MOLECULAR-CLONING AND CHARACTERIZATION OF A CDNA-ENCODING PEA MONODEHYDROASCORBATE REDUCTASE

Monodehydroascorbate radicals are generated in plant cells enzymatical ly by the hydrogen peroxide scavenging enzyme, ascorbate peroxidase, a nd nonenzymatically via the univalent oxidation of ascorbate by supero xide, hydroxyl, and various organic radicals. Regeneration of ascorbat e is achieved by monodehydroascorbate reductase (EC 1.6.5.4) using NAD (P)H as an electron donor or, alternatively, by a set of two coupled r eactions requiring dehydroascorbate reductase, glutathione reductase, glutathione, and NAD(P)H. As monodehydroascorbate reductase is a key e nzyme in maintaining reduced pools of ascorbate, an important antioxid ant, we undertook this study to learn more about its structure, functi on, and regulation. Herein we report the molecular cloning and charact erization of a cDNA encoding monodehydroascorbate reductase of pea (Pi sum sativum L.). The cDNA encodes a 433-amino acid polypeptide that sh ows, respectively, 73 and 87% identity with peptide fragments from soy bean and cucumber monodehydroascorbate reductase. Monodehydroascorbate reductase contains the NAD(P)H and FAD binding domains of other flavi n oxidoreductases. The cloned enzyme lacks a transit peptide, but the sequence of the carboxyl terminus is Ser-Lys-Ile, similar to the targe ting motif found in peroxisomal proteins. When expressed in Escherichi a coli fused to maltose-binding protein, monodehydroascorbate reductas e has enzymatic properties comparable with purified soybean and cucumb er monodehydroascorbate reductase. Northern blot analysis shows that t he monodehydroascorbate reductase transcript is 1.6 kilobase in size a nd is expressed at relatively low levels in all plant tissues examined .