Purification and characterization of recombinant FAD synthetase from Neurospora crassa

• DOI: 10.1016/j.bbrep.2021.101161
• Publication Date: 2021-11-01T10:44:53Z
• Authors: Nirupama Puvvada; Sridhar Gunde; Ch. Venkata Raman...; Raghu Gogada
• Keywords: Isothermal Titration Calorimetry; Enzyme Kinetics; Flavin adenine dinucleotide; Flavin mononucleotide; Molecular mass

FAD Synthetase (FADS) [EC 2.7.7.2], the second enzyme in flavin cofactor biosynthetic pathway converts FMN to FAD, plays an important role many redox reactions. Neurospora crassa FADS (NcFADS) was cloned and overexpressed E. coli cells. Recombinant NcFADS purified high yields of ∼8 mg per liter bacterial culture using a single step glutathione sepharose affinity chromatography. SDS-PAGE MALDI-MS revealed that has molecular mass ∼31 kDa. Enzyme kinetic analysis monitored by reverse phase HPLC demonstrate specific activity kcat 1356 nmol/min/mg 0.69sec-1 respectively. Steady state exhibited Km for is 2.7 μM MgATP-2 88.7 μM. Isothermal titration calorimetry experiments showed recombinant protein binds substrates with apparent Kd 20.8 16.6 MgATP-2. Biophysical characterization intrinsic fluorescence suggests folded conformation. Far-UV CD data suggest backbone predominantly helical Differential scanning shows Tm 53 °C ± 1. This first report on cloning, purification from N. crassa. The highest than any reported other source. results obtained this study expected pave way intensive research aimed understand basis extraordinarily turnover rate NcFADS.