Investigations of the Specificity of Oxidosqualene Cyclization: Errors are the Rule, Not the Exception
Bodager, Paul Gregory
Matsuda, Seiichi P.T.
Doctor of Philosophy
This thesis describes the characterization of oxidosqualene cyclases from numerous organisms, through heterologous expression in Saccharomyces cerevisiae, and extraction from organismal tissue. Oxidosqualene cyclases are a family of proteins which catalyze the cyclization of the linear substrate oxidosqualene into cyclic compounds known as triterpene alcohols, acting in both the primary and secondary metabolism of organisms. Detailed analyses of cyclase product profiles in both primary and secondary metabolism are used herein to develop a comprehensive discussion of cyclase product specificity. First, the characterization of two oxidosqualene cyclases of secondary metabolism from the plant Arabidopsis thaliana, LUP4 and LUP5, by heterologous expression, is described. While demonstrating quite different product specificity, both cyclases make a mixture of nearly 20 triterpene alcohols. The isolation of a novel triterpene alcohol, (20S)-dammara-12,24-dienol, is reported. Next, the characterization of six oxidosqualene cyclases of primary metabolism are detailed, including lanosterol synthases from S. cerevisiae, Trypanosoma cruzi, Trypanosoma brucei, Homo sapiens, Bos taurus, and cycloartenol synthase from A. thaliana. Despite no reports of minor product generation by lanosterol synthases prior to this work, each cyclase is shown to make minor “errors”. These cyclases make different sets of minor products, and produce the major product with varying accuracy. This work demonstrates that minor product formation is characteristic of oxidosqualene cyclization, and leads to the conclusion that no cyclase produces only a single product. Finally, lanosterol synthase product profiles are extended to in vivo systems, via the analysis of triterpene alcohols present in yeast culture, as well as in mammalian tissue. This analysis demonstrates that S. cerevisiae lanosterol synthase produces at least 16 products, including three generated through B-ring-chair intermediates, the first evidence of a non-mutant cyclase accessing B-ring-boat and B-ring-chair intermediates. Analysis of bovine brain extracts led to the discovery that 18 lanosterol synthase minor products are detectable in mammalian tissue, including two novel triterpene alcohols, protosta-20(22)E-dienol and CB-thalianol A. Finally, this analysis demonstrated that one lanosterol synthase minor product, parkeol, is metabolized by enzymes in the sterol biosynthetic pathway, demonstrating that enzymatic errors generate a previously hidden level of chemical diversity in primary metabolism.
Oxidosqualene Cyclase; Lanosterol Synthase; Sterol Biosynthesis, Triterpene, Triterpenoid