PURIFICATION AND CHARACTERIZATION OF OXOPANTOYL LACTONE REDUCTASE FROM HIGHER-PLANTS - ROLE IN PANTOTHENATE SYNTHESIS

Oxopantoyl lactone reductase has been purified to homogeneity from a crude extract of spinach leaves (Spinacia oleracea L.) using affinity chromatography on Red-Agarose and several subsequent ion exchange steps. The enzyme is monomeric with a relative molecular mass between 33,000 to 36,000. Affinity-purified antibodies directed against the homogenous enzyme have been used to determine the amount of oxopantoyl lactone reductase in the crude leaf extract as well as the chloroplast stroma. The overall purification factor has been determined to be 22,000. The subcellular location of the enzyme is chloroplastic. The final specific activity (strictly NADPH-dependent) is 4.5 mu mole min(-1).mg(-1). The enzyme is also able to reduce isatin, bornanedione and acenaphthenequinone. The enzyme activity is strongly and uncompetitively inhibited by 2-keto-4-hydroxybutyrolactone and substituted 4,5-dioxopyrrolidines. An oxopantoate reductase associated with acetohydroxy acid isomeroreductase could be detected in the plant extract. Using a specific inhibitor of this latter enzyme or oxopyrrolidines, complementation studies with branched chain amino-acids and pantothenate have shown that oxopantoyl lactone reductase is likely to be involved in pantothenate biosynthesis. Furthermore, pantoyl lactone, the putative product of the reaction, together with beta-alanine and ATP, has been shown to be the substrate of pantothenate synthase using a novel assay for pantothenate.