Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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Record number 551790
Title Distant Non-Obvious Mutations Influence the Activity of a Hyperthermophilic Pyrococcusfuriosus Phosphoglucose Isomerase
Author(s) Subramanian, Kalyanasundaram; Mitusińska, Karolina; Raedts, John; Almourfi, Feras; Joosten, Henk Jan; Hendriks, Sjon; Sedelnikova, Svetlana E.; Kengen, Servé W.M.; Hagen, Wilfred R.; Góra, Artur; Martins Dos Santos, Vitor A.P.; Baker, Patrick J.; Oost, John van der; Schaap, Peter J.
Source Biomolecules 9 (2019)6. - ISSN 2218-273X
DOI https://doi.org/10.3390/biom9060212
Department(s) Systems and Synthetic Biology
Microbiology
BacGen
WIMEK
VLAG
Publication type Refereed Article in a scientific journal
Publication year 2019
Keyword(s) Comulator - cupin phosphoglucose isomerase - Protein engineering - Pyrococcus furiosus - solvent access
Abstract

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.

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