- Human riboflavin kinase: Species-specific traits in the biosynthesis of the FMN cofactor
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Human riboflavin kinase (HsRFK) catalyzes vitamin B2 (riboflavin) phosphorylation to flavin mononucleotide (FMN), obligatory step in flavin cofactor synthesis. HsRFK expression is related to protection from oxidative stress, amyloid-β toxicity, and some malignant cancers progression. Its downregulation alters expression profiles of clock-controlled metabolic-genes and destroys flavins protection on stroke treatments, while its activity reduction links to protein-energy malnutrition and thyroid hormones decrease. We explored specific features of the mechanisms underlying the regulation of HsRFK activity, showing that both reaction products regulate it through competitive inhibition. Fast-kinetic studies show that despite HsRFK binds faster and preferably the reaction substrates, the complex holding both products is kinetically most stable. An intricate ligand binding landscape with all combinations of substrates/products competing with the catalytic complex and exhibiting moderate cooperativity is also presented. These data might contribute to better understanding the molecular bases of pathologies coursing with aberrant HsRFK availability, and envisage that interaction with its client-apoproteins might favor FMN release. Finally, HsRFK parameters differ from those of the so far evaluated bacterial counterparts, reinforcing the idea of species-specific mechanisms in RFK catalysis. These observations support HsRFK as potential therapeutic target because of its key functions, while also envisage bacterial RFK modules as potential antimicrobial targets.
- Anoz-Carbonell, Ernesto,Rivero, Maribel,Polo, Victor,Velázquez-Campoy, Adrián,Medina, Milagros
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- Spectroscopic study of molecular associations between FMN and β-carbolines
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The spectrophotometric and thermodynamic properties of molecular complexes of flavin mononucleotide (FMN) (riboflavin 5'-phosphate) with some β-carboline derivatives have been investigated in aqueous solution.The molecular associations have been examined by means of electronic absorption spectra, since in each a new charge-transfer band has been located, and also the variation of the fluorescenece emission of FMN on the solutions has been observed.The formation constants for the molecular complexes were determined from absorption data using the Foster-Hammick-Wardley method.The quenching phenomenon observed in FMN fluorescence is related to the concentration of the β-carboline derivatives, allowing the calculation of the quenching constants for FMN-β-carboline complexes.Thermodynamic parameters have been determined from the values of association constants for the molecular complexes at various temperatures.The influence of substituents in the β-carboline molecule on the stability of the complexes formed was also investigated.
- Codoner, Armando,Medina, Piedad,Jover, Enrique,Sanchez, Jesus A.
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- Catalytic role of a conserved cysteine residue in the desulfonation reaction by the alkanesulfonate monooxygenase enzyme
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Detailed kinetic studies were performed in order to determine the role of the single cysteine residue in the desulfonation reaction catalyzed by SsuD. Mutation of the conserved cysteine at position 54 in SsuD to either serine or alanine had little effect on FMNH2 binding. The kcat/Km value for the C54S SsuD variant increased 3-fold, whereas the kcat/Km value for C54A SsuD decreased 6-fold relative to wild-type SsuD. An initial fast phase was observed in kinetic traces obtained for the oxidation of flavin at 370 nm when FMNH2 was mixed against C54S SsuD (kobs, 111 s- 1) in oxygenated buffer that was 10-fold faster than wild-type SsuD (kobs, 12.9 s- 1). However, there was no initial fast phase observed in similar kinetic traces obtained for C54A SsuD. This initial fast phase was previously assigned to the formation of the C4a-(hydro)peroxyflavin in studies with wild-type SsuD. There was no evidence for the formation of the C4a-(hydro)peroxyflavin with either SsuD variant when octanesulfonate was included in rapid reaction kinetic studies, even at low octanesulfonate concentrations. The absence of any C4a-(hydro)peroxyflavin accumulation correlates with the increased catalytic activity of C54S SsuD. These results suggest that the conservative serine substitution is able to effectively take the place of cysteine in catalysis. Conversely, decreased accumulation of the C4a-(hydro)peroxyflavin intermediate with the C54A SsuD variant may be due to decreased activity. The data described suggest that Cys54 in SsuD may be either directly or indirectly involved in stabilizing the C4a-(hydro)peroxyflavin intermediate formed during catalysis through hydrogen bonding interactions.
- Carpenter, Russell A.,Zhan, Xuanzhi,Ellis, Holly R.
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- Photochemically induced dynamic nuclear polarization in a C450A mutant of the LOV2 domain of the Avena sativa blue-light receptor phototropin
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Phototropin is a blue-light receptor involved in the phototropic response of higher plants. The photoreceptor comprises a protein kinase domain and two structurally similar flavin-mononucleotide (FMN) binding domains designated LOV1 and LOV2. Blue-light irradiation of recombinant LOV2 domains induces the formation of a covalent adduct of the thiol group of a functional cysteine in the cofactor-binding pocket to C(4a) of the FMN. Cysteine-to-alanine mutants of LOV domains are unable to form that adduct but generate an FMN radical upon illumination. The recombinant C450A mutant of the LOV2 domain of Avena sativa phototropin was reconstituted with universally and site-selectively 13C-labeled FMN and the 13C NMR signals were unequivocally assigned. 13C NMR spectra were acquired in darkness and under blue-light irradiation. The chemical shifts and the coupling patterns of the signals were not affected by irradiation. However, under blue-light exposure, exceptionally strong nuclear-spin polarization was developed in the resonances belonging to certain carbons of the FMN's isoalloxazine moiety. An enhancement of the NMR absorption was observed for the signals of C(5a), C(7), and C(9). NMR lines in emission were detected for the signals belonging to C(2), C(4), C(4a), C(6), C(8), and C(9a). The signal of C(10a) remained in absorption but was slightly attenuated. In contrast, the intensities of the NMR signals belonging to the carbons of the ribityl side chain of FMN were not affected by light. The observation of spin-polarized 13C-nuclei in the NMR spectra of the mutant LOV2 domain is clear evidence for radical-pair intermediates in the reaction steps following optical sample excitation.
- Richter, Gerald,Weber, Stefan,Roemisch, Werner,Bacher, Adelbert,Fischer, Markus,Eisenreich, Wolfgang
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- Site-Selective Synthesis of 15N- and 13C-Enriched Flavin Mononucleotide Coenzyme Isotopologues
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Flavin mononucleotide (FMN) is a coenzyme for numerous proteins involved in key cellular and physiological processes. Isotopically labeled flavin is a powerful tool for studying the structure and mechanism of flavoenzyme-catalyzed reactions by a variety of techniques, including NMR, IR, Raman, and mass spectrometry. In this report, we describe the preparation of labeled FMN isotopologues enriched with 15N and 13C isotopes at various sites in the pyrazine and pyrimidine rings of the isoalloxazine core of the cofactor from readily available precursors by a five-step chemo-enzymatic synthesis.
- Neti, Syam Sundar,Poulter, C. Dale
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p. 5087 - 5092
(2016/07/06)
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- Preparation of riboflavin 5'-phosphate (FMN) and its sodium salt in a lactone
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An improved process for preparing riboflavin 5'-phosphate (FMN) or its monosodium salt by reacting riboflavin with excess phosphorus oxychloride in a solvent, hydrolyzing of the resulting reaction mixture and, if desired, partially neutralizing with aqueous sodium hydroxide solution, comprises reacting the riboflavin with from 1.2 to 3 moles of phosphorus oxychloride per mole of riboflavin in a suitable lactone, especially γ-butyrolactone, as solvent. The process is particularly advantageous when the reaction mixture is hydrolyzed at from 80° to 90° C. continuously or batchwise.
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