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Open AccessArticle

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

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Laboratory of Systems and Synthetic Biology, Wageningen University, Stippeneng 4, 6708 WE Wageningen, The Netherlands
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Biotechnology Center, Silesian University of Technology, ul. Krzywoustego 8, 44-100 Gliwice, Poland
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Faculty of Chemistry, Silesian University of Technology, ul. Strzody 9, 44-100 Gliwice, Poland
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Laboratory of Microbiology, Wageningen University, Stippeneng 4, 6708 WE Wageningen, The Netherlands
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Saudi Human Genome Project, National Center of Genome Technology, King Abdulaziz City for Science and Technology (KACST), Riyadh 11442, Saudi Arabia
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Bio-Prodict, Nieuwe Marktstraat 54E, 6511 AA Nijmegen, The Netherlands
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The Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, UK
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Department of Biotechnology, Delft University of Technology, Van der Maasweg 9, 2629 HZ Delft, The Netherlands
*
Authors to whom correspondence should be addressed.
These authors contributed equally to this work.
Biomolecules 2019, 9(6), 212; https://doi.org/10.3390/biom9060212
Received: 1 March 2019 / Revised: 20 May 2019 / Accepted: 28 May 2019 / Published: 31 May 2019
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. View Full-Text
Keywords: Protein engineering; Comulator; cupin phosphoglucose isomerase; Pyrococcus furiosus; solvent access Protein engineering; Comulator; cupin phosphoglucose isomerase; Pyrococcus furiosus; solvent access
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Subramanian, K.; Mitusińska, K.; Raedts, J.; Almourfi, F.; Joosten, H.-J.; Hendriks, S.; Sedelnikova, S.E.; Kengen, S.W.M.; Hagen, W.R.; Góra, A.; Martins dos Santos, V.A.P.; Baker, P.J.; van der Oost, J.; Schaap, P.J. Distant Non-Obvious Mutations Influence the Activity of a Hyperthermophilic Pyrococcus furiosus Phosphoglucose Isomerase. Biomolecules 2019, 9, 212.

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