Partial Purification and Characterization of Indol-3-Ylacetylglucosemyo-Inositol Indol-3-Ylacetyltransferase (Indoleacetic Acid-Inositol Synthase)

Abstract

A procedure is described for the purification of the enzyme indol-3-ylacetylglucose:myo-inositol indol-3-ylacetyltransferase (IAA-myo-inositol synthase). This enzyme catalyzes the transfer of indol-3-ylacetate from 1-0-indol-3-ylacetyl-0-d-glucose to myoinositol to form indol-3-ylacetyl-myo-inositol and glucose. A hexokinase or glucose oxidase based assay system is described. The enzyme has been purified approximately 16,000-fold, has an isoelectric point of pH 6.1 and yields three catalytically inactive bands upon acrylamide gel electrophoresis of the native protein.The enzyme shows maximum transferase activity with myo-inositol but shows some transferase activity with scyllo-inositol and myo-inosose-2. No transfer of IAA occurs with myo-inositol-dgalactopyranose, cyclohexanol, mannitol, or glycerol as acyl acceptor. The affinity of the enzyme for 1-0-indol-3-ylacetyl-ft-dglucose is, Km = 30 micromolar, and for myo-inositol is, Km = 4 millimolar. The enzyme does not catalyze the exchange incorporation of glucose into IAA-glucose indicating the reaction mechanism involves binding of IAA glucose to the enzyme with subsequent hydrolytic cleavage of the acyl moiety by the hydroxyl of myo-inositol to form IAA myo-inositol ester.This work deals with the partial purification and characterization of indol-3-ylacetylglucose:myo-inositol indol-3-ylacetyltransferase (IAInos synthase).3 The enzyme catalyzes the reaction between -0-indol-3-ylacetyl-fl-D-glucose and myoinositol to form indol-3-ylacetyl-myo-inositol and glucose according to the following equation:The reaction is important because mature kernels of Zea mays sweet corn contain 99% of their endogenous IAA as ester conjugates and less than 1% as the free acid (8). half of the esters are conjugates of IAA and myo-inositol or IAA and myo-inositol glycosides (8). The kernels contain small amounts of the isomeric conjugates of IAA and glucose, 2-0, 4-0, and 6-0-IAGlu (13). 1-0-IAGlu can be demonstrated to occur naturally in small amounts if precautions are used to prevent acyl migration (J Cohen, personal communication). An IAA glucose conjugate was the first IAA ester conjugate to be isolated from plants and was found following application of labeled IAA to plants (21,35).1-0-IAGlu is an acyl alkyl acetal4 and is chemically distinct, from an IAA ester of glucose. In aqueous solution, especially at a pH of 7 or above, the IAA moiety migrates to the position farthest from the carbonyl, that is, the 4-0 and 6-0 position. Owing to this facile acyl migration (1 1, 13,19,20,[25][26][27]) the bond energy of the acyl alkyl acetal is not readily determined but the equilibrium of the reaction:is of the order of Keq = 10-' (27) thus placing the free energy of hydrolysis of IAA glucose at about 1400 calories above that ofthe phosphato glucose bond of UDPG and many thousands of calories above that of an ester bond.These observations concerning energetics may explain the thermodynamics of the manner in which liquid endosperm of maize can contain 0.1 mm IAA ester. Once 1-0...

Purification and Biochemical Characterization of Indole-3-acetyl-aspartic Acid Synthetase from Immature Seeds of Pea (Pisum sativum)

“…A similar effect of the thiol reagents was observed for maize synthase of 1-O-IAA-glucose (Leznicki and Bandurski 1988). However, another enzyme involved in the biosynthesis of IAA-ester conjugates in maize endosperm, IAA-inositol synthase, was found to be strongly inhibited by DTT and ME (Kesy and Bandurski 1990). The inhibition of enzyme activity by DTT was also reported for both plant (Ludwig-Müller and others 1996;Campanella and others 2008) and bacterial IAA-amidohydrolases (Chou and others 1996;Chou and Huang 2005).…”

Acetylcholine as a Signaling System to Environmental Stimuli in Plants: III. Asymmetric solute distribution controlled by ACh in gravistimulated maize seedlings

“…This enzyme catalyzes the reaction between l-O-IAA-glucose and myo-inositol to form IAAinositol and glucose (Kesy and Bandurski, 1990). The immunoreacted IAA-inositol synthase in the lower half of the vascular system of coleoptile node and mesocotyl was darker than that in the upper half.…”

“…Since the synthesis of IAA-glucose, followed by transacylation to myo-inositol, represent the first steps in the IAA conjugation process, the enzymes catalyzing these steps are candidates for the regulation of IAA concentration by converting hormonally active free IAA into growth-inactive IAA ester (Bandurski et al, 1977;Bandurski et aI., 1990), as occur in the tropic responses to gravity and light. The IAA-inositol synthase catalyzes the transfer of IAA-acetate from 1-0-IAA-glucose to myo-inositol to form IAA-inositol and glucose (Kesy and Bandurski, 1990). This reaction is important because mature kernels of maize contain 99% of their endogenous IAA as ester conjugates and less than 1 % as the free acid (Cohen and Bandurski, 1982).…”

Novel 1-O-indole-3-acetyl-β-d-glucose-dependent acyltransferase transferring indoleacetyl moiety to some mono- di- and oligosaccharides

“…In the previous study, the substrate specificity of 1-OIAGlc-dependent myo-inositol acyltransferase (1-O-IAGlc- InosAc) was tested only toward several alcohols, but not toward sugars (Kęsy and Bandurski 1990), which is why the separation of 1-O-IAGlc-SugAc from 1-O-IAGlc-InosAc and from other potential 1-O-IAGlc-dependent acyltransferases has a major significance. In our preliminary experiments, the several sugar acceptors of IAA moiety were tested, however, in all qualitative assays and during purification of 1-O-IAGlc-SugAc, a melibiose was used as a sole acceptor.…”

“…The synthesis of IA-myo-inositol (IAInos) was observed for the first time in extract of immature kernels of maize Bandurski 1980, 1982), and more recently, 1-O-IAGlc-dependent myo-inositol acyltransferase (1-OIAGlc-InosAc) was partially purified and characterized (Kęsy and Bandurski 1990). Not long ago, an electrophoretically homogenous enzyme, as well the amino acid sequence of 21-aa peptide was obtained (Kowalczyk et al 2003), which was currently utilized to select a cDNA clone (submitted for publication).…”