Flavin Homeostasis Research Articles

AtDREB2G is involved in the regulation of riboflavin biosynthesis in response to low-temperature stress and abscisic acid treatment in Arabidopsis thaliana

Riboflavin (RF) serves as a precursor to flavin mononucleotide and flavin adenine dinucleotide, which are crucial cofactors in various metabolic processes. Strict regulation of cellular flavin homeostasis is imperative, yet information regarding the factors governing this regulation remains largely elusive. In this study, we first examined the impact of external flavin treatment on the Arabidopsis transcriptome to identify novel regulators of cellular flavin levels. Our analysis revealed alterations in the expression of 49 putative transcription factors. Subsequent reverse genetic screening highlighted a member of the dehydration-responsive element binding (DREB) family, AtDREB2G, as a potential regulator of cellular flavin levels. Knockout mutants of AtDREB2G (dreb2g) exhibited reduced flavin levels and decreased expression of RF biosynthetic genes compared to wild-type plants. Conversely, conditional overexpression of AtDREB2G led to an increase in the expression of RF biosynthetic genes and elevated flavin levels. In wild-type plants, exposure to low temperatures and abscisic acid treatment stimulated enhanced flavin levels and upregulated the expression of RF biosynthetic genes, concomitant with the induction of AtDREB2G. Notably, these responses were significantly attenuated in dreb2g mutants. Our findings establish AtDREB2G is involved in the positive regulation of flavin biosynthesis in Arabidopsis, particularly under conditions of low temperature and abscisic acid treatment.

Regulation of respiratory complex I assembly by FMN cofactor targeting

Respiratory complex I plays a crucial role in the mitochondrial electron transport chain and shows promise as a therapeutic target for various human diseases. While most studies focus on inhibiting complex I at the Q-site, little is known about inhibitors targeting other sites within the complex. In this study, we demonstrate that diphenyleneiodonium (DPI), a N-site inhibitor, uniquely affects the stability of complex I by reacting with its flavin cofactor FMN. Treatment with DPI blocks the final stage of complex I assembly, leading to the complete and reversible degradation of complex I in different cellular models. Growing cells in medium lacking the FMN precursor riboflavin or knocking out the mitochondrial flavin carrier gene SLC25A32 results in a similar complex I degradation. Overall, our findings establish a direct connection between mitochondrial flavin homeostasis and complex I stability and assembly, paving the way for novel pharmacological strategies to regulate respiratory complex I.

Mycobacterium bovis BCG dodecin gene codes a functional protein despite of a start codon mutation

Dodecin is a dodecamer involved in flavin homeostasis, with interesting temperature and osmolarity endurance features in Mycobacterium tuberculosis. A single nucleotide polymorphism in the gene's start codon in BCG, converting ATG to ACG, is predicted to generate a N-terminal shorter isoform, lacking the first 7 amino acids. We previously reported that the shortened recombinant protein has reduced extremophilic features. Here we investigate if within the mycobacterial context dodecin can be produced from both alleles, carrying ATG and ACG start codons. Reporter gene assays using mcherry cloned downstream and in phase to both M.tb and BCG “upstream” regions confirms production of functional proteins. Complementation with both dod alleles similarly enhances M. smegmatis growth after entry into logarithmic phase and exposure to hydrogen peroxide, possibly implicating this protein in oxidative stress response mechanisms. Altogether these data indicate that BCG dodecin is indeed produced, notwithstanding in lower levels compared to M.tb, conferring similar phenotypes, even with the SNP altering the M.tb ATG start codon to the BCG ACG. This protein might be an interesting drug target for the development of new therapeutics against tuberculosis.

Increased demand for FAD synthesis in differentiated and stem pancreatic cancer cells is accomplished by modulating FLAD1 gene expression: the inhibitory effect of Chicago Sky Blue.

