Structural insights into the bifunctional enzyme human FAD synthase.

Human flavin adenine dinucleotide synthase (hFADS) is a bifunctional, multi-domain enzyme that exhibits both flavin mononucleotide adenylyltransferase and pyrophosphatase activities. Here we report the crystal structure of full-length hFADS2 and its C-terminal PAPS domain in complex with flavin adenine dinucleotide (FAD), and dissect the structural determinants underlying the contribution of each individual domain, within isoforms 1 and 2, to each of the two enzymatic activities. Structural and functional characterization performed on complete or truncated constructs confirmed that the C-terminal domain tightly binds FAD and catalyzes its synthesis, while the combination of the N-terminal molybdopterin-binding and KH domains is the minimal essential substructure required for the hydrolysis of FAD and other ADP-containing dinucleotides. hFADS2 associates in a stable C2-symmetric dimer, in which the packing of the KH domain of one protomer against the N-terminal domain of the other creates the adenosine-specific active site responsible for the hydrolytic activity.

Micronutrient Deficiency in Inherited Metabolic Disorders Requiring Diet Regimen: A Brief Critical Review.

Many inherited metabolic disorders (IMDs), including disorders of amino acid, fatty acid, and carbohydrate metabolism, are treated with a dietary reduction or exclusion of certain macronutrients, putting one at risk of a reduced intake of micronutrients. In this review, we aim to provide available evidence on the most common micronutrient deficits related to specific dietary approaches and on the management of their deficiency, in the meanwhile discussing the main critical points of each nutritional supplementation. The emerging concepts are that a great heterogeneity in clinical practice exists, as well as no univocal evidence on the most common micronutrient abnormalities. In phenylketonuria, for example, micronutrients are recommended to be supplemented through protein substitutes; however, not all formulas are equally supplemented and some of them are not added with micronutrients. Data on pyridoxine and riboflavin status in these patients are particularly scarce. In long-chain fatty acid oxidation disorders, no specific recommendations on micronutrient supplementation are available. Regarding carbohydrate metabolism disorders, the difficult-to-ascertain sugar content in supplementation formulas is still a matter of concern. A ketogenic diet may predispose one to both oligoelement deficits and their overload, and therefore deserves specific formulations. In conclusion, our overview points out the lack of unanimous approaches to micronutrient deficiencies, the need for specific formulations for IMDs, and the necessity of high-quality studies, particularly for some under-investigated deficits.

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.

Over-Production of the Human SLC7A10 in E. coli and Functional Assay in Proteoliposomes.

The human SLC7A10 transporter, also known as ASC-1, catalyzes the transport of some neutral amino acids. It is expressed in astrocytes, neurons, and adipose tissues, playing roles in learning, memory processes, and lipid metabolism, thus being involved in neurological and metabolic pathologies. Structure/function studies on this transporter are still in their infancy. In this study, we present a methodology for producing the recombinant human transporter in E. coli. Its transport function was assayed in proteoliposomes following the uptake of radiolabeled L-serine. After the testing of several growth conditions, the hASC-1 transporter was successfully expressed in BL21(DE3) codon plus RIL in the presence of 0.5% glucose and induced with 0.05 mM IPTG. After solubilization with C12E8 and cholesteryl hemisuccinate and purification by Ni-chelating chromatography, hASC-1 was reconstituted in proteoliposomes. In this experimental system it was able to catalyze an Na+-independent homologous antiport of L-serine. A Km for L-serine transport of 0.24 mM was measured. The experimental model developed in this work represents a reproducible system for the transport assay of hASC-1 in the absence of interferences. This tool will be useful to unveil unknown transport properties of hASC-1 and for testing ligands with possible application in human pharmacology.

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.HIGHLIGHTSFAD 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.

Combined isobutyryl-CoA and multiple acyl-CoA dehydrogenase deficiency in a boy with altered riboflavin homeostasis.

