cDNA isolation and characterization of (+)-germacrene a synthase fromIxerls dentata form.albiflora Hara

Abstract

Ixeris dentata form.albiflora Hara, a perennial medicinal herb, accumulates various sesquiterpenes whose first committed step in biosynthesis is the cyclization of farnesyl diphosphate by terpene synthase. To isolate the synthase geneldGAS, we amplified a 184-bp DNA fragment of sesquiterpene synthase gene fromI. dentata genomic DNA, using a homology-based PCR technique. Sequence information derived from the rapid amplification of cDNA ends was used to produce a 1956-bp full-length cDNA sequence. This included a 1755-b open reading frame for 584 amino acids, with a deduced size of 67.1 kDa and a pi of 5.16. The partially purified recombinant synthase had an optimum temperature and pH at 37°C and 7.5 to 8.0, respectively, as well as aK _m of 11.0 and 14.9 mM at 25 and 37°C, respectively. The expressed protein was inactive with geranyl diphosphate, but did catalyze the cyclization of farnesyl diphosphate to produce a sesquiterpene that was then identified through GC-MS and NMR analyses as (+)-germacrene A. When 44 residues were deleted from its N-terminal, the mutant lost 90% of its activity, suggesting that additional residues are necessary for full enzymatic activity. Transcript levels were comparable between roots and leaves, but began to decline in leaves near the onset of flowering.