Subcellular localization and purification of a p-hydroxyphenylpyruvate dioxygenase from cultured carrot cells and characterization of the corresponding cDNA

Abstract

p-Hydroxyphenylpyruvate dioxygenase catalyses the transformation of p-hydroxyphenylpyruvate into homogentisate. In plants this enzyme has a crucial role because homogentisate is the aromatic precursor of all prenylquinones. Furthermore this enzyme was recently identified as the molecular target for new families of potent herbicides. In this study we examine precisely the localization of p-hydroxyphenylpyruvate dioxygenase activity within carrot cells. Our results provide evidence that, in cultured carrot cells, p-hydroxyphenylpyruvate dioxygenase is associated with the cytosol. Purification and SDS/PAGE analysis of this enzyme revealed that its activity is associated with a polypeptide of 45–46 kDa. This protein specifically cross-reacts with an antiserum raised against the p-hydroxyphenylpyruvate dioxygenase of Pseudomonas fluorescens. Gel-filtration chromatography indicates that the enzyme behaves as a homodimer. We also report the isolation and nucleotide sequence of a cDNA encoding a carrot p-hydroxyphenylpyruvate dioxygenase. The nucleotide sequence (1684 bp) encodes a protein of 442 amino acid residues with a molecular mass of 48094 Da and shows specific C-terminal regions of similarity with other p-hydroxyphenylpyruvate dioxygenases. This cDNA encodes a functional p-hydroxyphenylpyruvate dioxygenase, as evidenced by expression studies with transformed Escherichia coli cells. Comparison of the N-terminal sequence of the 45–46 kDa polypeptide purified from carrot cells with the deduced peptide sequence of the cDNA confirms that this polypeptide supports p-hydroxyphenylpyruvate dioxygenase activity. Immunodetection studies of the native enzyme in carrot cellular extracts reveal that N-terminal proteolysis occurs during the process of purification. This proteolysis explains the difference in molecular masses between the purified protein and the deduced polypeptide.

Broad 4-Hydroxyphenylpyruvate Dioxygenase Inhibitor Herbicide Tolerance in Soybean with an Optimized Enzyme and Expression Cassette

“…Analysis of the M. truncatula genome sequence around the HPPD gene revealed that this legume species also has an N-terminal ORF extension that contains a predicted CTP. What is not clear is why Arabidopsis and some other nonlegumes would encode only cytosolic HPPD (Garcia et al, 1997(Garcia et al, , 1999. Perhaps the movement of homogentisate into the chloroplast serves as a regulatory step for the plastoquinone and tocopherol pathways.…”

“…HPPD was predicted to have a chloroplast-targeting peptide (CTP). Subcellular fractionation supported a cytosolic location of the carrot (Daucus carota) cell enzyme (Garcia et al, 1997). The same authors determined that after PSORT analysis failed to identify a targeting signal within the Arabidopsis (Arabidopsis thaliana) HPPD amino acid sequence, native Arabidopsis HPPD heterologously expressed in tobacco (Nicotiana benthamiana) was located exclusively in the cytosol (Garcia et al, 1999).…”

Tetrahydrofolate biosynthesis in plants: Molecular and functional characterization of dihydrofolate synthetase and three isoforms of folylpolyglutamate synthetase in Arabidopsis thaliana

“…Protoplasts were prepared by enzymatic digestion of 6-day-old cell suspension cultures, using the procedure described in ref. 21. Protoplasts were gently ruptured by passing through a 20-m nylon mesh and subsequently through a 10-m nylon mesh.…”

Complementation of the Arabidopsispds1Mutation with the Gene Encodingp-Hydroxyphenylpyruvate Dioxygenase

“…Genes and cDNAs encoding HPPDase have been identified from several mammalian, fungal, bacterial, and plant sources (Gershwin et al, 1987;Endo et al, 1992Endo et al, , 1995Hummel et al, 1992;Ruetschi et al, 1993;Coon et al, 1994;Denoya et al, 1994;Wilson et al, 1994;Wintermeyer et al, 1994;Kaneko et al, 1995;Wyckoff et al, 1995;Garcia et al, 1997) and show between 25% and 95% identity at the amino acid level. A computer search of the plant DNA databases, including 20,000 random Arabidopsis cDNAs (Newman et al, 1994), was conducted using human and bacterial HPPDase sequences as the query.…”

“…1). HPPDase is generally present at low levels in plant tissues and has only recently been purified to homogeneity from a plant source (Garcia et al, 1997).…”