In this report, we describe the case of an 11-year-old boy, who came to our attention for myalgia and muscle weakness, associated with inappetence and vomiting. Hypertransaminasemia was also noted, with ultrasound evidence of hepatomegaly. Biochemical investigations revealed acylcarnitine and organic acid profiles resembling those seen in MADD, that is, multiple acyl-CoA dehydrogenase deficiencies (OMIM #231680) a rare inherited disorder of fatty acids, amino acids, and choline metabolism. The patient carried a single pathogenetic variant in the ETFDH gene (c.524G>A, p.Arg175His) and no pathogenetic variant in the riboflavin (Rf) homeostasis related genes (SLC52A1, SLC52A2, SLC52A3, SLC25A32, FLAD1). Instead, compound heterozygosity was found in the ACAD8 gene (c.512C>G, p.Ser171Cys; c.822C>A, p.Asn274Lys), coding for isobutyryl-CoA dehydrogenase (IBD), whose pathogenic variants are associated to IBD deficiency (OMIM #611283), a rare autosomal recessive disorder of valine catabolism. The c.822C>A was never previously described in a patient. Subsequent further analyses of Rf homeostasis showed reduced levels of flavins in plasma and altered FAD-dependent enzymatic activities in erythrocytes, as well as a significant reduction in the level of the plasma membrane Rf transporter 2 in erythrocytes. The observed Rf/flavin scarcity in this patient, possibly associated with a decreased ETF:QO efficiency might be responsible for the observed MADD-like phenotype. The patient's clinical picture improved after supplementation of Rf, l-carnitine, Coenzyme Q10, and also 3OH-butyrate. This report demonstrates that, even in the absence of genetic defects in genes involved in Rf homeostasis, further targeted molecular analysis may reveal secondary and possibly treatable biochemical alterations in this pattern.

Impact of natural mutations on the riboflavin transporter 2 and their relevance to human riboflavin transporter deficiency 2.

Riboflavin transporter deficiency 2 (RTD2) is a rare neurological disorder caused by mutations in the Solute carrier family 52 member 2 (Slc52a2) gene encoding human riboflavin transporter 2 (RFVT2). This transporter is ubiquitously expressed and mediates tissue distribution of riboflavin, a water-soluble vitamin that, after conversion into FMN and FAD, plays pivotal roles in carbohydrate, protein, and lipid metabolism. The 3D structure of RFVT2 has been constructed by homology modeling using three different templates that are equilibrative nucleoside transporter 1 (ENT1), Fucose: proton symporter, and glucose transporter type 5 (GLUT5). The structure has been validated by several approaches. All known point mutations of RFVT2, associated with RTD2, have been localized in the protein 3D model. Six of these mutations have been introduced in the recombinant protein for functional characterization. The mutants W31S, S52F, S128L, L312P, C325G, and M423V have been expressed in E. coli, purified, and reconstituted into proteoliposomes for transport assay. All the mutants showed impairment of function. The Km for riboflavin of the mutants increased from about 3 to 9 times with respect to that of WT, whereas Vmax was only marginally affected. This agrees with the improved outcome of most RTD2 patients after administration of high doses of riboflavin.

Mimicking human riboflavin responsive neuromuscular disorders by silencing flad-1 gene in C. elegans: Alteration of vitamin transport and cholinergic transmission.

Riboflavin (Rf), or vitamin B2, is the precursor of FMN and FAD, redox cofactors of several dehydrogenases involved in energy metabolism, redox balance and other cell regulatory processes. FAD synthase, coded by FLAD1 gene in humans, is the last enzyme in the pathway converting Rf into FAD. Mutations in FLAD1 gene are responsible for neuromuscular disorders, in some cases treatable with Rf. In order to mimic these disorders, the Caenorhabditis elegans (C. elegans) gene orthologue of FLAD1 (flad-1) was silenced in a model strain hypersensitive to RNA interference in nervous system. Silencing flad-1 resulted in a significant decrease in total flavin content, paralleled by a decrease in the level of the FAD-dependent ETFDH protein and by a secondary transcriptional down-regulation of the Rf transporter 1 (rft-1) possibly responsible for the total flavin content decrease. Conversely an increased ETFDH mRNA content was found. These biochemical changes were accompanied by significant phenotypical changes, including impairments of fertility and locomotion due to altered cholinergic transmission, as indicated by the increased sensitivity to aldicarb. A proposal is made that neuronal acetylcholine production/release is affected by alteration of Rf homeostasis. Rf supplementation restored flavin content, increased rft-1 transcript levels and eliminated locomotion defects. In this aspect, C. elegans could provide a low-cost animal model to elucidate the molecular rationale for Rf therapy in human Rf responsive neuromuscular disorders and to screen other molecules with therapeutic potential.

Subcellular Localization of Fad1p in Saccharomyces cerevisiae: A Choice at Post-Transcriptional Level?

