cDNA-cloning and functional expression of hydroxyphenylpyruvate dioxygenase from cell suspension cultures of Coleus blumei

The full-length cDNA of hydroxyphenylpyruvate dioxygenase (HPPD; E.C. 1.13.11.27) was cloned from Coleus blumei (Lamiaceae) by polymerase chain reaction with primer sequences deduced from already known HPPDs. The cloned cDNA had a length of 1657 base pairs containing an open reading frame (ORF) of 1308 base pairs which coded for a protein of 436 amino acid residues with a-calculated molecular mass of 47 736 Da. Identities of 62.7-76.0% on amino acid level and 64.8-74.9% on nucleotide level were shown to HPPDs from other plant sources. HPPD transforms 4-hydroxyphenylpyruvate to homogentisate and, therefore, competes for the same substrate as hydroxyphenylpyruvate reductase (HPPR), an enzyme of rosmarinic acid (RA) biosynthesis from C blumei. The ORF of HPPD was ligated into the expression vector pTrc99A and transferred into Escherichia coli DH5alpha. HPPD was functionally expressed after induction with isopropyl-beta-D-thiogalactoside (IPTG). Highest specific activities of HPPD were observed in bacterial protein extracts 5 h after induction. At the same time point the highest homogentisate concentration (110 nmol/ml) was measured in the cell-free media. The excretion of homogentisate was accompanied by a successive browning of the medium due to the formation of oxidation and polymerization products of homogentisate. The enzyme characteristics of the heterologously expressed C blumei HPPD were determined. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.