Distant Non-Obvious Mutations Influence the Activity of a Hyperthermophilic Pyrococcusfuriosus Phosphoglucose Isomerase

Kalyanasundaram Subramanian*, Karolina Mitusińska, John Raedts, Feras Almourfi, Henk Jan Joosten, Sjon Hendriks, Svetlana E. Sedelnikova, Servé W.M. Kengen, Wilfred R. Hagen, Artur Góra, Vitor A.P. Martins Dos Santos, Patrick J. Baker, John van der Oost, Peter J. Schaap

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

5 Citations (Scopus)


The cupin-type phosphoglucose isomerase (PfPGI) from the hyperthermophilic archaeon Pyrococcus furiosus catalyzes the reversible isomerization of glucose-6-phosphate to fructose-6-phosphate. We investigated PfPGI using protein-engineering bioinformatics tools to select functionally-important residues based on correlated mutation analyses. A pair of amino acids in the periphery of PfPGI was found to be the dominant co-evolving mutation. The position of these selected residues was found to be non-obvious to conventional protein engineering methods. We designed a small smart library of variants by substituting the co-evolved pair and screened their biochemical activity, which revealed their functional relevance. Four mutants were further selected from the library for purification, measurement of their specific activity, crystal structure determination, and metal cofactor coordination analysis. Though the mutant structures and metal cofactor coordination were strikingly similar, variations in their activity correlated with their fine-tuned dynamics and solvent access regulation. Alternative, small smart libraries for enzyme optimization are suggested by our approach, which is able to identify non-obvious yet beneficial mutations.

Original languageEnglish
Article number212
Issue number6
Publication statusPublished - 31 May 2019


  • Comulator
  • cupin phosphoglucose isomerase
  • Protein engineering
  • Pyrococcus furiosus
  • solvent access

Fingerprint Dive into the research topics of 'Distant Non-Obvious Mutations Influence the Activity of a Hyperthermophilic Pyrococcusfuriosus Phosphoglucose Isomerase'. Together they form a unique fingerprint.

Cite this