FAD synthase is the last enzyme in the pathway that converts riboflavin into FAD. In Saccharomyces cerevisiae, the gene encoding for FAD synthase is FAD1, from which a sole protein product (Fad1p) is expected to be generated. In this work, we showed that a natural Fad1p exists in yeast mitochondria and that, in its recombinant form, the protein is able, per se, to both enter mitochondria and to be destined to cytosol. Thus, we propose that FAD1 generates two echoforms-that is, two identical proteins addressed to different subcellular compartments. To shed light on the mechanism underlying the subcellular destination of Fad1p, the 3' region of FAD1 mRNA was analyzed by 3'RACE experiments, which revealed the existence of (at least) two FAD1 transcripts with different 3'UTRs, the short one being 128 bp and the long one being 759 bp. Bioinformatic analysis on these 3'UTRs allowed us to predict the existence of a cis-acting mitochondrial localization motif, present in both the transcripts and, presumably, involved in protein targeting based on the 3'UTR context. Here, we propose that the long FAD1 transcript might be responsible for the generation of mitochondrial Fad1p echoform.

Functional Study of the Human Riboflavin Transporter 2 Using Proteoliposomes System.

Riboflavin is essential for cell viability. The biologically active forms of riboflavin, FMN and FAD, participate in many biochemical redox reactions including the metabolism of carbohydrates, amino acids, and lipids. Differently from bacteria, fungi, and plants which synthesize riboflavin, higher organisms have lost the ability to synthesize the vitamin and must absorb it from food and intestinal microflora production. The riboflavin flux through cell membranes occurs via specific transporters belonging to the SLC52 family. Three members of this family have been identified so far which show poor homology with the riboflavin transporters of Saccharomyces cerevisiae or bacteria. Alterations of RFVTs are causative of severe diseases. Indeed, under pathological stress, humans are susceptible of developing riboflavin deficiency. Such a deficiency in pregnancy induces fetus abnormalities, and has been indicated as a risk factor for anemia, cancer, cardiovascular diseases, and neurodegeneration. Moreover, inherited diseases are also of interest; the most well-described is the Brown-Vialetto-van Laere syndrome, a rare neurological disorder characterized by infancy onset sensorineural deafness and pontobulbar palsy. Numerous polymorphisms of Slc52a2 and Slc52a3 genes associated with this syndrome have been discovered. In spite of their important metabolic role and their relevance to human health, the riboflavin transporters are still poorly characterized. Bacterial overexpression, purification, and protein reconstitution in liposomes represent an up-to-date methodology for obtaining functional data information. The methodology for reconstituting the RFVT2 into proteoliposomes and performing transport assay is described. These methods will be suitable for investigating the functional defects of the variants of RFVTs associated with human pathologies.

Purification of Recombinant Human 6His-FAD Synthase (Isoform 2) and Quantitation of FAD/Protein Monomer Ratio by UV-Vis Spectra.

Here we describe a protocol for a one-step purification of a soluble form of human FAD synthase (isoform 2; hFADS2), overexpressed as a 6-His-tagged fusion protein in Escherichia coli, with a yield of about 15 mg from 1 L of transformed bacterial culture.Following a desalting procedure, the protein is obtained in its FAD-bound form (about 0.8 molecules of FAD per 1 protein monomer). A simple method is also proposed here, for the rapid estimation of the [FAD ]/[protein monomer] ratio, starting from the typical flavoprotein spectrum of the purified protein fraction.The procedure described gives the protein at a quite high grade of purity (about 95%) and in its bifunctional (2.7.7.2/3.6.1.18) enzymatically active form, useful for further kinetical and molecular characterization.

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.

Alteration of Flavin Cofactor Homeostasis in Human Neuromuscular Pathologies.

The aim of this short review chapter is to provide a brief summary of the relevance of riboflavin (Rf or vitamin B2) and its derived cofactors flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) for human neuromuscular bioenergetics.Therefore, as a completion of this book we would like to summarize what kind of human pathologies could derive from genetic disturbances of Rf transport, flavin cofactor synthesis and delivery to nascent apoflavoproteins, as well as by alteration of vitamin recycling during protein turnover.

Development of Novel Experimental Models to Study Flavoproteome Alterations in Human Neuromuscular Diseases: The Effect of Rf Therapy.

Inborn errors of Riboflavin (Rf) transport and metabolism have been recently related to severe human neuromuscular disorders, as resulting in profound alteration of human flavoproteome and, therefore, of cellular bioenergetics. This explains why the interest in studying the "flavin world", a topic which has not been intensively investigated before, has increased much over the last few years. This also prompts basic questions concerning how Rf transporters and FAD (flavin adenine dinucleotide) -forming enzymes work in humans, and how they can create a coordinated network ensuring the maintenance of intracellular flavoproteome. The concept of a coordinated cellular "flavin network", introduced long ago studying humans suffering for Multiple Acyl-CoA Dehydrogenase Deficiency (MADD), has been, later on, addressed in model organisms and more recently in cell models. In the frame of the underlying relevance of a correct supply of Rf in humans and of a better understanding of the molecular rationale of Rf therapy in patients, this review wants to deal with theories and existing experimental models in the aim to potentiate possible therapeutic interventions in Rf-related neuromuscular diseases.