FLAD1, along with its FAD synthase (FADS, EC 2.7.7.2) product, is crucial for flavin homeostasis and, due to its role in the mitochondrial respiratory chain and nuclear epigenetics, is closely related to cellular metabolism. Therefore, it is not surprising that it could be correlated with cancer. To our knowledge, no previous study has investigated FLAD1 prognostic significance in pancreatic ductal adenocarcinoma (PDAC). Thus, in the present work, the FAD synthesis process was evaluated in two PDAC cell lines: (a) PANC-1- and PANC-1-derived cancer stem cells (CSCs), presenting the R273H mutation in the oncosuppressor p53, and (b) MiaPaca2 and MiaPaca2-derived CSCs, presenting the R248W mutation in p53. As a control, HPDE cells expressing wt-p53 were used. FADS expression/activity increase was found with malignancy and even more with stemness. An increased FAD synthesis rate in cancer cell lines is presumably demanded by the increase in the FAD-dependent lysine demethylase 1 protein amount as well as by the increased expression levels of the flavoprotein subunit of complex II of the mitochondrial respiratory chain, namely succinate dehydrogenase. With the aim of proposing FADS as a novel target for cancer therapy, the inhibitory effect of Chicago Sky Blue on FADS enzymatic activity was tested on the recombinant 6His-hFADS2 (IC50 = 1.2 μm) and PANC-1-derived CSCs' lysate (IC50 = 2-10 μm). This molecule was found effective in inhibiting the growth of PANC-1 and even more of its derived CSC line, thus assessing its role as a potential chemotherapeutic drug.

Retrograde response to mitochondrial dysfunctions associated to LOF variations in FLAD1 exon 2: unraveling the importance of RFVT2

Flavin adenine dinucleotide (FAD) synthase (EC 2.7.7.2), encoded by human flavin adenine dinucleotide synthetase 1 (FLAD1), catalyzes the last step of the pathway converting riboflavin (Rf) into FAD. FLAD1 variations were identified as a cause of LSMFLAD (lipid storage myopathy due to FAD synthase deficiency, OMIM #255100), resembling Multiple Acyl-CoA Dehydrogenase Deficiency, sometimes treatable with high doses of Rf; no alternative therapeutic strategies are available. We describe here cell morphological and mitochondrial alterations in dermal fibroblasts derived from a LSMFLAD patient carrying a homozygous truncating FLAD1 variant (c.745C > T) in exon 2. Despite a severe decrease in FAD synthesis rate, the patient had decreased cellular levels of Rf and flavin mononucleotide and responded to Rf treatment. We hypothesized that disturbed flavin homeostasis and Rf-responsiveness could be due to a secondary impairment in the expression of the Rf transporter 2 (RFVT2), encoded by SLC52A2, in the frame of an adaptive retrograde signaling to mitochondrial dysfunction. Interestingly, an antioxidant response element (ARE) is found in the region upstream of the transcriptional start site of SLC52A2. Accordingly, we found that abnormal mitochondrial morphology and impairments in bioenergetics were accompanied by increased cellular reactive oxygen species content and mtDNA oxidative damage. Concomitantly, an active response to mitochondrial stress is suggested by increased levels of PPARγ-co-activator-1α and Peroxiredoxin III. In this scenario, the treatment with high doses of Rf might compensate for the secondary RFVT2 molecular defect, providing a molecular rationale for the Rf responsiveness in patients with loss of function variants in FLAD1 exon 2. HIGHLIGHTS FAD synthase deficiency alters mitochondrial morphology and bioenergetics; FAD synthase deficiency triggers a mitochondrial retrograde response; FAD synthase deficiency evokes nuclear signals that adapt the expression of RFVT2.

Cofactors and pathogens: Flavin mononucleotide and flavin adenine dinucleotide (FAD) biosynthesis by the FAD synthase from Brucella ovis.

