Large Scale Synthesis and Purification of Flavin Adenine Dinucleotide

DeLuca, Chester; Kaplan, Nathan O.

doi:10.1016/s0021-9258(18)65165-8

Cited by 27 publications

(11 citation statements)

References 17 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We synthesized FCD by dissolving 0.1 g of FMN and 35 mg of CMP in 1 mL of trifluoroacetic anhydride (TFAA). The procedure was based on the previously described preparation of FAD from FMN and AMP (14). After the reactants dissolved (∼1 min), the sample was kept in the dark for 18 h. Following incubation, a stream of N 2 gas was used to evaporate the solvent.…”

Section: Kda)mentioning

confidence: 99%

Archaeal RibL: A New FAD Synthetase That Is Air Sensitive

Mashhadi

Grochowski

et al. 2010

Biochemistry

View full text Add to dashboard Cite

FAD synthetases catalyze the transfer of the AMP portion of ATP to FMN to produce FAD and pyrophosphate (PP(i)). Monofunctional FAD synthetases exist in eukaryotes, while bacteria have bifunctional enzymes that catalyze both the phosphorylation of riboflavin and adenylation of FMN to produce FAD. Analyses of archaeal genomes did not reveal the presence of genes encoding either group, yet the archaea contain FAD. Our recent identification of a CTP-dependent archaeal riboflavin kinase strongly indicated the presence of a monofunctional FAD synthetase. Here we report the identification and characterization of an archaeal FAD synthetase. Methanocaldococcus jannaschii gene MJ1179 encodes a protein that is classified in the nucleotidyl transferase protein family and was previously annotated as glycerol-3-phosphate cytidylyltransferase (GCT). The MJ1179 gene was cloned and its protein product heterologously expressed in Escherichia coli. The resulting enzyme catalyzes the adenylation of FMN with ATP to produce FAD and PP(i). The MJ1179-derived protein has been designated RibL to indicate that it follows the riboflavin kinase (RibK) step in the archaeal FAD biosynthetic pathway. Aerobically isolated RibL is active only under reducing conditions. RibL was found to require divalent metals for activity, the best activity being observed with Co(2+), where the activity was 4 times greater than that with Mg(2+). Alkylation of the two conserved cysteines in the C-terminus of the protein resulted in complete inactivation. RibL was also found to catalyze cytidylation of FMN with CTP, making the modified FAD, flavin cytidine dinucleotide (FCD). Unlike other FAD synthetases, RibL does not catalyze the reverse reaction to produce FMN and ATP from FAD and PP(i). Also in contrast to other FAD synthetases, PP(i) inhibits the activity of RibL.

show abstract

Section: Kda)mentioning

confidence: 99%

Archaeal RibL: A New FAD Synthetase That Is Air Sensitive

Mashhadi

Grochowski

et al. 2010

Biochemistry

View full text Add to dashboard Cite

show abstract

“…The primers used for the cloning of wild-type and mutants and the plasmids created are given in the supplementary information Table S2 and S3 The reaction mixture was incubated at 37°C overnight and visualized using fluoresce-based thin layer chromatography (TLC) (Merck Silica gel 60 F 254 ) using the long wavelength (365 nm) of handheld UV lamp (Spectroline E-series UV lamp) nm with the solvent systemwater: acetic acid: n-butanol in 5:1:4 ratios. 20 The retention time of riboflavin and FMN in this solvent system was 0.45 and 0.2, respectively. The enzymatic reaction product was further analyzed by high performance liquid chromatography (Agilent HPLC system ( 1260Infinity II) equipped with UV-Vis DAD detector on a C-18 reverse-phase column (Phenomenex, Gemini 5µm NX-C18 110 Å, 250 × 4.6 mm).…”

Section: Molecular Cloning Overexpression and Purificationmentioning

confidence: 76%

Characterization of a novel mesophilic CTP-dependent riboflavin kinase and rational engineering to create its thermostable homologs

Kumar

Singh

Hazra

2021

Preprint

View full text Add to dashboard Cite

Flavins play a central role in cellular metabolism as cofactors that are engaged in a wide range of oxidation-reduction reactions in living organisms. Several interesting variations exist in the flavin biosynthesis pathway among the domains of life, and the analysis of enzymes on this pathway have resulted in unique structural and mechanistic insights. The CTP-dependent riboflavin kinase in archaea is one such example - unlike most kinase enzymes that use adenosine triphosphate for conducting phosphorylation reactions, riboflavin kinases from tarchaea utilizes cytidine triphosphate (CTP) to phosphorylate riboflavin to produce flavin mononucleotide (FMN). In this study, we present the characterization of a mesophilic archaeal riboflavin kinase homolog from Methanococcus maripaludis (MmpRibK), which is linked closely in sequence to the thermophilic RFK homolog from Methanocaldococcus jannaschii (MjRibK). We reconstitute the activity of the CTP-dependent MmpRibK and analyse the molecular factors that contribute to the uncommon properties of this class of enzymes. Furthermore, based on the high degree of sequence similarity between the mesophilic MmpRibK and the thermophilic MjRibK, we establish a set of the residues that are responsible for the thermostability of the enzyme without any loss in activity or substrate specificity. Our work contributes to the molecular understanding of flavin biosynthesis in archaea through the characterization of the first mesophilic CTP-dependent riboflavin kinase. Finally, it validates the role of salt bridges and rigidifying amino acid residues in imparting thermostability to enzymes, with implications in enzyme engineering and biotechnological applications.

show abstract

“…As an improvement, a one-step condensation of adenosine monophosphate (AMP) with salts of FMN in the presence of trifluoroacetic acid anhydride (TFAA) as a condensing agent was devised [23] . This strategy, used for the synthesis of FAD and its nucleobase analogues, has extremely poor yields in the range of 0.5-1% owing to the self-condensation side reactions of the substrates [10] . Yet, being a one-step process with readily available starting materials, it has been used in a few instances to create nucleobase cofactor analogues [24,25] .…”

Section: Resultsmentioning

confidence: 99%

Efficient chemical and enzymatic syntheses of FAD nucleobase analogues and their analysis as enzyme cofactors

Shah

Kumar

Rohan

et al. 2023

Preprint

View full text Add to dashboard Cite

Flavin adenine dinucleotide (FAD), an essential cofactor in cellular metabolism, catalyses a wide range of redox reactions. The organic synthesis of FAD is typically conducted by coupling flavin mononucleotide (FMN) and adenosine monophosphate. The reported synthesis routes have certain limitations such as multiple reaction steps, low yields, and/or difficult-to-obtain starting materials. In this study, we report the synthesis of FAD nucleobase analogues using chemical and enzymatic methods with readily available starting materials achieved in 1-3 steps with moderate yields (10-51%). Further, we demonstrate that Escherichia coli glutathione reductase can use these analogues to catalyse the reduction of glutathione. Finally, we show that FAD nucleobase analogues can also be synthesized inside a cell from cellular substrates FMN and nucleoside triphosphates. This lays the foundation for their use in studying the molecular role of FAD in cellular metabolism and as biorthogonal reagents in biotechnology and synthetic biology applications.

show abstract

Large Scale Synthesis and Purification of Flavin Adenine Dinucleotide

Cited by 27 publications

References 17 publications

Archaeal RibL: A New FAD Synthetase That Is Air Sensitive

Archaeal RibL: A New FAD Synthetase That Is Air Sensitive

Characterization of a novel mesophilic CTP-dependent riboflavin kinase and rational engineering to create its thermostable homologs

Efficient chemical and enzymatic syntheses of FAD nucleobase analogues and their analysis as enzyme cofactors

Contact Info

Product

Resources

About