A Key Role in Catalysis and Enzyme Thermostability of a Conserved Helix H5 Motif of Human Glutathione Transferase A1-1

Evangelia G. Chronopoulou; Lana Mutabdzija; Nirmal Poudel; Anastassios C. Papageorgiou; Nikolaos E. Labrou

doi:10.3390/ijms24043700

A Key Role in Catalysis and Enzyme Thermostability of a Conserved Helix H5 Motif of Human Glutathione Transferase A1-1

Evangelia G. Chronopoulou^*, Lana Mutabdzija, Nirmal Poudel, Anastassios C. Papageorgiou, Nikolaos E. Labrou

^*Corresponding author for this work

Turku Bioscience

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Abstract

Glutathione transferases (GSTs) are promiscuous enzymes whose main function is the detoxification of electrophilic compounds. These enzymes are characterized by structural modularity that underpins their exploitation as dynamic scaffolds for engineering enzyme variants, with customized catalytic and structural properties. In the present work, multiple sequence alignment of the alpha class GSTs allowed the identification of three conserved residues (E137, K141, and S142) at α-helix 5 (H5). A motif-directed redesign of the human glutathione transferase A1-1 (hGSTA1-1) was performed through site-directed mutagenesis at these sites, creating two single- and two double-point mutants (E137H, K141H, K141H/S142H, and E137H/K141H). The results showed that all the enzyme variants displayed enhanced catalytic activity compared to the wild-type enzyme hGSTA1-1, while the double mutant hGSTA1-K141H/S142H also showed improved thermal stability. X-ray crystallographic analysis revealed the molecular basis of the effects of double mutations on enzyme stability and catalysis. The biochemical and structural analysis presented here will contribute to a deeper understanding of the structure and function of alpha class GSTs.

Original language	English
Article number	3700
Number of pages	20
Journal	International Journal of Molecular Sciences
Volume	24
Issue number	4
DOIs	https://doi.org/10.3390/ijms24043700
Publication status	Published - 12 Feb 2023
MoE publication type	A1 Journal article-refereed

Funding

E.G.C. thanks ΙΚΥ Scholarship Programs for the financial assistance provided. This work was performed within the grants Strengthening PostDoctoral Research. The sector falls under the Operational Programme “Human Resources Development Program, Education and Lifelong Learning” with priority axes 6,8,9 and is co-funded by the European Social Fund—ESF and the Greek government. A.C.P. thanks Biocenter Finland and Academy of Finland for infrastructure support and the staff at EMBL-Hamburg for assistance during data collection. N.P. thanks Magnus Ehrnrooth Foundation for financial support. Access to EMBL-Hamburg was provided by iNEXT (project number 653706 funded by the Horizon 2020 programme of the European Union).

Funders	Funder number
EMBL-Hamburg
Greek government
Horizon 2020 Framework Programme
European Commission
Academy of Finland
Magnus Ehrnrooth foundation	653706
European Social Fund
Biocenter Finland

Keywords

alpha class glutathione transferase
conserved amino acids
motif-based protein engineering
site-directed mutagenesis
thermostability

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10.3390/ijms24043700

ijms-24-03700-v2Final published version, 8.81 MBLicence: CC BY

https://urn.fi/URN:NBN:fi-fe202402147129

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abstract = "Glutathione transferases (GSTs) are promiscuous enzymes whose main function is the detoxification of electrophilic compounds. These enzymes are characterized by structural modularity that underpins their exploitation as dynamic scaffolds for engineering enzyme variants, with customized catalytic and structural properties. In the present work, multiple sequence alignment of the alpha class GSTs allowed the identification of three conserved residues (E137, K141, and S142) at α-helix 5 (H5). A motif-directed redesign of the human glutathione transferase A1-1 (hGSTA1-1) was performed through site-directed mutagenesis at these sites, creating two single- and two double-point mutants (E137H, K141H, K141H/S142H, and E137H/K141H). The results showed that all the enzyme variants displayed enhanced catalytic activity compared to the wild-type enzyme hGSTA1-1, while the double mutant hGSTA1-K141H/S142H also showed improved thermal stability. X-ray crystallographic analysis revealed the molecular basis of the effects of double mutations on enzyme stability and catalysis. The biochemical and structural analysis presented here will contribute to a deeper understanding of the structure and function of alpha class GSTs.",

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AB - Glutathione transferases (GSTs) are promiscuous enzymes whose main function is the detoxification of electrophilic compounds. These enzymes are characterized by structural modularity that underpins their exploitation as dynamic scaffolds for engineering enzyme variants, with customized catalytic and structural properties. In the present work, multiple sequence alignment of the alpha class GSTs allowed the identification of three conserved residues (E137, K141, and S142) at α-helix 5 (H5). A motif-directed redesign of the human glutathione transferase A1-1 (hGSTA1-1) was performed through site-directed mutagenesis at these sites, creating two single- and two double-point mutants (E137H, K141H, K141H/S142H, and E137H/K141H). The results showed that all the enzyme variants displayed enhanced catalytic activity compared to the wild-type enzyme hGSTA1-1, while the double mutant hGSTA1-K141H/S142H also showed improved thermal stability. X-ray crystallographic analysis revealed the molecular basis of the effects of double mutations on enzyme stability and catalysis. The biochemical and structural analysis presented here will contribute to a deeper understanding of the structure and function of alpha class GSTs.

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A Key Role in Catalysis and Enzyme Thermostability of a Conserved Helix H5 Motif of Human Glutathione Transferase A1-1

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