New Insights into Terpene Synthase Catalysis for Rational Enzyme Engineering

Abstract

This doctoral dissertation consists of 4 reviews and 16 research articles that present mechanistic studies on terpene biosynthesis. Specifically, sesquiterpenes biosynthetically derived from germacrene A, hedycaryol and germacrene B are summarised in three reviews. All compounds are described together with their structures, natural sources, determination of their absolute configurations, biosynthetic pathways and NMR data. For the original research, the studied terpenes included the new compounds isoishwarane, (1S,5S,7R,10R)-guaia-4(15)-en-11-ol, kitaviridene and the sesterviridenes, besides known hedycaryol, (4S,7R)-germacra-(1(10)E,5E)-dien-11-ol, (1S,7R,10R)-guaia-4-en-11-ol, selina-4(15),7(11)-diene, patchoulol, α-humulene, spiroviolene and the non-canonical compounds sodorifen and geosmin. For all these compounds the biosynthesis was extensively studied through conversion of isotopically labelled precursors with the respective terpene synthases, in conjunction with DFT calculations or QM/MM MD simulations, culminating in the proposal of rational biosynthetic pathways for each investigated compound. <br /> The fourth review article summarises site-directed mutagenesis studies on terpene synthases, contributing to a deeper understanding of these enzymes with respect to the functions of residues and motifs in the active site cavity. Two experimental studies regarding the structure-based site-directed mutagenesis were conducted on the diterpene synthase for cattleyene (CyS) and three sesquiterpene synthases for presilphiperfolan-8β-ol (BcBOT), protoillud-6-ene (DbPROS) and longiborneol (CLM1), respectively, demonstrating the potential of enzyme engineering to expand the chemical space that can be reached with terpene synthase catalysis. <br /> Taken together, the results presented in this thesis significantly expand our knowledge on the complex mechanisms of terpene synthase mediated cyclisation reactions and the structural requirements for efficient catalysis by this remarkable class of biocatalysts.