Purification and characterization of pterocarpan hydroxylase,a flavoprotein monooxygenase from the fungus Ascochyta rabiei involved in pterocarpan phytoalexin metabolism

Crude protein extracts from the chickpea (Cicer arietinum) pathogenic fungus Ascochyta rabiei catalyze the hydroxylation of the pterocarpan phytoalexins medicarpin and maackiain to the corresponding 1a-hydroxy-1,4-diene-3-one derivatives. The enzyme reaction depends on NAD(P)H and molecular oxygen. Low amounts of FAD are necessary for maximal enzyme activity. The pterocarpan hydroxylase is a new flavoprotein monooxygenase with a molecular weight of 58 kDa in SDS-PAGE. The soluble enzyme can utilize NADH and NADPH with similar values for K _m and V _max respectively. The pterocarpan hydroxylase and a pterocarpan reductase (M _r 29 kDa; Höhl and Barz 1987) are constitutively expressed by A. rabiei isolates.Abbreviations AAS atomic absorption spectroscopy - BCS bathocuproindisulfonate - BSA bovine serum albumin - FAD flavin-adenine dinucleotide - FMN flavin-mononucleotide - M _r molecular weight - PAGE polyacrylamide gelelectrophoresis - pda pisatin demethylating ability - SDS sodium dodecylsulfate - Tris tris(hydroxymethyl)aminomethane