Monoterpene biosynthesis in lemon (Citrus limon). cDNA isolation and functional analysis of four monoterpene synthases.
Citrus limon possesses a high content and large variety of monoterpenoids, especially in the glands of the fruit flavedo. The genes responsible for the production of these monoterpenes have never been isolated. By applying a random sequencing approach to a cDNA library from mRNA isolated from the peel of young developing fruit, four monoterpene synthase cDNAs were isolated that appear to be new members of the previously reported tpsb family. Based on sequence homology and phylogenetic analysis, these sequences cluster in two separate groups. All four cDNAs could be functionally expressed in Escherichia coli after removal of their plastid targeting signals. The main products of the enzymes in assays with geranyl diphosphate as substrate were (+)-limonene (two cDNAs) (-)-beta-pinene and gamma-terpinene. All enzymes exhibited a pH optimum around 7; addition of Mn(2+) as bivalent metal ion cofactor resulted in higher activity than Mg(2+), with an optimum concentration of 0.6 mm. K(m) values ranged from 0.7 to 3.1 microm. The four enzymes account for the production of 10 out of the 17 monoterpene skeletons commonly observed in lemon peel oil, corresponding to more than 90% of the main components present.
Lücker
Joost
J
Business Unit Cell Cybernetics, Plant Research International, 6700 AA Wageningen, the Netherlands.
El Tamer
Mazen K
MK
Schwab
Wilfried
W
Verstappen
Francel W A
FW
van der Plas
Linus H W
LH
Bouwmeester
Harro J
HJ
Verhoeven
Harrie A
HA
D000595:Amino Acid Sequence; D000081008:Bicyclic Monoterpenes; D001643:Bridged Bicyclo Compounds / Q000378:metabolism; D002957:Citrus / Q000235:genetics / Q000378:metabolism*; D000081005:Cyclohexane Monoterpenes; D053138:Cyclohexenes; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics; D008401:Gas Chromatography-Mass Spectrometry; D019753:Intramolecular Lyases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000077222:Limonene; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D008969:Molecular Sequence Data; D039821:Monoterpenes; D010802:Phylogeny; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013237:Stereoisomerism; D013729:Terpenes / Q000378:metabolism*
D000081008:Bicyclic Monoterpenes; D001643:Bridged Bicyclo Compounds; D000081005:Cyclohexane Monoterpenes; D053138:Cyclohexenes; D018076:DNA, Complementary; D039821:Monoterpenes; D011994:Recombinant Proteins; D013729:Terpenes; D008345:Manganese; C010789:beta-pinene; C018669:gamma-terpinene; D000077222:Limonene; D019753:Intramolecular Lyases; C040265:pinene cyclase I; D008274:Magnesium