The major protein of guayule rubber particles is a cytochrome P450. Characterization based on cDNA cloning and spectroscopic analysis of the solubilized enzyme and its reaction products.
Guayule plants accumulate large quantities of rubber within parenchyma cells of their stembark tissues. This rubber is packed within discrete organelles called rubber particles composed primarily of a lipophilic, cis-polyisoprene core, small amounts of lipids, and several proteins, the most abundant of which is the M(r) 53,000 rubber particle protein (RPP). We have cloned and sequenced a full-length cDNA for RPP and show that it has 65% amino acid identity and 85% similarity to a cytochrome P450 known as allene oxide synthase (AOS), recently identified from flaxseed. RPP contains the same unusual heme-binding region and possesses a similar defective I-helix region as AOS, suggesting an equivalent biochemical function. Spectral analysis of solubilized RPP verifies it as a P450, and enzymatic assays reveal that it also metabolizes 13(S)-hydroperoxy-(9Z,11E)-octadecadienoic acid into the expected ketol fatty acids at rates comparable with flaxseed AOS. RPP is unusual in that it lacks the amino-terminal membrane anchor and the established organelle targeting sequences found on other conventional P450s. Together, these factors place RPP in the CYP74 family of P450s and establish it as the first P450 localized in rubber particles and the first eukaryotic P450 to be identified outside endoplasmic reticulum, mitochondria, or plastids.
Pan
Z
Z
Department of Botany, Arizona State University, Tempe 85287-1601, USA.
Durst
F
F
Werck-Reichhart
D
D
Gardner
H W
HW
Camara
B
B
Cornish
K
K
Backhaus
R A
RA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018076:DNA, Complementary; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D012408:Rubber; D017386:Sequence Homology, Amino Acid; D012996:Solutions; D013056:Spectrophotometry, Ultraviolet
D018076:DNA, Complementary; D010940:Plant Proteins; D012996:Solutions; D012408:Rubber; D003577:Cytochrome P-450 Enzyme System; C093121:RPP30 protein, Parthenium argentatum