Chrysanthemyl diphosphate synthase_Isolation of the gene and characterization of the recombinant non

Chrysanthemyl diphosphate synthase_Isolation of the gene and characterization of the recombinant non

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Chrysanthemyl diphosphate synthase: Isolation of the gene and characterization of the recombinant non-head-to-tail monoterpene synthase from Chrysanthemum cinerariaefolium Susan B. Rivera*, Bradley D. Swedlund ²‡ , Gretchen J. King ²§ , Russell N. Bell ²‡ , Charles E. Hussey, Jr. ² , Donna M. Shattuck-Eidens ²‡ , Wislawa M. Wrobel ² , Galen D. Peiser ² , and C. Dale Poulter* *Department of Chemistry, University of Utah, Salt Lake City, UT 84112; and ² Agridyne Technologies, Salt Lake City, UT 84112 Edited by Rodney B. Croteau, Washington State University, Pullman, WA, and approved January 18, 2001 (received for review November 15, 2000) Chrysanthemyl diphosphate synthase (CPPase) catalyzes the con- densation of two molecules of dimethylallyl diphosphate to pro- duce chrysanthemyl diphosphate (CPP), a monoterpene with a non-head-to-tail or irregular c1 * -2-3 linkage between isoprenoid units. Irregular monoterpenes are common in Chrysanthemum cinerariaefolium and related members of the Asteraceae family. In C. cinerariaefolium, CPP is an intermediate in the biosynthesis of the pyrethrin ester insecticides. CPPase was purified from imma- ture chrysanthemum flowers, and the N terminus of the protein was sequenced. A C. cinerariaefolium l cDNA library was screened by using degenerate oligonucleotide probes based on the amino acid sequence to identify a CPPase clone that encoded a 45-kDa preprotein. The first 50 aa of the ORF constitute a putative plastidial targeting sequence. Recombinant CPPase bearing an N-terminal polyhistidine affinity tag in place of the targeting sequence was purified to homogeneity from an overproducing Escherichia coli strain by Ni 2 1 chromatography. Incubation of recombinant CPPase with dimethylallyl diphosphate produced CPP. The diphosphate ester was hydrolyzed by alkaline phosphatase, and the resulting monoterpene alcohol was analyzed by GC y MS to confirm its structure. The amino acid sequence of CPPase aligns closely with that of the chain elongation prenyltransferase farnesyl diphosphate synthase rather than squalene synthase or phytoene synthase, which catalyze c1 * -2-3 cyclopropanation reactions similar to the CPPase reaction. O ver 30,000 isoprenoid compounds have now been identified (1). The vast majority of these compounds have ‘‘regular’’ 1 9 -4, or head-to-tail, linkages between isoprenoid units (2). The pathway for biosynthesis of regular isoprenoids from the basic five-carbon precursors isopentenyl diphosphate (IPP) and dim- ethylallyl diphosphate (DMAPP) is well established. More com- plex hydrocarbon skeletons are typically generated by prenyl transfer reactions, where successive molecules of IPP are added to a growing allylic diphosphate chain through 1 9 -4 linkages (3, 4). ‘‘Irregular,’’ or non-head-to-tail, isoprenoids are encountered less frequently. The most prominent examples are the 1 9 -1 isoprenoid compounds squalene (SQ) and phytoene (PT), which are precursors for biosynthesis of sterols and carotenoids (2). However, the greatest variety of non-head-to-tail structures are
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This note was uploaded on 08/01/2009 for the course HORT hor-11-12 taught by Professor Park during the Spring '09 term at A.T. Still University.

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Chrysanthemyl diphosphate synthase_Isolation of the gene and characterization of the recombinant non

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