The biosynthesis of the flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), cofactors used by 2% of proteins, occurs through the sequential action of two ubiquitous activities: a riboflavinkinase (RFK) that phosphorylates the riboflavin (RF) precursor to FMN, and a FMN:adenylyltransferase (FMNAT) that transforms FMN into FAD. In most mammals two different monofunctional enzymes have each of these activities, but in prokaryotes a single bifunctional enzyme, FAD synthase (FADS), holds them. Differential structural and functional traits for RFK and FMNAT catalysis between bacteria and mammals, as well as within the few bacterial FADSs so far characterized, has envisaged the potentiality of FADSs from pathogens as targets for the development of species‐specific inhibitors. Here, we particularly characterize the FADS from the ovine pathogen Brucella ovis (BoFADS), causative agent of brucellosis. We show that BoFADS has RFK activity independently of the media redox status, but its FMNAT activity (in both forward and reverse senses) only occurs under strong reducing conditions. Moreover, kinetics for flavin and adenine nucleotides binding to the RFK site show that BoFADS binds preferentially the substrates of the RFK reaction over the products and that the adenine nucleotide must bind prior to flavin entrapment. These results, together with multiple sequence alignments and phylogenetic analysis, point to variability in the less conserved regions as contributing to the species‐specific features in prokaryotic FADSs, including those from pathogens, that allow them to adopt alternative strategies in FMN and FAD biosynthesis and overall flavin homeostasis.

M. bovis BCG Moreau N-Terminal Loss Leads to a Less Stable Dodecin With Lower Flavin Binding Capacity.

Tuberculosis still remains a concerning health problem worldwide. Its etiologic agent, Mycobacterium tuberculosis, continues to be the focus of research to unravel new prophylactic and therapeutic strategies against this disease. The only vaccine in use against tuberculosis is based on the in vitro attenuated strain, M. bovis BCG. Dodecin is a dodecameric complex important for flavin homeostasis in Archea and Eubacteria, and the M. tuberculosis protein is described as thermo- and halostable. M. bovis BCG Moreau, the Brazilian vaccine strain, has a single nucleotide polymorphism in the dodecin start codon, leading to a predicted loss of seven amino acids at the protein N-terminal end. In this work we aimed to characterize the effect of this mutation in the BCG Moreau protein features. Our recombinant protein assays show that the predicted BCG homolog is less thermostable than M.tb’s but maintains its dodecamerization ability, although with a lower riboflavin-binding capacity. These data are corroborated by structural analysis after comparative modeling, showing that the predicted BCG dodecin complex has a lower interaction energy among its monomers and also a distinct electrostatic surface near the flavin binding pocket. However, western blotting assays with the native proteins were unable to detect significant differences between the BCG Moreau and M.tb orthologs, indicating that other factors may be modulating protein structure/function in the bacterial context.

Continuous and Discontinuous Approaches to Study FAD Synthesis and Degradation Catalyzed by Purified Recombinant FAD Synthase or Cellular Fractions.

Riboflavin, or vitamin B2, is the precursor of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), essential redox (and sometimes non-redox) cofactors of a large number of flavoenzymes involved in energetic metabolism, protein folding, apoptosis, chromatin remodeling, and a number of other cell regulatory processes.The cellular and subcellular steady-state concentrations of flavin cofactors, which are available for flavoprotein biogenesis and assembly, depend on carrier-mediated transport processes and on coordinated synthesizing/destroying enzymatic activities, catalyzed by enzymes whose catalytic and structural properties are still matter of investigation.Alteration of flavin homeostasis has been recently correlated to human pathological conditions, such as neuromuscular disorders and cancer, and therefore we propose here protocols useful to detect metabolic processes involved in FAD forming and destroying.Our protocols exploit the chemical-structural differences between riboflavin, FMN , and FAD , which are responsible for differences in the spectroscopic properties (mainly fluorescence) of the two cofactors(FMN and FAD); therefore, in our opinion, when applicable measurements of fluorescence changes in continuo represent the elective techniques to follow FAD synthesis and degradation. Thus, after procedures able to calibrate flavin concentrations (Subheading 3.1), we describe simple continuous and rapid procedures, based on the peculiar optical properties of free flavins, useful to determine the rate of cofactor metabolism catalyzed by either recombinant enzymes or natural enzymes present in cellular lysates/subfractions (Subheading 3.2).Fluorescence properties of free flavins can also be useful in analytical determinations of the three molecular flavin forms, based on HPLC separation, with a quite high sensitivity. Assaying at different incubation times the molecular composition of the reaction mixture is a discontinuous experimental approach to measure the rate of FAD synthesis/degradation catalyzed by cell lysates or recombinant FAD synthase (Subheading 3.3). Continuous and discontinuous approaches can, when necessary, be performed in parallel.

