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 beta-pinene synthase named Cl(-)betaPINS, a gamma-terpinene synthase named ClgammaTS, 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(-)betaPINS and ClgammaTS and between Cl(+)LIMS2 and ClgammaTS 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(-)betaPINS and ClgammaTS, determines the specificity for the formation of beta-pinene or gamma-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 ClgammaTS. 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 ClgammaTS. Molecular modeling was used to further pinpoint the amino acids responsible for the differences in product specificity of ClgammaTS and Cl(-)betaPINS.
El Tamer
Mazen K
MK
Plant Research International B.V, P.O. Box 16, 6700 AA Wageningen, The Netherlands.
Lücker
Joost
J
Bosch
Dirk
D
Verhoeven
Harrie A
HA
Verstappen
Francel W A
FW
Schwab
Wilfried
W
van Tunen
Arjen J
AJ
Voragen
Alphons G J
AG
de Maagd
Ruud A
RA
Bouwmeester
Harro J
HJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D000081008:Bicyclic Monoterpenes; D001643:Bridged Bicyclo Compounds / Q000378:metabolism; D002384:Catalysis; D002957:Citrus / Q000201:enzymology*; D000081005:Cyclohexane Monoterpenes; D053138:Cyclohexenes; D019753:Intramolecular Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000077222:Limonene; D008958:Models, Molecular; D008969:Molecular Sequence Data; D039821:Monoterpenes / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary / Q000502:physiology; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013729:Terpenes / Q000378:metabolism
D000081008:Bicyclic Monoterpenes; D001643:Bridged Bicyclo Compounds; D000081005:Cyclohexane Monoterpenes; D053138:Cyclohexenes; D039821:Monoterpenes; D011994:Recombinant Proteins; D013729:Terpenes; C010789:beta-pinene; C018669:gamma-terpinene; D000077222:Limonene; D019753:Intramolecular Lyases; C040265:pinene cyclase I