4S-LIMONENE SYNTHASE FROM THE OIL GLANDS OF SPEARMINT (MENTHA-SPICATA) - CDNA ISOLATION, CHARACTERIZATION, AND BACTERIAL EXPRESSION OF THE CATALYTICALLY ACTIVE MONOTERPENE CYCLASE

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, a nd four clones were isolated. Three of these cDNA isolates were full-l ength and were functionally expressed in Escherichia coli, yielding a peptide that is immunologically recognized by polyclonal antibodies ra ised against the purified limonene synthase from spearmint and that is catalytically active in generating from geranyl pyrophosphate a produ ct distribution identical to that of the native enzyme (principally li monene 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 pep tide of approximately 90 amino acids and a mature protein of about 510 residues corresponding to the native enzyme. Several nucleotide diffe rences in the 5'-untranslated region of all three full-length clones s uggest the presence of several limonene synthase genes and/or alleles in the allotetraploid spearmint genome. Sequence comparisons with a se squiterpene cyclase, epi-aristolochene synthase from tobacco, and a di terpene cyclase, casbene synthase from castor bean, demonstrated a sig nificant degree of similarity between the three terpenoid cyclase type s, the first three example of this large family of catalysts to be des cribed from higher plants.