Date of Award


Degree Type


Degree Name

Doctor of Philosophy (PhD)

Graduate Group


First Advisor

David W. Christianson


Terpene cyclases convert acyclic isoprenoid precursors into complex cyclic terpenoid compounds. Hydrophobic active site contours in terpene cyclases direct cyclization reactions through a cascade of carbocation intermediates by serving as templates for terpenoid product structure. Both local molecular structure at the active site and global protein structure encompassing domain organization and oligomerization state have effects on catalytic function in terpene cyclases. The goal of this work is characterization of the effects of local structural changes to the active site of a terpene cyclase, and a thorough understanding of the structure-function relationship of active site structure and domain organization in two terpene cyclases, geosmin synthase (ScGS) and epi-isozizaene synthase (EIZS) from Streptomyces coelicolor.

Geosmin synthase (ScGS) is a bifunctional class I sesquiterpene cyclase that catalyzes the conversion of FPP to germacradienol, germacrene D, and geosmin in unique cyclization and cyclization-fragmentation reactions occurring in separate active sites. We determined the X-ray crystal structure of the N-terminal domain of ScGS and homology models of the C-terminal domain of ScGS, and used small-angle X-ray scattering (SAXS) to propose models of domain association in this system. Product analysis by gas chromatography-mass spectrometry (GC-MS) in this system indicates residues that are important for catalysis in the C-terminal domain cyclization-fragmentation reaction.

EIZS is a promiscuous terpene cyclase and produces epi-isozizaene as a major product, along with five other sesquiterpene products. Single mutations at the active site of EIZS can drastically change the proportions and identities of sesquiterpene products. Mutagenesis of key residues at the active site to polar side chains results in mutant EIZS enzymes with altered catalytic properties. The X-ray crystal structures of EIZS F95N and F95C were determined and demonstrate that EIZS mutants containing polar residues at the active site do not exhibit global structural changes when compared to the wild type enzyme. GC-MS was used to analyze the products of eight new EIZS mutants, demonstrating formation of thirteen new sesquiterpene products not previously observed in this system. These results demonstrate that polar mutations are structurally and catalytically tolerated at the active site of EIZS.

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