Cinnamic acid 4-hydroxylase mechanism-based inactivation by psoralen derivatives: cloning and characterization of a C4H from a psoralen producing plant-Ruta graveolens-exhibiting low sensitivity to psoralen inactivation.
Cinnamate 4-hydroxylase (C4H, EC 1.14.13.11) complete cDNA was cloned from the leaves of Ruta graveolens, a psoralen producing plant. The recombinant enzyme (classified CYP73A32) was expressed in Saccharomyces cerevisiae. Mechanism-based inactivation was investigated using various psoralen derivatives. Only psoralen and 8-methoxypsoralen were found to inactivate C4H. The inactivation was dependent on the presence of NADPH, time of pre-incubation, and inhibitor concentration. Inactivation stoichiometry was 0.9 (+/-0.2) for CYP73A1 and 1.1 (+/-0.2) for CYP73A32. SDS-PAGE analysis demonstrated that [3H]psoralen was irreversibly bound to the C4H apoprotein. K(i) and k(inact) for psoralen and 8-methoxypsoralen inactivation on the two C4H revealed a lower sensitivity for CYP73A32 compared to CYP73A1. Inactivation kinetics were also determined for CYP73A10, a C4H from another furocoumarin-producing plant, Petroselinum crispum. This enzyme was found to behave like CYP73A32, with a weak sensitivity to psoralen and 8-MOP inactivation. Cinnamic acid hydroxylation is a key step in the biosynthesis of phenylpropanoid compounds, psoralen derivatives included. Our results suggest a possible evolution of R. graveolens and P. crispum C4H that might tolerate substantial levels of psoralen derivatives in the cytoplasmic compartment without a depletive effect on C4H and the general phenylpropanoid metabolism.
Gravot
Antoine
A
UMR 1121 Agronomie Environnement INPL-INRA, ENSAIA 2, av. de la Forêt de Haye, 54505 Vandoeuvre-lés-Nancy Cedex, France.
Larbat
Romain
R
Hehn
Alain
A
Lièvre
Karine
K
Gontier
Eric
E
Goergen
Jean Louis
JL
Bourgaud
Frédéric
F
D000595:Amino Acid Sequence; D001059:Apoproteins / Q000378:metabolism; D003001:Cloning, Molecular; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D005363:Ficusin / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D011564:Furocoumarins / Q000096:biosynthesis / Q000737:chemistry / Q000494:pharmacology; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008730:Methoxsalen / Q000494:pharmacology; D008861:Microsomes / Q000201:enzymology; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors* / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D011869:Radioligand Assay; D032105:Ruta / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D050564:Trans-Cinnamate 4-Monooxygenase; D014316:Tritium
D001059:Apoproteins; D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D011564:Furocoumarins; D007527:Isoenzymes; D014316:Tritium; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D050564:Trans-Cinnamate 4-Monooxygenase; D005363:Ficusin; D008730:Methoxsalen