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

Leone, Piero; Tolomeo, Maria; Barile, Maria

doi:10.1007/978-1-0716-1286-6_7

Cited by 8 publications

(8 citation statements)

References 61 publications

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“…As soon as the coenzyme is finally produced and transported, the likelihood of FMN binding to complex I NDUFV1 subunit would be regulated, among other factors, by the availability of FMN in the mitochondrial matrix and by the competition with other FMN binding enzymes. An exact quantification of matrix flavin coenzymes is challenging for the tendency that these compounds have to be degraded during sample handling 119,120 ; nevertheless, various estimations of FMN matrix concentration can be found in the literature, albeit often expressed in different units of measurement (mol/g of protein, g/g, molarity). Based upon the quantifications made in a variety of biological samples, FAD seems to be the main flavin compound present in the cell, meanwhile FMN occurs to be less abundant; riboflavin is often found only in traces or completely absent 111,121–123 .…”

Section: Complex I and The Mitochondrial Flavin Cofactor Homeostasismentioning

confidence: 99%

The ins and outs of the flavin mononucleotide cofactor of respiratory complex I

Curtabbi

Enríquez

2022

IUBMB Life

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The flavin mononucleotide (FMN) cofactor of respiratory complex I occupies a key position in the electron transport chain. Here, the electrons coming from NADH start the sequence of oxidoreduction reactions, which drives the generation of the proton-motive force necessary for ATP synthesis. The overall architecture and the general catalytic proprieties of the FMN site are mostly well established. However, several aspects regarding the complex I flavin cofactor are still unknown. For example, the flavin binding to the N-module, the NADH-oxidizing portion of complex I, lacks a molecular description. The dissociation of FMN from the enzyme is beginning to emerge as an important regulatory mechanism of complex I activity and ROS production. Finally, how mitochondria import and metabolize FMN is still uncertain. This review summarizes the current knowledge on complex I flavin cofactor and discusses the open questions for future research.

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Section: Complex I and The Mitochondrial Flavin Cofactor Homeostasismentioning

confidence: 99%

The ins and outs of the flavin mononucleotide cofactor of respiratory complex I

Curtabbi

Enríquez

2022

IUBMB Life

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“…For flavin content analysis, 120 μl of either fresh or defrozen plasma were added with 2 mM NaF (as phosphatase inhibitor) and then deproteinized by treatment with 10% perchloric acid as described in the studies by Giancaspero and colleagues. 24 , 25 Rf, FMN, and FAD were measured in neutralized perchloric acid extracts of plasma samples by HPLC as described in the study by Leone et al 26 …”

Section: Methodsmentioning

confidence: 99%

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

et al. 2022

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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

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“…The reaction mixture was assembled in 20 μL volume with 3 μM α- 32 P-ATP, 50 μM ATP, 50 μM FMN and 5 μM human FADS in a final buffer of 50 mM Tris-HCl pH 7.4, 100 mM NaCl, 12 mM MgCl 2 and 1 mM DTT 3 , with the addition of 0.1 Units (1 μL) inorganic pyrophosphatase in the proper conditions.…”

Section: Methodsmentioning

confidence: 99%

“…When attempting to test PPase with FAD synthetase (FADS) to recreate 32 P-FAD from FMN and 32 P-ATP, we observed that addition of PPase created an aberrant 32 P product that migrated to ~10.5 cm, and could represent AMP, ADP or cyclic-FMN (Figure S3). 21 The appearance of a PPase-dependent aberrant product indicated that its utility may depend on the specific reaction.…”

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confidence: 99%

Improved Yield for the Enzymatic Synthesis of Radiolabeled Nicotinamide Adenine Dinucleotide

Eller

Goyal

Cambronne

2022

Preprint

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Labeled β-nicotinamide adenine dinucleotide (NAD) analogs have been critical for uncovering new biochemical connections and quantitating enzymatic activity. They function as tracers for enzymology, flux analyses, and in situ measurements. Nevertheless, there is limited availability of specific types of analogs, especially radiolabeled NAD analogs. Here, we describe an improved enzymatic synthesis reaction for 32P- NAD+ with a yield of 98% ± 1%, using lowered concentrations of reactants and standard equipment. This represents the highest reported yield for the enzymatic synthesis of NAD+ to date. With the high yield we were able to directly use the re-action product to generate derivatives, such as 32P-NADP. The high-yield enzymatic synthesis is versatile for a broad variety of labels and NAD derivatives. Its advantages include lowered concentrations of reactants, providing sufficient amounts of product for downstream applications, and minimizing intermediate purification steps.

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

Cited by 8 publications

References 61 publications

The ins and outs of the flavin mononucleotide cofactor of respiratory complex I

The ins and outs of the flavin mononucleotide cofactor of respiratory complex I

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

Improved Yield for the Enzymatic Synthesis of Radiolabeled Nicotinamide Adenine Dinucleotide

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