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

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Abstract

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.

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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

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Tenhaken, R., Salmen, H.C. & Barz, W. Purification and characterization of pterocarpan hydroxylase, a flavoprotein monooxygenase from the fungus Ascochyta rabiei involved in pterocarpan phytoalexin metabolism. Arch. Microbiol. 155, 353–359 (1991). https://doi.org/10.1007/BF00243455

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