Flavins Act as a Critical Liaison Between Metabolic Homeostasis and Oxidative Stress in the Retina.

Derivatives of the vitamin riboflavin, FAD and FMN, are essential cofactors in a multitude of bio-energetic reactions, indispensable for lipid metabolism and also are requisites in mitigating oxidative stress. Given that a balance between all these processes contributes to the maintenance of retinal homeostasis, effective regulation of riboflavin levels in the retina is paramount. However, various genetic and dietary factors have brought to fore pathological conditions that co-occur with a suboptimal level of flavins in the retina. Our focus in this review is to, comprehensively summarize all the possible metabolic and oxidative reactions which have been implicated in various retinal pathologies and to highlight the contribution flavins may have played in these. Recent research has found a sensitive method of measuring flavins in both diseased and healthy retina, presence of a novel flavin binding protein exclusively expressed in the retina, and the presence of flavin specific transporters in both the inner and outer blood-retina barriers. In light of these exciting findings, it is even more imperative to shift our focus on how the retina regulates its flavin homeostasis and what happens when this is disrupted.

Riboflavin metabolism: role in mitochondrial function

Riboflavin, known as vitamin B2, a water-soluble vitamin, is an essential nutrient in vertebrates, hence adequate dietary intake is imperative. Riboflavin plays a role in a variety of metabolic pathways, serving primarily as an integral component of its crucial biologically active forms, the flavocoenzymes flavin adenine dinucleotide and flavin mononucleotide. These flavocoenzymes ensure the functionality of numerous flavoproteins including dehydrogenases, oxidases, monooxygenases, and reductases, which play pivotal roles in mitochondrial electron transport chain, β-oxidation of fatty acids, redox homeostasis, citric acid cycle, branched-chain amino acid catabolism, chromatin remodeling, DNA repair, protein folding, and apoptosis. Unsurprisingly, impairment of flavin homeostasis in humans has been linked to various diseases including neuromuscular and neurological disorders, abnormal fetal development, and cardiovascular diseases. This review presents an overview of riboflavin metabolism, its role in mitochondrial function, primary and secondary flavocoenzyme defects associated with mitochondrial dysfunction, and the role of riboflavin supplementation in these conditions.

Disruption of flavin homeostasis in isolated rat liver mitochondria

It has been shown that spontaneous release of non-covalent flavins (from flavoenzymes) begins after isolation of mitochondria from rat liver, which is hydrolyzed to riboflavin. This process is stopped by 1 mM EDTA in the incubation medium. In the presence of NADH, deflavinization of flavoproteins leads to formation of superoxide by at least of three processes. The first of these occurs in complex I as a result of the spontaneous release of FMN from the active center. This process is inhibited by adenosine and guanosine phosphates, as well as NAD, but amplified by nicotinamide. The second process is associated with enzymatic hydrolysis of FAD and FMN to riboflavin; it is blocked by EDTA, AMP, NA, NAD. The third process is associated with non-enzymatic hydrolysis of FAD by iron ions in matrix; it is blocked by EDTA and AMP.

Disorders of riboflavin metabolism

Riboflavin (vitamin B2), a water-soluble vitamin, is an essential nutrient in higher organisms as it is not endogenously synthesised, with requirements being met principally by dietary intake. Tissue-specific transporter proteins direct riboflavin to the intracellular machinery responsible for the biosynthesis of the flavocoenzymes flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). These flavocoenzymes play a vital role in ensuring the functionality of a multitude of flavoproteins involved in bioenergetics, redox homeostasis, DNA repair, chromatin remodelling, protein folding, apoptosis, and other physiologically relevant processes. Hence, it is not surprising that the impairment of flavin homeostasis in humans may lead to multisystem dysfunction including neuromuscular disorders, anaemia, abnormal fetal development, and cardiovascular disease. In this review, we provide an overview of riboflavin absorption, transport, and metabolism. We then focus on the clinical and biochemical features associated with biallelic FLAD1 mutations leading to FAD synthase deficiency, the only known primary defect in flavocoenzyme synthesis, in addition to providing an overview of clinical disorders associated with nutritional deficiency of riboflavin and primary defects of riboflavin transport. Finally, we give a brief overview of disorders of the cellular flavoproteome. Because riboflavin therapy may be beneficial in a number of primary or secondary disorders of the cellular flavoproteome, early recognition and prompt management of these disorders is imperative.

