Purification and characterization of recombinant FAD synthetase from Neurosporacrassa

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