cDNA isolation and characterization of guaiadiene synthase from lxeris dentata form. albiflora hara

Abstract

various sesquiterpenes, whose first committed step in biosynthesis is cyclization of farnesyl diphosphate by terpene synthase. To isolate the synthase gene, we amplified 184-bp DNA fragment of a sesquiterpene synthase from I. dentata genomic DNA using homology-based PCR technique. Cloning and sequencing of the PCR product showed two groups of partial sequences of sesquiterpene synthases, which suggest the presence of at least two enzymes. The sequence information derived from rapid amplification of cDNA ends was used to produce 1956 bp of a full-length cDNA sequence including 1755 bp of an open reading frame for 584 amino acids with a deduced size of 67.1 kDa and a pI of 5.16. The deduced amino acid sequence displayed 38% identity with 5-epi-aristolochene synthase of Nicotiana tabacum. The partially purified recombinant synthase has temperature and pH optimum at 37 ¡É and pH 7.5 - 8.0, and Km of 4.3 and 14.9 M at 25 and 37 ¡É, respectively. Expressed protein was inactive with geranyl diphosphate, but catalyzed the cyclization of farnesyl diphosphate to produce a sesquiterpene, which was identified through GC-MS and NMR analyses as guaiadiene. This is the first report on cDNA isolation of the sesquiterpene synthase producing a guaiane-type skeleton.