8V3K

F95S-F198S epi-Isozizaene Synthase: complex with 3 Mg2+, inorganic pyrophosphate, and benzyl triethyl ammonium cation

Summary for 8V3K

• Entry DOI: 10.2210/pdb8v3k/pdb
• Descriptor: Epi-isozizaene synthase, GLYCEROL, MAGNESIUM ION, ... (7 entities in total)
• Functional Keywords: terpene, terpenoid, isoprenoid, cyclase, lyase
• Biological source: Streptomyces coelicolor A3(2)
• Total number of polymer chains: 1
• Total formula weight: 44315.25

Authors

Christianson, D.W.,Eaton, S.A. (deposition date: 2023-11-28, release date: 2024-01-24, Last modification date: 2024-04-03)

Primary citation

Eaton, S.A.,Christianson, D.W.
Structure-Based Engineering of a Sesquiterpene Cyclase to Generate an Alcohol Product: Conversion of epi -Isozizaene Synthase into alpha-Bisabolol Synthase.
Biochemistry, 63:797-805, 2024

PubMed Abstract: The sesquiterpene cyclase -isozizaene synthase (EIZS) from catalyzes the metal-dependent conversion of farnesyl diphosphate (FPP) into the complex tricyclic product -isozizaene. This remarkable transformation is governed by an active site contour that serves as a template for catalysis, directing the conformations of multiple carbocation intermediates leading to the final product. Mutagenesis of residues defining the active site contour remolds its three-dimensional shape and reprograms the cyclization cascade to generate alternative cyclization products. In some cases, mutagenesis enables alternative chemistry to quench carbocation intermediates, e.g., through hydroxylation. Here, we combine structural and biochemical data from previously characterized EIZS mutants to design and prepare F95S-F198S EIZS, which converts EIZS into an α-bisabolol synthase with moderate fidelity (65% at 18 °C, 74% at 4 °C). We report the complete biochemical characterization of this double mutant as well as the 1.47 Å resolution X-ray crystal structure of its complex with three Mg ions, inorganic pyrophosphate, and the benzyltriethylammonium cation, which partially mimics a carbocation intermediate. Most notably, the two mutations together create an active site contour that stabilizes the bisabolyl carbocation intermediate and positions a water molecule for the hydroxylation reaction. Structural comparison with a naturally occurring α-bisabolol synthase reveals common active site features that direct α-bisabolol generation. In showing that EIZS can be redesigned to generate a sesquiterpene alcohol product instead of a sesquiterpene hydrocarbon product, we have expanded the potential of EIZS as a platform for the development of designer cyclases that could be utilized in synthetic biology applications.

PubMed: 38420671
DOI: 10.1021/acs.biochem.3c00681

Experimental method

X-RAY DIFFRACTION (1.47 Å)