Poster: Late and Moved Abstracts

Abs # 1415: Effect of substitution mutations of cysteine residues on cytosolic monodehydroascorbate reductase from cucumber

Presenter:	Sano, Satoshi , satsano@kpu.ac.jp
Authors	Sano, Satoshi  (A)   Morishita, Yoshihiko  (A)   Tao, Satoru  (A)   Saito, Kazumi  (A)
Affiliations:	(A): Graduate School of Agriculture, Kyoto Prefectural University

When Ascorbate (AsA) acts as an antioxidant in cells, in most cases, monodehydroascorbate (MDA) radical is formed as a primary oxidant product. The regeneration of AsA from MDA is indispensable to protect cells against oxidative stress. In plant, MDA reductase (MDAR), an FAD-enzyme, catalyzes the reduction of MDA using NAD(P)H, via a ping-pong mechanism. The first step of this reaction is the reduction of the enzyme-bound FAD by NAD(P)H, which is known to be inhibited by SH reagents. Two Cys residues, corresponding to Cys69 and Cys198 of cucumber enzyme, are conserved in cytosolic MDAR. To understand how these Cys residues function, MDARs in which Cys residues were substituted by either Ala or Ser were produced in E. coli using the cDNA of the cytosolic cucumber MDAR and were designated C69A, C69S, C198A and C198S. While the specific activities of C198A and C198S were comparable to that of the wild-type enzyme, C69A and C69S had lower specific activities, which were 81% and 47% of that of the wild type respectively. In C69A, especially, the dissociation of the FAD from the apoprotein seems to cause the decrease of the activity. However there was no difference in the Km values for NAD(P)H and the rate constants for the reduction of the FAD by NADH between the mutated enzymes and the wild-type enzyme. Moreover, the absorption spectra of these enzymes showed that the reduction of the enzyme-bound FAD by NADH and its inhibition by pCMB, an SH reagent, were not affected by the substitution of Cys residues. These results might be accounted by the following: (1) Cys residues in MDAR do not participate in the reduction of the enzyme-bound FAD by NAD(P)H directly, (2) SH reagents inhibit the FAD reduction by modification of amino acid residue(s) other than Cys.