Structural, functional, and mechanistic studies or terpene synthases
2007
Mahrez, W.
Monoterpene synthases are the key enzymes in the monoterpene biosynthetic pathway, as they catalyze the cyclisation of the ubiquitous geranyl diphosphate to the specific monoterpene skeletons. Monoterpene synthases are nuclear encoded preproteins that are destined to be imported in the plastids, where they are proteolyticaly processed into their mature forms. Attempts to get activity from Salvia fruticosa cDNA library clone 215 were unsuccessful. Nevertheless, subcloning of clone P20, a gene with close homology to sesquiterpene cyclases isolated from the S. pomifera EST library, into another strong expression vector "pRSETa modified" was successful in confirming the sesquiterpene synthase activity of this enzyme. The alignment and comparison of the primary structure of SfCinS1 with those of monoterpene synthases of only very closely related species and computer modeling of its structure onto the crystal structure of bornyl diphosphate synthase revealed a number of residues that were conserved between the different synthases and others which varied and could play a role in product specificity. These variable residues that appear to be clustered in two regions were the target for site-directed mutagenesis to assess their role in product specificity. Substitution of Asn338 with 5 other residues allowed the production of 5 additional terpene synthases with remarkably different product profiles. Furthermore, new volatile products not previously detected using the cell extract for the enzymatic assay were obtained from analysis using purified enzyme variants.
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Эту запись предоставил Mediterranean Agronomic Institute of Chania