TRUNCATION OF LIMONENE SYNTHASE PREPROTEIN PROVIDES A FULLY ACTIVE PSEUDOMATURE FORM OF THIS MONOTERPENE CYCLASE AND REVEALS THE FUNCTION OF THE AMINO-TERMINAL ARGININE PAIR

The monoterpene cyclase limonene synthase transforms geranyl diphospha te to a monocyclic olefin and constitutes the simplest model for terpe noid cyclase catalysis. (-)-4S-Limonene synthase preprotein from spear mint bears a long plastidial targeting sequence. Difficulty expressing the full-length preprotein in Escherichia coli is encountered because of host codon usage, inclusion body formation, and the tight associat ion of bacterial chaperones with the transit peptide. The purified pre protein is also kinetically impaired relative to the mixture of N-bloc ked native proteins produced in vivo by proteolytic processing in plas tids. Therefore, the targeting sequence, that precedes a tandem pair o f arginines (R58R59) which is highly conserved in the monoterpene synt hases, was removed. Expression of this truncated protein, from a vecto r that encodes a tRNA for two rare arginine codons (pSBET), affords a soluble, tractable 'pseudomature' form of the enzyme that is catalytic ally more efficient than the native species. Truncation up to and incl uding R58, or substitution of R59, yields enzymes that are incapable o f converting the natural substrate geranyl diphosphate, via the enzyma tically formed tertiary allylic isomer 3S-linalyl diphosphate, to (-)- limonene. However, these enzymes are able to cyclize exogenously suppl ied 3S-linalyl diphosphate to the olefinic product. This result indica tes a role for the tandem arginines in the unique diphosphate migratio n step accompanying formation of the intermediate 3S-linalyl diphospha te and preceding the final cyclization reaction catalyzed by the monot erpene synthases. The structural basis for this coupled isomerization- cyclization reaction sequence can be inferred by homology modeling of (-)-4S-limonene synthase based on the three-dimensional structure of t he sesquiterpene cyclase epiaristolochene synthase.