Date of Award
Doctor of Philosophy
Biochemistry, Biophysics and Molecular Biology
Reuben J. Peters
Labdane-related diterpenoids (LRDs) are a large group of natural products (over 7,000 known) with broad applications whose chemical complexity is significantly contributed by various diterpene skeletons which are provided by class II diterpene cyclases (DTCs) and class I diterpene synthases (DTSs). Recent results have highlighted the structural diversity that can be generated by DTCs. However, the specificity of subsequently acting DTSs largely restricts the diterpene diversity. The extreme promiscuity of the terpentetriene synthase from Kitasatospora griseola (KgTS) and the sclareol synthase from Salvia sclarea (SsSS) not only enables combinatorial biosynthesis of diterpenes but also extends the current diterpene library with new enzymatic activity to produce elaborated 'non-natural' diterpenoids further. The structure-activity relationship of diterpene substrates has been systematically investigated with the additional screening of the other four DTSs, where it showed that the elaborated size, shape and geometry of substrates are all critical in enzyme recognition. The striking plasticity of DTSs has also been illustrated in this thesis, where a limited number of mutants were able to alternate enzymatic products. Specifically, a single isoleucine substitution for an asparagine in alcohol synthase SsSS was found to completely abolish water attack leading to olefin products. Furthermore, the previously defined primary effector residue from angiosperm DTSs was extended to land plant kingdom and the mechanistic roles of this residue was also investigated. Further studies have led to the discovery of a secondary residue interactively acting with the primary residue to tune product outcome. This residue pair has been well conserved in vascular plant KSLs and also ancestor KSs adding another piece of evidence into the evolution of DTSs though the functional roles of each residue have been changed under particular protein background.
Jia, Meirong, "Substrate promiscuity and functional plasticity investigations of class I diterpene synthases" (2016). Graduate Theses and Dissertations. 15941.