Cloning and Characterization of Oxidosqualene Cyclases from Kalanchoe daigremontiana

Abstract

The first committed step in triterpenoid biosynthesis is the cyclization of oxidosqualene to polycyclic alcohols or ketones C 30 H 50 O. It is catalyzed by single oxidosqualene cyclase (OSC) enzymes that can carry out varying numbers of carbocation rearrangements and, thus, generate triterpenoids with diverse carbon skeletons. OSCs from diverse plant species have been cloned and characterized, the large majority of them catalyzing relatively few rearrangement steps. It was recently predicted that special OSCs must exist that can form friedelin, the pentacyclic triterpenoid whose formation involves the maximum possible number of rearrangement steps. The goal of the present study, therefore, was to clone a friedelin synthase from Kalanchoe daigremontiana, a plant species known to accumulate this triterpenoid in its leaf surface waxes. Five OSC cDNAs were isolated, encoding proteins with 761-779 amino acids and sharing between 57.4 and 94.3% nucleotide sequence identity. Heterologous expression in yeast and GC-MS analyses showed that one of the OSCs generated the steroid cycloartenol together with minor side products, whereas the other four enzymes produced mixtures of pentacyclic triterpenoids dominated by lupeol (93%), taraxerol (60%), glutinol (66%), and friedelin (71%), respectively. The cycloartenol synthase was found expressed in all leaf tissues, whereas the lupeol, taraxerol, glutinol, and friedelin synthases were expressed only in the epidermis layers lining the upper and lower surfaces of the leaf blade. It is concluded that the function of these enzymes is to form respective triterpenoid aglycones destined to coat the leaf exterior, probably as defense compounds against pathogens or herbivores.

Friedelin Synthase from Maytenus ilicifolia: Leucine 482 Plays an Essential Role in the Production of the Most Rearranged Pentacyclic Triterpene

“…4). As previously described, friedelin, glutinol and lupeol synthases from K. daigremontiana (Kd FRS, Kd GLS and Kd LUS) form one branch, while taraxerol synthase from the same species ( Kd TAS) is closely related to β-amyrin synthases from Betula platyphylla (BPY) and from Panax ginseng (PNY1 and PNY2)3. Nevertheless, the Mi CAS1 enzyme clusters closely to the cycloartenol synthases clade.…”

“…All of the sequences showed the presence of the Asp-Cys-Thr-Ala-Glu (DCTAE) motif, which contains the catalytic aspartic acid residue (initiator of oxidosqualene cyclization-substrate carbocation formation)4243. They also showed the four QW motifs (conserved motifs rich in aromatic amino acids, starting with Q-Gln and ending with W-Trp), which are suggested to be important for enzyme structure maintenance344. The presence of the characteristic Gly-Tyr-Asn (GYN) residues and the conserved Met-Trp-Cys-His-Cys-Arg (MWCHCR) motif from cycloartenol synthases as well as the Ser-Phe (SF) residues and conserved Met-X-Cys-Y-Cys-Arg (MXCYCR) motif from pentacyclic triterpene synthases at the predicted positions are in agreement with the functional classification of Mi CAS1 and Mi FRS, respectively3435 (Fig.…”

“…1). Among plant pentacyclic triterpenes, germanicol is the molecule with the smallest number of rearrangements, while friedelin 1 has the highest number234.…”

“…Thus, the structural features related to its activity have not been addressed3. Therefore, to investigate the structural basis underlying the product specificity, we successfully cloned and expressed OSC genes from Maytenus ilicifolia .…”

β‐Amyrin synthase from Conyza blinii expressed in Saccharomyces cerevisiae

“…The tags Cb54088, Cb70382, Cb827, and Cb874 were phylogenetically related to Ricinus communis LUS 20. Cb72002 was similar to LUS from Kalanchoe daigremontiana , which produces lupeol and β‐amyrin in a ratio of 13 : 1 21. In addition, another four tags Cb34533, Cb35585, Cb38895, and Cb46070 were homologous to DS from the Panax species, which is involved in the ginsenoside biosynthetic pathway 22, 23.…”