Thermoplasma volcanium is a thermoacidophilic archaea which thrives at around 60°C as well as pH 2.0. Archaea are uniquely adapted to thrive in extreme environments. One adaptation involves their unique membrane lipids that consist of polyisoprenoids, which are generated by the mevalonate pathway. Part of the stability of the isoprenoid membrane lipids comes from their glycerol-1-phosphate (G1P) ether linkages. These ether linkages are more stable than the ester linkages typically found in the phospholipid bilayers of other organisms. Geranylgeranylglyceryl phosphate synthase (GGGPS) is a key player in the archaeal biosynthesis pathway of the isoprenoid lipids; GGGPS catalyzes the formation of the ether linkages between G1P and an isoprenoid diphosphate. The FX-cloning method was used to clone the GGGPS gene from T. volcanium into the p7XNH3 plasmid. Recombinant GGGPS with an N-terminal 10x-His tag was expressed in BL21(DE3) cells and three mutants were generated, each containing a single amino acid mutation, Y170A, E172A, and R139A. The Y170 and E172 residues are thought to be required for catalysis, whereas R139 is thought to be involved in the quaternary structure of the enzyme. Each mutant was purified using a His-trap column and the purified GGGPS was crystallized using the hanging drop method in 0.1 M Tris (pH 8) using ammonium sulfate as the precipitant. Successful crystallization will allow for comparison between the wild type GGGPS crystal structure and the mutant GGGPS crystal structures to further examine the implications of the single amino acid changes.

Exploration of single amino acid changes in GGGPS from Thermoplasma volcanium

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.