Characterization and subcellular compartmentation of recombinant 4-hydroxyphenylpyruvate dioxygenase from Arabidopsis in transgenic tobacco

4-Hydroxyphenylpyruvate dioxygenase (4HPPD) catalyzes the formation of homo gentisate (2,5-dihydroxyphenylacetate) from p-hydroxyphenylpyruvate and mol ecular oxygen. In plants this enzyme activity is involved in two distinct m etabolic processes, the biosynthesis of prenylquinones and the catabolism o f tyrosine. We report here the molecular and biochemical characterization o f an Arabidopsis 4HPPD and the compartmentation of the recombinant protein in chlorophyllous tissues. We isolated a 1508-bp cDNA with one large open r eading frame of 1338 bp. Southern analysis strongly suggested that this Ara bidopsis 4HPPD is encoded by a single-copy gene. We investigated the bioche mical characteristics of this 4HPPD by overproducing the recombinant protei n in Escherichia coli JM105. The subcellular localization of the recombinan t 4HPPD in chlorophyllous tissues was examined by overexpressing its comple te coding sequence in transgenic tobacco (Nicotiana tabacum), using Agrobac terium tumefaciens transformation. We. performed western analyses for the i mmunodetection of protein extracts from purified chloroplasts and total lea f extracts and for the immunocytochemistry on tissue sections. These analys es clearly revealed that 4HPPD was confined to the cytosol compartment, not targeted to the chloroplast. Western analyses confirmed the presence of a cytosolic form of 4HPPD in cultured green Arabidopsis cells.