A serine carboxypeptidase-like acyltransferase catalyzes synthesis of indole-3-acetic (IAA) ester conjugate in rice (Oryza sativa)

Abstract

Indole-3-acetic acid (IAA) conjugation is one of mechanisms responsible for regulation of free auxin levels in plants. A new member of the serine carboxypeptidase-like (SCPL) acyltransferases family from Oryza sativa has been cloned and characterized. 1-O-indole-3-acetyl-β-D-glucose (1-O-IAGlc): myo-inositol acyltransferase (IAInos synthase) is an enzyme of IAA ester conjugates biosynthesis pathway that catalyzes transfer of IAA moiety from 1-O-IAGlc to myo-inositol forming IA-myo-inositol (IAInos). The OsIAA-At cDNA has been cloned and expressed using yeast and bacterial expression systems. Proteins produced in Saccharomyces cerevisiae and Escherichia coli contained 483 and 517 amino acids, respectively. The enzyme functionally expressed in both expression systems exhibits 1-O-IAGlc-dependent acyltransferase activity. Analysis of amino acid sequence confirmed that rice IAInos synthase belongs to the SCPL protein family. Recombinant IAInos synthases produced in yeast and bacterial expression systems have been partially characterized and their properties have been compared to those of the native enzyme obtained from 6-days-old rice seedlings by biochemical approach. The oligosaccharide component of the protein enzyme is not necessary for its catalytic activity. The native enzyme showed the lowest specific activity of 5.01 nmol min−1 mg−1 protein, whereas the recombinant enzymes produced in yeast and bacteria showed specific activity of 18.75 nmol min−1 mg−1 protein and 18.09 nmol min−1 mg−1 protein, respectively. The KM values for myo-inositol were similar for all three forms of the enzyme: 1.38, 0.83, 1.0 mM for native, bacterial and yeast protein, respectively. Both recombinant forms of IAInos synthase and the native enzyme also have the same optimal pH of 7.4 and all of them are inhibited by phenylmethylsulfonyl fluoride (PMSF), specific inhibitor of serine carboxypeptidases.