Flavin homeostasis in the mouse retina during aging and degeneration

Involvement of flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) in cellular homeostasis has been well established for tissues other than the retina. Here, we present an optimized method to effectively extract and quantify FAD and FMN from a single neural retina and its corresponding retinal pigment epithelium (RPE). Optimizations led to detection efficiency of 0.1 pmol for FAD and FMN while 0.01 pmol for riboflavin. Interestingly, levels of FAD and FMN in the RPE were found to be 1.7- and 12.5-fold higher than their levels in the retina, respectively. Both FAD and FMN levels in the RPE and retina gradually decline with age and preceded the age-dependent drop in the functional competence of the retina as measured by electroretinography. Further, quantifications of retinal levels of FAD and FMN in different mouse models of retinal degeneration revealed differential metabolic requirements of these two factors in relation to the rate and degree of photoreceptor degeneration. We also found twofold reductions in retinal levels of FAD and FMN in two mouse models of diabetic retinopathy. Altogether, our results suggest that retinal levels of FAD and FMN can be used as potential markers to determine state of health of the retina in general and more specifically the photoreceptors.

The Dimer-of-Trimers Assembly Prevents Catalysis at the Transferase Site of Prokaryotic FAD Synthase

Flavin mononucleotide (FMN) and flavin-adenine dinucleotide (FAD) are essential flavoprotein cofactors. A riboflavin kinase (RFK) activity catalyzes riboflavin phosphorylation to FMN, which can then be transformed into FAD by an FMN:adenylyltransferase (FMNAT) activity. Two enzymes are responsible for each one of these activities in eukaryotes, whereas prokaryotes have a single bifunctional enzyme, FAD synthase (FADS). FADS folds in two independent modules: the C-terminal with RFK activity and the N-terminal with FMNAT activity. Differences in structure and chemistry for the FMNAT catalysis among prokaryotic and eukaryotic enzymes pointed to the FMNAT activity of prokaryotic FADS as a potential antimicrobial target, making the structural model of the bacterial FMNAT module in complex with substrates relevant to understand the FADS catalytic mechanism and to the discovery of antimicrobial drugs. However, such a crystallographic complex remains elusive. Here, we have used molecular docking and molecular dynamics simulations to generate energetically stable interactions of the FMNAT module of FADS from Corynebacterium ammoniagenes with ATP/Mg2+ and FMN in both the monomeric and dimer-of-trimers assemblies reported for this protein. For the monomer, we have identified the residues that accommodate the reactive phosphates in a conformation compatible with catalysis. Interestingly, for the dimer-of-trimers conformation, we have found that the RFK module negatively influences FMN binding at the interacting FMNAT module. These results agree with calorimetric data of purified samples containing nearly 100% monomer or nearly 100% dimer-of-trimers, indicating that FMN binds to the monomer but not to the dimer-of-trimers. Such observations support regulation of flavin homeostasis by quaternary C. ammoniagenes FADS assemblies.

Flavin Storage and Sequestration by Mycobacterium tuberculosis Dodecin.

Dodecins are small flavin binding proteins occurring in archaea and bacteria. They are remarkable for binding dimers of flavins with their functional relevant aromatic isoalloxazine rings deeply covered. Bacterial dodecins are widely spread and found in a large variety of pathogens, among them Pseudomonas aeruginosa, Streptococcus pneumonia, Ralstonia solanacearum, and Mycobacterium tuberculosis ( M.tuberculosis). In this work, we seek to understand the function of dodecins from M.tuberculosis dodecin. We describe flavin binding in thermodynamic and kinetic properties and achieve mechanistic insight in dodecin function by applying spectroscopic and electrochemical methods. Intriguingly, we reveal a significant pH dependence in the affinity and specificity of flavin binding. Our data give insight in M.tuberculosis dodecin function and advance the current understanding of dodecins as flavin storage and sequestering proteins. We suggest that the dodecin in M.tuberculosis may specifically be important for flavin homeostasis during the elaborate lifestyle of this organism, which calls for the evaluation of this protein as drug target.

