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

Research output: Contribution to journalArticleAcademicpeer-review

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.

LanguageEnglish
JournalBiomolecules
Volume9
Issue number6
DOIs
Publication statusPublished - 31 May 2019

Fingerprint

Glucose-6-Phosphate Isomerase
Protein Engineering
Metals
Glucose-6-Phosphate
Mutation
Libraries
Bioinformatics
Isomerization
Pyrococcus furiosus
Purification
Proteins
Crystal structure
Archaea
Computational Biology
Amino Acids
Enzymes
fructose-6-phosphate

Keywords

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

Cite this

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. ; van der Oost, John ; Schaap, Peter J. / Distant Non-Obvious Mutations Influence the Activity of a Hyperthermophilic Pyrococcusfuriosus Phosphoglucose Isomerase. In: Biomolecules. 2019 ; Vol. 9, No. 6.
@article{28cfcb933c744928972e79d2db4b5a16,
title = "Distant Non-Obvious Mutations Influence the Activity of a Hyperthermophilic Pyrococcusfuriosus Phosphoglucose Isomerase",
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.",
keywords = "Comulator, cupin phosphoglucose isomerase, Protein engineering, Pyrococcus furiosus, solvent access",
author = "Kalyanasundaram Subramanian and Karolina Mitusińska and John Raedts and Feras Almourfi and Joosten, {Henk Jan} and Sjon Hendriks and Sedelnikova, {Svetlana E.} and Kengen, {Serv{\'e} W.M.} and Hagen, {Wilfred R.} and Artur G{\'o}ra and {Martins Dos Santos}, {Vitor A.P.} and Baker, {Patrick J.} and {van der Oost}, John and Schaap, {Peter J.}",
year = "2019",
month = "5",
day = "31",
doi = "10.3390/biom9060212",
language = "English",
volume = "9",
journal = "Biomolecules",
issn = "2218-273X",
publisher = "MDPI AG",
number = "6",

}

Distant Non-Obvious Mutations Influence the Activity of a Hyperthermophilic Pyrococcusfuriosus Phosphoglucose Isomerase. / 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.; van der Oost, John; Schaap, Peter J.

In: Biomolecules, Vol. 9, No. 6, 31.05.2019.

Research output: Contribution to journalArticleAcademicpeer-review

TY - JOUR

T1 - Distant Non-Obvious Mutations Influence the Activity of a Hyperthermophilic Pyrococcusfuriosus Phosphoglucose Isomerase

AU - Subramanian, Kalyanasundaram

AU - Mitusińska, Karolina

AU - Raedts, John

AU - Almourfi, Feras

AU - Joosten, Henk Jan

AU - Hendriks, Sjon

AU - Sedelnikova, Svetlana E.

AU - Kengen, Servé W.M.

AU - Hagen, Wilfred R.

AU - Góra, Artur

AU - Martins Dos Santos, Vitor A.P.

AU - Baker, Patrick J.

AU - van der Oost, John

AU - Schaap, Peter J.

PY - 2019/5/31

Y1 - 2019/5/31

N2 - 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.

AB - 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.

KW - Comulator

KW - cupin phosphoglucose isomerase

KW - Protein engineering

KW - Pyrococcus furiosus

KW - solvent access

U2 - 10.3390/biom9060212

DO - 10.3390/biom9060212

M3 - Article

VL - 9

JO - Biomolecules

T2 - Biomolecules

JF - Biomolecules

SN - 2218-273X

IS - 6

ER -