Abstract
The committed step in the biosynthesis of monoterpenes in mint (Mentha) species is the cyclization of geranyl pyrophosphate to the olefin (-)-4S-limonene catalyzed by limonene synthase (cyclase). Internal amino acid sequences of the purified enzyme from spearmint oil glands were utilized to design three distinct oligonucleotide probes. These probes were subsequently employed to screen a spearmint leaf cDNA library, and four clones were isolated. Three of these cDNA isolates were full-length and were functionally expressed in Escherichia coli, yielding a peptide that is immunologically recognized by polyclonal antibodies raised against the purified limonene synthase from spearmint and that is catalytically active in generating from geranyl pyrophosphate a product distribution identical to that of the native enzyme (principally limonene with small amounts of the coproducts alpha- and beta-pinene and myrcene). The longest open reading frame is 1800 nucleotides and the deduced amino acid sequence contains a putative plastidial transit peptide of approximately 90 amino acids and a mature protein of about 510 residues corresponding to the native enzyme. Several nucleotide differences in the 5'-untranslated region of all three full-length clones suggest the presence of several limonene synthase genes and/or alleles in the allotetraploid spearmint genome. Sequence comparisons with a sesquiterpene cyclase, epi-aristolochene synthase from tobacco, and a diterpene cyclase, casbene synthase from castor bean, demonstrated a significant degree of similarity between these three terpenoid cyclase types, the first three examples of this large family of catalysts to be described from higher plants.
Highlights
The committed step in the biosynthesisof monoter- known, and essentially all are biosynthesizedfromgeranyl penes in mint (Mentha) species is the cyclization of pyrophosphate,theubiquitous Clo intermediate of the isogeranyl pyrophosphate to the olefin (-)-4S-limonene prenoid pathway (1,Z)
Internal toas “cyclases,”catalyze thereactions by whichgeranyl aminoacidsequences of thepurifiedenzymefrom pyrophosphate is cyclized to the various monoterpene carbon spearmint oil glands wereutilized to design threedistinct oligonucleotide probes
Research on monoterpencyeclases has been spearmint and thaist catalytically active in generatingstimulated by the possibleregulatory importance of these fromgeranylpyrophosphate a productdistribution enzymes that function aat branch point inisoprenoid metabidentical to thaotf the native enzyme (principallliym- olism [3], as well as by the commercialsignificance of the onene with small amountsof the coproducts a- and 8- essential oils [4] and aromatic resins [5] and the ecological pinene and myrcene)
Summary
Polyvinylidene difluoride membranes (Immobilon-PSQ,Millipore) were first wetted in methanol and equilibrated in 25 mM Tris, 190 mM glycine (pH 8.3) containing 20% (v/v) diterpenecyclase(casbene synthase from castor bean) has methanol and thengel blotted in the same buffer [33].Electroblotting alsobeenaccomplished.' In this paper, the isolation and was carried out for 90 min at a constant current of 100 mA using a functional expression of a (-)-4S-limonene synthase cDNA from spearmint is described This first cloning of a monoterpene cyclase was made possibbylerecently developed methods. The supernatantwas desalted into 15 mM potassium phosphate buffer (pH 6.0) containing 10% (v/v) glycerol,150 mM KCl, and 1 mM dithiothreitol and applied to a DEAE-Sepharose Fast Flow column (5 X 100mm, Pharmacia fast protein liquid chromatography system) that was eluted with a linear gradient from 150 to 600 mM KC1 in loading buffer This limonene cyclase activity expressed in E. coli eluted as a single peak between 365 and 465 mM KCl, as with the native enzyme from Mentha.
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