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Structural characterization of geranylgeranyl pyrophosphate synthase GACE1337 from the hyperthermophilic archaeon Geoglobus acetivorans

Abstract

A novel type 1 geranylgeranyl pyrophosphate synthase GACE1337 has been identified within the genome of a newly identified hyperthermophilic archaeon Geoglobus acetivorans. The enzyme has been cloned and over-expressed in Escherichia coli. The recombinant enzyme has been biochemically and structurally characterized. It is able to catalyze the synthesis of geranylgeranyl pyrophosphate as a major product and of farnesyl pyrophosphate in smaller amounts, as measured by gas chromatography–mass spectrometry at an elevated temperature of 60 °C. Its ability to produce two products is consistent with the fact that GACE1337 is the only short-chain isoprenyl diphosphate synthase in G. acetivorans. Attempts to crystallize the enzyme were successful only at 37 °C. The three-dimensional structure of GACE1337 was determined by X-ray diffraction to 2.5 Å resolution. A comparison of its structure with those of related enzymes revealed that the Geoglobus enzyme has the features of both type I and type III geranylgeranyl pyrophosphate synthases, which allow it to regulate the product length. The active enzyme is a dimer and has three aromatic amino acids, two Phe, and a Tyr, located in the hydrophobic cleft between the two subunits. It is proposed that these bulky residues play a major role in the synthetic reaction by controlling the product elongation.

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Abbreviations

IDS:

Isoprenyl diphosphate synthase

IPP:

Isopentenyl pyrophosphate

DMAPP:

Dimethylallyl pyrophosphate

GPP:

Geranyl pyrophosphate

FPP:

Farnesyl pyrophosphate

GGPP:

Geranylgeranyl pyrophosphate

FARM:

The first aspartate-rich motif

GFPP:

Geranylfarnesyl pyrophosphate

GACE1337:

Geranylgeranyl pyrophosphate synthase from archaeon G. acetivorans

GC-MS:

Gas chromatography–mass spectrometry

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Acknowledgements

This work was supported in part by the Russian Science Foundation Project 14-24-00172 (purification and structure determination), the Russian Foundation for Basic Research 16-04-01037a (structure refinement and analysis), the ERA-IB project THERMOGENE (cloning and expression) funded through the ERA-NET Scheme of the seventh EU Framework Programme by the Russian Foundation for Assistance to Small Innovative Enterprises, and by the Russian Federal Space Agency (crystallization and subsequent X-ray diffraction experiments). JAL acknowledges the THERMOGENE project and funding from the BBSRC, UK BB/L002035/1.

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Petrova, T.E., Boyko, K.M., Nikolaeva, A.Y. et al. Structural characterization of geranylgeranyl pyrophosphate synthase GACE1337 from the hyperthermophilic archaeon Geoglobus acetivorans. Extremophiles 22, 877–888 (2018). https://doi.org/10.1007/s00792-018-1044-5

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Keywords

  • Prenyltransferase
  • Biocatalysis
  • Enzyme structure
  • Archaea
  • Structure activity relationship