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

摘要

The monoterpene cyclase limonene synthase transforms geranyl diphosphate to a monocyclic olefin and constitutes the simplest model for terpenoid cyclase catalysis. (-)-4S-Limonene synthase preprotein from spearmint 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 association of bacterial chaperones with the transit peptide. The purified preprotein is also kinetically impaired relative to the mixture of N-blocked native proteins produced in vivo by proteolytic processing in plastids. Therefore, the targeting sequence, that precedes a tandem pair of arginines (R58R59) which is highly conserved in the monoterpene synthases, was removed. Expression of this truncated protein, from a vector that encodes a tRNA for two rare arginine codons (pSBET), affords a soluble, tractable 'pseudomature' form of the enzyme that is catalytically more efficient than the native species. Truncation up to and including R58, or substitution of R59, yields enzymes that are incapable of converting the natural substrate geranyl diphosphate, via the enzymatically formed tertiary allylic isomer 3S-linalyl diphosphate, to (-)-limonene. However, these enzymes are able to cyclize exogenously supplied 3S-linalyl diphosphate to the olefinic product. This result indicates a role for the tandem arginines in the unique diphosphate migration step accompanying formation of the intermediate 3S-linalyl diphosphate and preceding the final cyclization reaction catalyzed by the monoterpene 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 the sesquiterpene cyclase epiaristolochene synthase. [References: 60]