Abstract
Ascorbate functions as an antioxidant to be oxidized to monodehydroascorbate (MDA) radical. Its regeneration is essential to avoid damages by oxidative stress. In plant, MDA reductase (MDAR) containing FAD catalyzes the reduction of MDA by ping-pong mechanism using NAD(P)H. The first step of this reaction is the reduction of FAD by NAD(P)H, which is inhibited by thiol reagents. Cytosolic MDAR has two Cys residues at the conserved positions, but it has not been clarified which of Cys residues participates in its activity. Mutated MDARs whose Cys residues were substituted to Ala or Ser were designated C69A, C69S, C198A and C198S respectively. The C69S showed 32% of the activity of the native enzyme and lability of binding with FAD. The Km values for NAD(P)H of mutants were comparable to those of the native enzyme. Now we are analyzing the rapid reaction between NADH and FAD on these enzymes.