Evidence for Posttranslational Protein Flavinylation in the Syphilis Spirochete Treponema pallidum: Structural and Biochemical Insights from the Catalytic Core of a Periplasmic Flavin-Trafficking Protein.

ABSTRACTThe syphilis spirochete Treponema pallidum is an important human pathogen but a highly enigmatic bacterium that cannot be cultivated in vitro. T. pallidum lacks many biosynthetic pathways and therefore has evolved the capability to exploit host-derived metabolites via its periplasmic lipoprotein repertoire. We recently reported a flavin-trafficking protein in T. pallidum (Ftp_Tp; TP0796) as the first bacterial metal-dependent flavin adenine dinucleotide (FAD) pyrophosphatase that hydrolyzes FAD into AMP and flavin mononucleotide (FMN) in the spirochete’s periplasm. However, orthologs of Ftp_Tp from other bacteria appear to lack this hydrolytic activity; rather, they bind and flavinylate subunits of a cytoplasmic membrane redox system (Nqr/Rnf). To further explore this dichotomy, biochemical analyses, protein crystallography, and structure-based mutagenesis were used to show that a single amino acid change (N55Y) in Ftp_Tp converts it from an Mg2+-dependent FAD pyrophosphatase to an FAD-binding protein. We also demonstrated that Ftp_Tp has a second enzymatic activity (Mg2+-FMN transferase); it flavinylates protein(s) covalently with FMN on a threonine side chain of an appropriate sequence motif using FAD as the substrate. Moreover, mutation of a metal-binding residue (D284A) eliminates Ftp_Tp’s dual activities, thereby underscoring the role of Mg2+ in the enzyme-catalyzed reactions. The posttranslational flavinylation activity that can target a periplasmic lipoprotein (TP0171) has not previously been described. The observed activities reveal the catalytic flexibility of a treponemal protein to perform multiple functions. Together, these findings imply mechanisms by which a dynamic pool of flavin cofactor is maintained and how flavoproteins are generated by Ftp_Tp locally in the T. pallidum periplasm.

Bacterial flavin homeostasis: the role of an FAD pyrophosphatase/FMN transferase

Treponema pallidum, an obligate parasite of humans and the causative agent of syphilis, has evolved the capacity to exploit host-derived metabolites for its survival. Flavin-containing compounds are essential cofactors that are required for metabolic processes in all living organisms, and riboflavin is a direct precursor of the cofactors FMN and FAD. Unlike many pathogenic bacteria, Treponema pallidum cannot synthesize riboflavin; we recently described a flavin-uptake mechanism composed of an ABC-type transporter [1]. However, there is a paucity of information about flavin utilization in bacterial periplasms. We have identified the TP0796 lipoprotein as a previously uncharacterized Mg2+-dependent FAD pyrophosphatase/FMN transferase within the ApbE superfamily [2,3]. Biochemical and structural investigations revealed that the enzyme has a unique bimetal Mg2+ catalytic center. Furthermore, the pyrophosphatase activity is product-inhibited by AMP, indicating a possible role for this molecule in modulating FMN and FAD levels in the treponemal periplasm. The ApbE superfamily was previously thought to be involved in thiamine biosynthesis, but our characterization of TP0796 prompts a renaming of this superfamily as a periplasmic flavin-trafficking protein (Ftp). Treponemal Ftp (Ftp_Tp) is the first structurally and biochemically characterized metal-dependent FAD pyrophosphatase/FMN transferase in bacteria. We have shown in vitro and in vivo that Ftps from several types of pathogenic bacteria are capable of flavinylating proteins through covalent attachment of FMN via a phosphoester bond to threonine residues of an appropriate sequence signature. Progress on the structural characterization of a product of this post-translational modification will be presented. This new paradigm for a bacterial flavin utilization pathway may prove to be useful for future inhibitor design.

Alteration of ROS homeostasis and decreased lifespan in S. cerevisiae elicited by deletion of the mitochondrial translocator FLX1.

