Germacrene C synthase from Lycopersicon esculentum cv. VFNT cherry tomato: cDNA isolation, characterization, and bacterial expression of the multiple product sesquiterpene cyclase.
Germacrene C was found by GC-MS and NMR analysis to be the most abundant sesquiterpene in the leaf oil of Lycopersicon esculentum cv. VFNT Cherry, with lesser amounts of germacrene A, guaia-6,9-diene, germacrene B, beta-caryophyllene, alpha-humulene, and germacrene D. Soluble enzyme preparations from leaves catalyzed the divalent metal ion-dependent cyclization of [1-3H]farnesyl diphosphate to these same sesquiterpene olefins, as determined by radio-GC. To obtain a germacrene synthase cDNA, a set of degenerate primers was constructed based on conserved amino acid sequences of related terpenoid cyclases. With cDNA prepared from leaf epidermis-enriched mRNA, these primers amplified a 767-bp fragment that was used as a hybridization probe to screen the cDNA library. Thirty-one clones were evaluated for functional expression of terpenoid cyclase activity in Escherichia coli by using labeled geranyl, farnesyl, and geranylgeranyl diphosphates as substrates. Nine cDNA isolates expressed sesquiterpene synthase activity, and GC-MS analysis of the products identified germacrene C with smaller amounts of germacrene A, B, and D. None of the expressed proteins was active with geranylgeranyl diphosphate; however, one truncated protein converted geranyl diphosphate to the monoterpene limonene. The cDNA inserts specify a deduced polypeptide of 548 amino acids (Mr = 64,114), and sequence comparison with other plant sesquiterpene cyclases indicates that germacrene C synthase most closely resembles cotton delta-cadinene synthase (50% identity).
Colby
S M
SM
U.S. Department of Agriculture/University of California, Berkeley, Plant Gene Expression Center and Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA.
Crock
J
J
Dowdle-Rizzo
B
B
Lemaux
P G
PG
Croteau
R
R
D019883:Alkyl and Aryl Transferases / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D000595:Amino Acid Sequence; D019755:Carbon-Carbon Lyases / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D003001:Cloning, Molecular; D017124:Conserved Sequence; D017931:DNA Primers; D004926:Escherichia coli; D018551:Solanum lycopersicum / Q000201:enzymology*; D008969:Molecular Sequence Data; D010938:Plant Oils; D011106:Polyisoprenyl Phosphates / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D012717:Sesquiterpenes / Q000737:chemistry / Q000378:metabolism
D017931:DNA Primers; D010938:Plant Oils; D011106:Polyisoprenyl Phosphates; D011994:Recombinant Proteins; D012717:Sesquiterpenes; C004808:farnesyl pyrophosphate; D019883:Alkyl and Aryl Transferases; C111249:germacrene C synthase; D019755:Carbon-Carbon Lyases; C051130:trichodiene synthetase