The cofactors FMN and FAD participate in numerous processes in all organisms, including mitochondrial electron transport, photosynthesis, fatty-acid oxidation, and metabolism of vitamin B6, vitamin B12 and folates . While monofunctional riboflavin kinase is found in eukaryotes, some bacteria have a bifunctional enzyme that exhibits both this activity and that of EC 2.7.7.2, FMN adenylyltransferase . A divalent metal cation is required for activity (with different species preferring Mg2+, Mn2+ or Zn2+). In Bacillus subtilis, ATP can be replaced by other phosphate donors but with decreasing enzyme activity in the order ATP > dATP > CTP > UTP .
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SYSTEMATIC NAME
IUBMB Comments
ATP:riboflavin 5'-phosphotransferase
The cofactors FMN and FAD participate in numerous processes in all organisms, including mitochondrial electron transport, photosynthesis, fatty-acid oxidation, and metabolism of vitamin B6, vitamin B12 and folates [5]. While monofunctional riboflavin kinase is found in eukaryotes, some bacteria have a bifunctional enzyme that exhibits both this activity and that of EC 2.7.7.2, FMN adenylyltransferase [5]. A divalent metal cation is required for activity (with different species preferring Mg2+, Mn2+ or Zn2+). In Bacillus subtilis, ATP can be replaced by other phosphate donors but with decreasing enzyme activity in the order ATP > dATP > CTP > UTP [6].
substrate binding structure analysis using crystal structures of substrate-bound RFK. Binding of HsRFK substrates is the kinetically preferred process compared to product inhibition, kinetic modeling, overview
substrate binding structure analysis using crystal structures of substrate-bound RFK. Binding of HsRFK substrates is the kinetically preferred process compared to product inhibition, kinetic modeling, overview
Michaelis-Menten kinetics and modeling, cooperativity model, pre-steady-state kinetics/pre-steady-state stopped-flow kinetics and dissociation constants, overview. Isothermal titration calorimetry, and Gibbs free energy flow for the interaction of HsRFK with substrates and products. Thermodynamics modulates the ligand binding landscape of HsRFK. Cooperativity coefficients, ANP and FLV ligands cooperate in their binding to HsRFK
Michaelis-Menten kinetics and modeling, cooperativity model, pre-steady-state kinetics/pre-steady-state stopped-flow kinetics and dissociation constants, overview. Isothermal titration calorimetry, and Gibbs free energy flow for the interaction of HsRFK with substrates and products. Thermodynamics modulates the ligand binding landscape of HsRFK. Cooperativity coefficients, ANP and FLV ligands cooperate in their binding to HsRFK
enzyme RFK 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
the enzyme plays a critical role in the KD548-Fc-mediated reactive oxygen species accumulation and downstream signaling. The enzyme is essential in recruiting Nox1 to death receptor4/5
human riboflavin kinase is an essential enzyme that catalyzes the biosynthesis of the flavin mononucleotide (FMN) cofactor using riboflavin (RF, vitamin B2) and ATP as substrates. 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-beta toxicity, and some malignant cancers progression. HsRFK is also predicted as involved in a protein-protein association network that at the system level affects to different cellular processes
analysis of the crystallographic structure of HsRFK in complex with FMN and ADP in either the open (PDB ID 1P4M) or the closed conformation (PDB ID 1Q9S) of the flavin binding site overview
purified recombinant enzyme with bound products FMN and MgADP, hanging drop vapour diffusion method, 34 mg/ml protein in 50 mM Tris, pH 7.4, 0.3 M NaCl, 1 mM DTT, 20°C, mixing with equal volume of reservoir solution containing 0.1 M sodium acetate, pH 4.7, 30% PEG monomethyl ether 5000, and 0.2 M ammonium sulfate, followed by microseeding in reservoir solution containing 0.1 M sodium acetate, pH 4.4, 22.5% PEG monomethyl ether 5000, and 0.2 M ammonium sulfate, cryoprotection in 30% glycerol in reservoir solution, storage in liquid propane, X-ray diffraction structure determination and analysis at 2.4 A resolution
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PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant His-tagged enzyme RFK from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, tag cleavage by a protease, followed by ammonium sulfate fractionation, hydrophobic interaction chromatography, and dialysis. HsRFK is purified free of flavin and adenine ligands
HsRFK parameters differ from those of the so far evaluated bacterial counterparts, suggesting species-specific mechanisms in RFK catalysis. Thus, HsRFK is a potential therapeutic target because of its key functions, while bacterial RFK modules are potential antimicrobial targets