Friedelin in Maytenus ilicifolia Is Produced by Friedelin Synthase Isoforms.

Triterpenes are interesting compounds because they play an important role in cell homeostasis and a wide variety exhibiting defense functions is produced by plant secondary metabolism. Those same plant secondary metabolites also exhibit biological properties with promising therapeutic potential as anti-inflammatory and antitumor agents. Friedelin is a triterpene ketone with anti-inflammatory and gastroprotective activities and it is a precursor of relevant antitumor quinonemethides. Although many triterpene synthases have been described, only two friedelin synthases were characterized and there is no information about their genomic features and alleles. In the present work, we aimed to identify the gene and new isoforms of friedelin synthase in Maytenus ilicifolia leaves to be functionally characterized in Saccharomyces cerevisiae. The gene sequence analysis elucidated the exon/intron structure and confirmed the presence of single nucleotide polymorphisms with four non-synonymous mutations outside the active site of the enzyme. Therefore, two new isoforms were observed and the heterologous production of the enzymes in yeast showed similar production of friedelin. This first description of different alleles of the gene of friedelin synthase in M. ilicifolia can guide their validation as markers for friedelin-producer specimens.

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

Among the biologically active triterpenes, friedelin has the most-rearranged structure produced by the oxidosqualene cyclases and is the only one containing a cetonic group. In this study, we cloned and functionally characterized friedelin synthase and one cycloartenol synthase from Maytenus ilicifolia (Celastraceae). The complete coding sequences of these 2 genes were cloned from leaf mRNA, and their functions were characterized by heterologous expression in yeast. The cycloartenol synthase sequence is very similar to other known OSCs of this type (approximately 80% identity), although the M. ilicifolia friedelin synthase amino acid sequence is more related to ß-amyrin synthases (65-74% identity), which is similar to the friedelin synthase cloned from Kalanchoe daigremontiana. Multiple sequence alignments demonstrated the presence of a leucine residue two positions upstream of the friedelin synthase Asp-Cys-Thr-Ala-Glu (DCTAE) active site motif, while the vast majority of OSCs identified so far have a valine or isoleucine residue at the same position. The substitution of the leucine residue with valine, threonine or isoleucine in M. ilicifolia friedelin synthase interfered with substrate recognition and lead to the production of different pentacyclic triterpenes. Hence, our data indicate a key role for the leucine residue in the structure and function of this oxidosqualene cyclase.