally regarded as critical activation and detoxification pathways for arylamine carcinogenesis. In this study, we examined the in vitro hepatic metabolism of the carcinogens, 2-aminofluorene (2-AF) and 2-naphthylamine (2-NA), and the suspected carcinogen, 1-naphthylamine (1-NA), using high-pressure liquid chromatography. Hepatic microsomes from rats, dogs, and humans were shown to catalyze the N-oxidation of 2-AF and of 2-NA, but not of 1-NA; and the rates of 2-AF N-oxidation were 2- to 3-fold greater than the rates of 2-NA N-oxidation. In each species, rates of 1-hydroxylation of 2-NA and 2-hydroxylation of 1-NA were comparable and were 2- to 5-fold greater than 6-hydroxylation of 2-NA or 5- and 7-hydroxylation of 2-AF. Purified rat hepatic monooxygenases, cytochromes P-450UT-A, P-450UT-H, P-450PB-B, P-450PB-D, P-450BNF-B, and P-450ISF/BNF-G but not P-450PB-C or P-450PB/PCN-E, catalyzed several ring oxidations as well as the N-oxidation of 2-AF. Cytochromes P-450PB-B, P-450BNF-B, and P-450ISF/BNF-G were most active; however, only cytochrome P-450ISF/BNF-G, the isosafrole-induced isozyme, catalyzed the N-oxidation of 2-NA. The purified porcine hepatic flavin-containing monooxygenase, which was known to carry out the N-oxidation of 2-AF, was found to catalyze only ring oxidation of 1-NA and 2-NA. No activity for 1-NA N-oxidation was found with any of the purified enzymes. These data support the hypothesis that 1-NA is probably not carcinogenic. Furthermore, carcinogenic arylamines appear to be metabolized similarly in humans and experimental animals and perhaps selectively by a specific form of hepatic cytochrome P-450. Enzyme mechanisms accounting for the observed product distributions were evaluated by Hückel molecular orbital calculations on neutral, free radical, and cation intermediates. A reaction pathway is proposed that involves two consecutive one-electron oxidations to form a paired substrate cation-enzyme hydroxyl anion intermediate that collapses to ring and N-hydroxy products.

Riboflavin requirement for growth, tissue saturation and maximal flavin-dependent enzyme activity in young rainbow trout (Salmo gairdneri) at two temperatures.

Two temperatures (10 degrees and 15 degrees C) and two fish stocks differing in growth potential were used to determine the dietary riboflavin requirement of young rainbow trout (Salmo gairdneri) on the basis of growth parameters, tissue saturation and a flavin-dependent biochemical function. Two experiments were conducted with purified diets based on vitamin-free casein. In experiment 1, fry (initially 2.0 g/fish) were fed, for 16 wk at 15 degrees C, diets containing 0.6, 2.6, 3.6, 4.6, 5.6 or 6.6 mg riboflavin per kilogram diet. In experiment 2, fry (initially 1.7-1.8 g/fish) were held at either 10 degrees or 15 degrees C and fed, for 10 wk, diets containing 0.7, 2.7, 3.7, 4.7, 6.7 or 8.7 mg riboflavin per kilogram. The riboflavin requirements for maximal growth rate, liver flavin saturation, spleen and head kidney flavin saturation and maximal hepatic D-amino-acid oxidase activity were 3.6, 4.6, 6.6 and 5.6 mg/kg diet, respectively, in a diet containing 40% crude protein and 15% ether extract. The requirements were not affected by temperature or by genetically determined differences in maximal growth rate. When expressed on a dietary energy basis, the riboflavin requirements of the trout for maximal growth rate and liver flavin saturation appear similar to those of several homeothermic species.