Geraniol hydroxylase and hydroxygeraniol oxidase activities of the CYP76 family of cytochrome P450 enzymes and potential for engineering the early steps of the (seco)iridoid pathway.
The geraniol-derived (seco)iridoid skeleton is a precursor for a large group of bioactive compounds with diverse therapeutic applications, including the widely used anticancer molecule vinblastine. Despite of this economic prospect, the pathway leading to iridoid biosynthesis from geraniol is still unclear. The first geraniol hydroxylation step has been reported to be catalyzed by cytochrome P450 enzymes such as CYP76B6 from Catharanthus roseus and CYP76C1 from Arabidopsis thaliana. In the present study, an extended functional analysis of CYP76 family members was carried-out to identify the most effective enzyme to be used for pathway reconstruction. This disproved CYP76C1 activity and led to the characterization of CYP76C4 from A. thaliana as a geraniol 9- or 8-hydroxylase. CYP76B6 emerged as a highly specialized multifunctional enzyme catalyzing two sequential oxidation steps leading to the formation of 8-oxogeraniol from geraniol. This dual function was confirmed in planta using a leaf-disc assay. The first step, geraniol hydroxylation, was very efficient and fast enough to outcompete geraniol conjugation in plant tissues. When the enzyme was expressed in leaf tissues, 8-oxogeraniol was converted into further oxidized and/or reduced compounds in the absence of the next enzyme of the iridoid pathway.
Institut de Biologie Moléculaire des Plantes, CNRS UPR 2357-Université de Strasbourg, France.
René
R
Höfer
Lemeng
L
Dong
François
F
André
Jean-François
JF
Ginglinger
Raphael
R
Lugan
Carole
C
Gavira
Sebastien
S
Grec
Gerhard
G
Lang
Johan
J
Memelink
Sander
S
Van der Krol
Harro
H
Bouwmeester
Danièle
D
Werck-Reichhart
D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D057888:Iridoid Glucosides / Q000378:metabolism*; D010084:Oxidation-Reduction
D029681:Arabidopsis Proteins; D057888:Iridoid Glucosides; D003577:Cytochrome P-450 Enzyme System
Catharanthus roseus; Cytochrome P450; Geraniol oxidation; In planta enzyme assay; Multifunctional enzyme; Pathway reconstitution