Fungal Auxin Antagonist Hypaphorine Competitively Inhibits Indole-3-acetic Acid-Dependent Superoxide Generation by Horseradish Peroxidase

Abstract

Plant peroxidases (EC 1.11.1.7) including horseradish peroxidase (HRP-C), but not the nonplant peroxidases, are known to be highly specific indole-3-acetic acid (IAA) oxygenases which oxidize IAA in the absence of H2O2, and superoxide anion radicals (O•−2) are produced as by-products. Hypaphorine, a putative auxin antagonist isolated from ectomycorrhizal fungi, inhibited the IAA-dependent generation of O•−2 by HRP-C, which occurs in the absence of H2O2. Hypaphorine has no effect on the nonspecific heme-catalyzed O•−2 generation induced by high concentration of ethanol. It is probable that the inhibitory effect of hypaphorine on O•−2 generation is highly specific to the IAA-dependent reaction. The mode of inhibition of the IAA-dependent O•−2-generating reaction by hypaphorine was analyzed with a double-reciprocal plot and determined to be competitive inhibition, indicating that hypaphorine competes with IAA by binding to the putative IAA binding site on HRP-C. This implies the importance of structural similarity between hypaphorine and IAA. This work presented the first evidence for antagonism between IAA and a structurally related fungal alkaloid on binding to a purified protein which shares some structural similarity with auxin-binding proteins.