Purification and characterization of recombinant FAD synthetase from Neurospora crassa.

ABSTRACT: 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 ti

SUBMITTER: Puvvada N 

PROVIDER: S-EPMC8571487 | biostudies-literature | 2021 Dec

REPOSITORIES: biostudies-literature