Reconstitution in Proteoliposomes of the Recombinant Human Riboflavin Transporter 2 (SLC52A2) Overexpressed in E. coli.

BACKGROUND: the SLC52A2 gene encodes for the riboflavin transporter 2 (RFVT2). This transporter is ubiquitously expressed. It mediates the transport of Riboflavin across cell membranes. Riboflavin plays a crucial role in cells since its biologically active forms, FMN and FAD, are essential for the metabolism of carbohydrates, amino acids, and lipids. Mutation of the Riboflavin transporters is a risk factor for anemia, cancer, cardiovascular disease, neurodegeneration. Inborn mutations of SLC52A2 are associated with Brown-Vialetto-van Laere syndrome, a rare neurological disorder characterized by infancy onset. In spite of the important metabolic and physio/pathological role of this transporter few data are available on its function and regulation. METHODS: the human recombinant RFVT2 has been overexpressed in E. coli, purified and reconstituted into proteoliposomes in order to characterize its activity following the [3H]Riboflavin transport. RESULTS: the recombinant hRFVT2 showed a Km of 0.26 ± 0.07 µM and was inhibited by lumiflavin, FMN and Mg2+. The Riboflavin uptake was also regulated by Ca2+. The native protein extracted from fibroblast and reconstituted in proteoliposomes also showed inhibition by FMN and lumiflavin. CONCLUSIONS: proteoliposomes represent a suitable model to assay the RFVT2 function. It will be useful for screening the mutation of RFVT2.

The Expression of Riboflavin Transporters in Human Colorectal Cancer.

BACKGROUND/AIM: Riboflavin transport in enterocytes is mediated by three translocators: RFVT3 located on the apical membrane, and RFVT1 and RFVT2 on the basolateral membrane. The aim of this study was to investigate whether the expression levels of RFVTs are altered in human colorectal cancer (CRC). MATERIALS AND METHODS: In human colon adenocarcinoma cell lines (CaCo2, DLD-1, HT-29) and in tissues of patients with CRC, gene and protein expression levels were evaluated by real time-polymerase chain reaction and western blotting. Intracellular flavin content was determined by high-performance liquid chromatography. RESULTS: RFVT3 and RFVT2 gene and protein expression levels were higher in DLD-1 and HT-29 compared to Caco2 cells. In HT-29 cells, the RFVT1 protein level was drastically lower. These differences are presumably responsible for the higher total flavin content in DLD-1 and HT-29 cells. In tumor tissues of patients with CRC, RFVT1 content was reduced at both protein and mRNA levels compared to normal mucosa. RFVT3 and RFVT2 gene expression levels were increased, while protein expression was reduced, with a small reduction in riboflavin amount. CONCLUSION: This study provides first evidence that transcription/translation of RFVTs are profoundly altered in CRC.

Bacterial Production, Characterization and Protein Modeling of a Novel Monofuctional Isoform of FAD Synthase in Humans: An Emergency Protein?

FAD synthase (FADS, EC 2.7.7.2) is the last essential enzyme involved in the pathway of biosynthesis of Flavin cofactors starting from Riboflavin (Rf). Alternative splicing of the human FLAD1 gene generates different isoforms of the enzyme FAD synthase. Besides the well characterized isoform 1 and 2, other FADS isoforms with different catalytic domains have been detected, which are splice variants. We report the characterization of one of these novel isoforms, a 320 amino acid protein, consisting of the sole C-terminal 3'-phosphoadenosine 5'-phosphosulfate (PAPS) reductase domain (named FADS6). This isoform has been previously detected in Riboflavin-Responsive (RR-MADD) and Non-responsive Multiple Acyl-CoA Dehydrogenase Deficiency (MADD) patients with frameshift mutations of FLAD1 gene. To functionally characterize the hFADS6, it has been over-expressed in Escherichia coli and purified with a yield of 25 mg·L-1 of cell culture. The protein has a monomeric form, it binds FAD and is able to catalyze FAD synthesis (kcat about 2.8 min-1), as well as FAD pyrophosphorolysis in a strictly Mg2+-dependent manner. The synthesis of FAD is inhibited by HgCl2. The enzyme lacks the ability to hydrolyze FAD. It behaves similarly to PAPS. Combining threading and ab-initio strategy a 3D structural model for such isoform has been built. The relevance to human physio-pathology of this FADS isoform is discussed.