Domain swapping of Citrus limon monoterpene synthases: Impact on enzymatic activity and product specificity

Monoterpene cyclases are the key enzymes in the monoterpene biosynthetic pathway, as they catalyze the cyclization of the ubiquitous geranyl diphosphate (GDP) to the specific monoterpene skeletons. From Citrus limon, four monoterpene synthase-encoding cDNAs for a β-pinene synthase named Cl(-)βPINS, a γ-terpinene synthase named ClγTS, and two limonene synthases named Cl(+)LIMS1 and Cl(+)LIMS2 were recently isolated [J. Lücker et al., Eur. J. Biochem. 269 (2002) 3160]. The aim of our work in this study was to identify domains within these monoterpene synthase enzymes determining the product specificity. Domain swapping experiments between Cl(-)βPINS and ClγTS and between Cl(+)LIMS2 and ClγTS were conducted. We found that within the C-terminal domain of these monoterpene synthases, a region comprising 200 amino acids, of which 41 are different between Cl(-)βPINS and ClγTS, determines the specificity for the formation of β-pinene or γ-terpinene, respectively, while another region localized further downstream is required for a chimeric enzyme to yield products in the same ratio as in the wild-type ClγTS. For Cl(+)LIMS2, the two domains together appear to be sufficient for its enzyme specificity, but many chimeras were inactive probably due to the low homology with ClγTS. Molecular modeling was used to further pinpoint the amino acids responsible for the differences in product specificity of ClγTS and Cl(-)βPINS.