Bifunctional indole‐3‐acetyl transferase catalyses synthesis and hydrolysis of indole‐3‐acetyl‐myo‐inositol in immature endosperm of Zea mays

Abstract

1‐O‐(indole‐3‐acetyl)‐β‐d‐glucose: myo‐inositol indoleacetyl transferase (IA‐myo‐inositol synthase) is an important enzyme in IAA metabolism. This enzyme catalyses the transfer of the indole acetyl (IA) moiety from 1‐O‐(indole‐3‐acetyl)‐β‐d‐glucose to myo‐inositol to form IA‐myo‐inositol and glucose. IA‐myo‐inositol synthase was purified to an electrophoretically homogenous state from maize liquid endosperm by fractionation with ammonium sulphate, anion‐exchange, adsorption on hydroxylapatite, affinity chromatography on ConA‐Sepharose, preparative PAGE and isoelectric focusing. We thus obtained two enzyme preparations which differ in their Rf on 8% polyacrylamide gel. The preparation of Rf 0.36 contained a single 56.4 kDa polypeptide, whereas the preparation of Rf 0.39 consisted of two polypeptides of 56.4 and 53.5 kDa. Both purified preparations of IAInos synthase also exhibited the activity of an IAInos hydrolase, showing that the dual activity was associated with a single protein. Results of gel filtration and analytical SDS‐PAGE suggest that the native enzyme exists as both a monomeric (65 kDa) and homo‐ or heterodimeric form (110–130 kDa). Analysis of peptide maps and amino acid sequences of two 21 amino‐acid peptides showed that polypeptides of 56.4 and 53.5 kDa have the same primary structure and that the 3 kDa difference in molecular mass is probably caused by different glycosylation levels. Comparison of this partial and internal amino acid sequence with sequences of other plant acyltransferases indicated similarity to several proteins which belonged to the serine carboxypeptidase‐like (SCPL) acyltransferase family.