ABSTRACT
Friedelin, a pentacyclic triterpene found in the leaves of the Celastraceae species, demonstrates numerous biological activities and is a precursor of quinonemethide triterpenes, which are promising antitumoral agents. Friedelin is biosynthesized from the cyclization of 2,3-oxidosqualene, involving a series of rearrangements to form a ketone by deprotonation of the hydroxylated intermediate, without the aid of an oxidoreductase enzyme. Mutagenesis studies among oxidosqualene cyclases (OSCs) have demonstrated the influence of amino acid residues on rearrangements during substrate cyclization: loss of catalytic activity, stabilization, rearrangement control or specificity changing. In the present study, friedelin synthase from Maytenus ilicifolia (Celastraceae) was expressed heterologously in Saccharomyces cerevisiae. Site-directed mutagenesis studies were performed by replacing phenylalanine with tryptophan at position 473 (Phe473Trp), methionine with serine at position 549 (Met549Ser) and leucine with phenylalanine at position 552 (Leu552Phe). Mutation Phe473Trp led to a total loss of function; mutants Met549Ser and Leu552Phe interfered with the enzyme specificity leading to enhanced friedelin production, in addition to α-amyrin and ß-amyrin. Hence, these data showed that methionine 549 and leucine 552 are important residues for the function of this synthase.
Subject(s)
Alkyl and Aryl Transferases/metabolism , Maytenus/enzymology , Plant Proteins/metabolism , Triterpenes/metabolism , Alkyl and Aryl Transferases/chemistry , Alkyl and Aryl Transferases/genetics , Amino Acid Substitution , Biosynthetic Pathways , Cyclization , Genes, Plant , Leucine/chemistry , Maytenus/genetics , Methionine/chemistry , Models, Molecular , Mutagenesis, Site-Directed , Oleanolic Acid/analogs & derivatives , Oleanolic Acid/biosynthesis , Pentacyclic Triterpenes/metabolism , Plant Proteins/chemistry , Plant Proteins/genetics , Protein Structure, Secondary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Substrate SpecificityABSTRACT
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.
Subject(s)
Intramolecular Transferases/metabolism , Maytenus/enzymology , Plant Proteins/metabolism , Triterpenes/metabolism , Amino Acid Motifs , Binding Sites , Catalytic Domain , Intramolecular Transferases/chemistry , Intramolecular Transferases/classification , Intramolecular Transferases/genetics , Leucine/chemistry , Leucine/metabolism , Maytenus/genetics , Molecular Docking Simulation , Mutagenesis, Site-Directed , Oleanolic Acid/analogs & derivatives , Oleanolic Acid/chemistry , Oleanolic Acid/metabolism , Phylogeny , Plant Leaves/genetics , Plant Proteins/chemistry , Plant Proteins/classification , Plant Proteins/genetics , RNA, Plant/isolation & purification , RNA, Plant/metabolism , Sequence Alignment , Triterpenes/analysis , Triterpenes/chemistryABSTRACT
The absolute configuration and solution-state conformers of three peperomin-type secolignans isolated from Peperomia blanda (Piperaceae) are unambiguously determined by using vibrational circular dichroism (VCD) spectroscopy associated with density functional theory (DFT) calculations. Advantages of VCD over the electronic form of CD for the analysis of diastereomers are also discussed. This work extends our growing knowledge about secondary metabolites within the Piperaceae family species while providing a definitive and straightforward method to assess the absolute stereochemistry of secolignans.
