Nearly every aspect of sterol biosynthesis has been studied or exploited as means of regulating pathway flux, inhibiting specific enzymatic reactions, elucidating product cyclization or determining the evolutionary nature of the pathway itself. Despite enormous efforts in this area pathway regulation, product formation and the evolutionary origins of sterol biosynthesis remain unknown. Described herein is the use of molecular, synthetic and phylogenetic techniques to examine how oxidosqualene cyclases (OSC) (a specific enzyme within the sterol biosynthetic pathway) have evolved over time and how this class of enzymes controls the cyclization of oxidosqualene to a variety of triterpenes.
OSCs are a unique family of enzymes producing over 100 naturally occurring cyclization products ranging from mono to hexacyclic compounds. These enzymes display high sequence similarity and it is predicted that OSCs share a similar three-dimensional structure, while subtle residue changes in the active site alter catalysis. There are two subclasses of OSCs, protosteryl and dammarenyl, and they are defined by the tetracyclic intermediate cation formed during cyclization. Residues that are differentially conserved between protosteryl and dammarenyl type cyclases were identified as candidates imparting a specific catalytic function. Using DNA synthesis, libraries of chimeric enzymes were generated that blended the sequences of one protosteryl-type cyclase (cycloartenol synthase from Arabidopsis thaliana) and a dammarenyl-type cyclase (lupeol synthase from Olea europaea) and screened for the ability to produce protosteryl-type products. Using this approach we were able to narrow the number of residues imparting specific catalytic function from 759 to 15 candidate residues within the Arabidopsis thaliana cycloartenol synthase. This information prompted direct mutagenesis of several single residues to better our understanding of the active site environment.
To complement the DNA synthesis experiments, the second half of this thesis describes the cloning and characterization of the Methylococcus capsulatus lanosterol synthase, the Gemmata obscuriglobus parkeol synthase and several other bacterial/ancient cyclases. Sterols, once thought to be only eukaryotic in nature, are now being isolated from many prokaryotic organisms. Whether OSCs evolved in prokaryotes or were acquired later via horizontal gene transfer remains unknown. By studying both the residue conservation patterns and the product profiles of these bacterial cyclases, we have been able to formulate a working hypothesis that supports the evolution of sterols in prokaryotes. These primitive cyclases also retain residue conservation patterns that vary from eukaryotic enzymes indicating they direct product formation differently than more modern cyclases.