Crystallization and structure determination of DAO1 from Arabidopsis thaliana

Abstract

Indole-3-acetic acid (IAA), the major form of the plant hormone auxin, regulates almost every aspect of plant growth and development. Therefore, auxin homeostasis is an essential issue in plants. Various pathways of synthesis, transport, conjugation, and catabolism are involved in auxin homeostasis, but its catabolic pathway has remained elusive until recent studies elucidated the presence of DIOXYGENASE FOR AUXIN OXIDATION (DAO) from Oryza sativa and Arabidopsis thaliana. DAO, a member of the 2-oxoglutarate/Fe(II)-dependent oxygenase family, constitutes a major enzyme for IAA catabolism. It catalyzes, with the cosubstrate 2-oxoglutarate, the conversion of IAA into 2-oxoindole-3-acetic acid, a functionally inactive oxidative product of IAA. Here, I report a crystal structure of the unliganded DAO1 from A. thaliana (AtDAO1) and its complex with 2-oxoglutarate. AtDAO1 is structurally homologous with members of the 2-oxoglutarate/Fe(II)-dependent oxygenase family but exhibits unique features in the prime substrate-binding site. Using liquid chromatography-tandem mass spectrometry analyses of the reaction products from various mutant enzymes, I provide structural insights into a putative binding site for the prime substrate IAA, thereby suggesting possible structural determinants for the substrate specificity of AtDAO1 toward IAA.