Cloning and bacterial expression of a sesquiterpene cyclase from Hyoscyamus muticus and its molecular comparison to related terpene cyclases.
Genomic and cDNA clones for vetispiradiene synthase, a sesquiterpene cyclase found in Hyoscyamus muticus, were isolated using a combination of reverse transcription-polymerase chain reactions and conventional cloning procedures. RNA blot hybridization demonstrated an induction of mRNA consistent with the induction of cyclase enzyme activity in elicitor-treated cells, DNA blot hybridization indicated a gene family of 6 to 8 members, and bacterial expression of 3 cDNA clones indicated that each coded for a vetispiradiene synthase enzyme activity catalyzing the synthesis of a single reaction product. Intron-exon organization of the vetispiradiene synthase gene was identical with that previously described for 5-epi-aristolochene synthase (tobacco sesquiterpene cyclase) and casbene synthase (castor bean diterpene cyclase), and the vetispiradiene synthase amino acid sequence was 77% identical with and 81% similar to the tobacco sesquiterpene cyclase. Regions of the vetispiradiene synthase sequence centered around amino acids 60, 100, and 370 were conspicuously different relative to the tobacco sesquiterpene cyclase. The sequence similarity between the tobacco and H. muticus enzymes is suggested to be reflective of the conservation of several partial reactions common to both enzymes, and the differences may be reflective of a partial reaction unique to each enzyme.
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Agronomy Department, University of Kentucky, Lexington 40546-0091, USA.
Chappell
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D019883:Alkyl and Aryl Transferases; D000595:Amino Acid Sequence; D001483:Base Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary; D005091:Exons; D017343:Genes, Plant; D015698:Genomic Library; D007438:Introns; D007700:Kinetics; D008969:Molecular Sequence Data; D010944:Plants / Q000201:enzymology*; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D014166:Transferases / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics
D017931:DNA Primers; D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D014166:Transferases; D019883:Alkyl and Aryl Transferases; C092952:vetispiradiene synthase