2-3-oxidosqualene and 3-hydroxydiploptene

Triterpenes are one of the largest classes of plant metabolites and have important functions. A diverse array of triterpenoid skeletons are synthesized via the isoprenoid pathway by enzymatic cyclization of 2,3-oxidosqualene. The genomes of the lower plants Chlamydomonas reinhardtii and moss (Physcomitrella patens) contain just one oxidosqualene cyclase (OSC) gene (for sterol biosynthesis), whereas the genomes of higher plants contain nine to 16 OSC genes. Here we carry out functional analysis of rice OSCs and rigorous phylogenetic analysis of 96 OSCs from higher plants, including Arabidopsis thaliana, Oryza sativa, Sorghum bicolor and Brachypodium distachyon. The functional analysis identified an amino acid sequence for isoarborinol synthase (OsIAS) (encoded by Os11g35710/OsOSC11) in rice. Our phylogenetic analysis suggests that expansion of OSC members in higher plants has occurred mainly through tandem duplication followed by positive selection and diversifying evolution, and consolidated the previous suggestion that dicot triterpene synthases have been derived from an ancestral lanosterol synthase instead of directly from their cycloartenol synthases. The phylogenetic trees are consistent with the reaction mechanisms of the protosteryl and dammarenyl cations which parent a wide variety of triterpene skeletal types, allowing us to predict the functions of the uncharacterized OSCs.

Topics: Arabidopsis; Brachypodium; Cyclization; Evolution, Molecular; Gene Duplication; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Plant; Intramolecular Transferases; Multigene Family; Oryza; Phylogeny; Sorghum; Squalene; Triterpenes

1. A cell-free system from the bacterium Methylococcus capsulatus was incubated with [12-3H]-squalene; diploptene and diplopterol, normally present in the bacterium, were labelled. 2 The same cell-free system was incubated with (RS)-2,3-epoxy-2,3-dihydro-[3-3H]squalene. Several radioactive 3-hydroxytriterpenes were purifed. Lanosterol, which is normally present in this bacterium, was found labelled as well as 3-epilanosterol. In addition, radioactive 3 alpha-hydroxy and 3 beta-hydroxydiploptene were formed. 3. These data may be explained by the coexistence of two cyclases in M. capsulatus: a squalene/hopane cyclase and a squalene epoxide/lanosterol cyclase. The squalene cyclase exhibits the same lack of substrate specificity as those of Acetobacter pasteurianum and Tetrahymena pyriformis, i.e. in addition to its normal substrate squalene, it can cyclize the two enantiomers of squalene epoxide into 3-hydroxyhopanoids. 4. The presence of a squalene epoxide/lanosterol cyclase activity, which was suspected in view of the unique 3 beta-hydroxy 4 alpha-methyl steroids of M. capsulatus, was demonstrated by the labelling of lanosterol. More surprisingly 3-epilanosterol was also present and labelled. We showed that this does not derive from lanosterol by isomerization via a 3-oxo compound. Therefore the squalene expoxide cyclase of M. capsulatus, like the one of eukaryotes cyclizes the (3S) enantiomer of squalene epoxide into lanosterol. But it is definitely less substrate-specific as it can also cyclize the (3R) enantiomer into 3-epilanosterol.

Topics: Cyclization; Lanosterol; Lyases; Methylococcaceae; Squalene; Stereoisomerism; Substrate Specificity; Triterpenes