Functional expression of cinnamate 4-hydroxylase from Ammi majus L.
Total RNA was isolated from dark-grown cell suspension cultures of Ammi majus L. that had been induced with fungal elicitor or treated with water for control and used as template with cytochrome P450-specific primers for DD-RT-PCR amplifications. A cDNA clone was generated from the elicited transcripts and assigned to cinnamate 4-monooxygenase based on sequence alignments and functional expression in yeast cells. Comparison of the translated polypeptide with database accessions of heterologous cytochrome P450 monooxygenases revealed a high degree of similarity (99.6%) with 98.6% identity to cinnamic acid 4-hydroxylase from parsley, documenting the close evolutionary relationship within the Apiaceae family. Maximal activity of the Ammi hydroxylase in yeast microsomes was determined at 25 degrees C and in the pH range of 6.5-7.0 reaching 2.5 pkat/mg on average. An apparent K(m) of 8.9 microM was determined for cinnamate. Preincubations with psoralen or 8-methoxypsoralen added up to 100 microM in the presence or absence of NADPH hardly affected the turnover rate. A. majus cell cultures accumulate sets of O-prenylated umbelliferones and linear furanocoumarins besides lignin-like compounds upon treatment with elicitor, and cinnamic acid 4-hydroxylase catalyzes the initial reaction leading from the general into the various phenylpropanoid branch pathways. Correspondingly, the hydroxylase transcript abundance was induced in the elicited cells.
Hübner
Silvia
S
Institut für Pharmazeutische Biologie, Philipps-Universität Marburg, Deutschhausstrasse 17 A, D-35037 Marburg, Germany.
Hehmann
Marc
M
Schreiner
Stephan
S
Martens
Stefan
S
Lukacin
Richard
R
Matern
Ulrich
U
D000595:Amino Acid Sequence; D031165:Ammi / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D003374:Coumarins / Q000737:chemistry / Q000378:metabolism; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology; D011564:Furocoumarins / Q000378:metabolism / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008861:Microsomes / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D028528:Petroselinum / Q000201:enzymology; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D050564:Trans-Cinnamate 4-Monooxygenase; D015003:Yeasts / Q000378:metabolism
D003374:Coumarins; D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D011564:Furocoumarins; D012313:RNA; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D050564:Trans-Cinnamate 4-Monooxygenase