This paper deals with the control exerted by the mitochondrial translocator FLX1, which catalyzes the movement of the redox cofactor FAD across the mitochondrial membrane, on the efficiency of ATP production, ROS homeostasis, and lifespan of S. cerevisiae. The deletion of the FLX1 gene resulted in respiration-deficient and small-colony phenotype accompanied by a significant ATP shortage and ROS unbalance in glycerol-grown cells. Moreover, the flx1Δ strain showed H2O2 hypersensitivity and decreased lifespan. The impaired biochemical phenotype found in the flx1Δ strain might be justified by an altered expression of the flavoprotein subunit of succinate dehydrogenase, a key enzyme in bioenergetics and cell regulation. A search for possible cis-acting consensus motifs in the regulatory region upstream SDH1-ORF revealed a dozen of upstream motifs that might respond to induced metabolic changes by altering the expression of Flx1p. Among these motifs, two are present in the regulatory region of genes encoding proteins involved in flavin homeostasis. This is the first evidence that the mitochondrial flavin cofactor status is involved in controlling the lifespan of yeasts, maybe by changing the cellular succinate level. This is not the only case in which the homeostasis of redox cofactors underlies complex phenotypical behaviours, as lifespan in yeasts.

FAD Synthesis and Degradation in the Nucleus Create a Local Flavin Cofactor Pool

FAD is a redox cofactor ensuring the activity of many flavoenzymes mainly located in mitochondria but also relevant for nuclear redox activities. The last enzyme in the metabolic pathway producing FAD is FAD synthase (EC 2.7.7.2), a protein known to be localized both in cytosol and in mitochondria. FAD degradation to riboflavin occurs via still poorly characterized enzymes, possibly belonging to the NUDIX hydrolase family. By confocal microscopy and immunoblotting experiments, we demonstrate here the existence of FAD synthase in the nucleus of different experimental rat models. HPLC experiments demonstrated that isolated rat liver nuclei contain ∼300 pmol of FAD·mg(-1) protein, which was mainly protein-bound FAD. A mean FAD synthesis rate of 18.1 pmol·min(-1)·mg(-1) protein was estimated by both HPLC and continuous coupled enzymatic spectrophotometric assays. Rat liver nuclei were also shown to be endowed with a FAD pyrophosphatase that hydrolyzes FAD with an optimum at alkaline pH and is significantly inhibited by adenylate-containing nucleotides. The coordinate activity of these FAD forming and degrading enzymes provides a potential mechanism by which a dynamic pool of flavin cofactor is created in the nucleus. These data, which significantly add to the biochemical comprehension of flavin metabolism and its subcellular compartmentation, may also provide the basis for a more detailed comprehension of the role of flavin homeostasis in biologically and clinically relevant epigenetic events.

An Arabidopsis FAD Pyrophosphohydrolase, AtNUDX23, is Involved in Flavin Homeostasis

Although flavins, riboflavin (RF), FMN and FAD, are essential for primary and secondary metabolism in plants, the metabolic regulation of flavins is still largely unknown. Recently, we found that an Arabidopsis Nudix hydrolase, AtNUDX23, has FAD pyrophosphohydrolase activity and is distributed in plastids. Levels of RF and FAD but not FMN in Arabidopsis leaves significantly increased under continuous light and decreased in the dark. The transcript levels of AtNUDX23 as well as genes involved in flavin metabolism (AtFADS, AtRibF1, AtRibF2, AtFMN/FHy, LS and AtRibA) significantly increased under continuous light. The pyrophosphohydrolase activity toward FAD was enhanced in AtNUDX23-overexpressing (OX-NUDX23) plants and reduced in AtNUDX23-suppressed (KD-nudx23) plants, compared with the control plants. Interestingly intracellular levels of RF, FMN and FAD significantly decreased in not only OX-NUDX23 but also KD-nudx23 plants. The transcript levels of the flavin metabolic genes also decreased in both plants. Similarly, the increase in intracellular levels on treatment with flavins caused a reduction in the transcript levels of genes involved in flavin metabolism. These results suggest that negative feedback regulation of the metabolism of flavins through the hydrolysis of FAD by AtNUDX23 in plastids is involved in flavin homeostasis in plant cells.