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

^*Tämän työn vastaava kirjoittaja

Turku Bioscience

Tutkimustuotos: Lehtiartikkeli › Artikkeli › Tieteellinen › vertaisarvioitu

1 Sitaatiot (Scopus)

3 Lataukset (Pure)

Abstrakti

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.

Alkuperäiskieli	Englanti
Artikkeli	3700
Sivumäärä	20
Julkaisu	International Journal of Molecular Sciences
Vuosikerta	24
Numero	4
DOI - pysyväislinkit	https://doi.org/10.3390/ijms24043700
Tila	Julkaistu - 12 helmik. 2023
OKM-julkaisutyyppi	A1 Julkaistu artikkeli, soviteltu

Rahoitus

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).

Rahoittajat	Rahoittajan numero
EMBL-Hamburg
Greek government
Horizon 2020 Framework Programme
European Commission
Academy of Finland
Magnus Ehrnroothin säätiö	653706
European Social Fund
Biocenter Finland

Pääsy asiakirjaan

10.3390/ijms24043700

ijms-24-03700-v2Lopullinen julkaistu versio, 8,81 MBLisenssi: 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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T1 - A Key Role in Catalysis and Enzyme Thermostability of a Conserved Helix H5 Motif of Human Glutathione Transferase A1-1

AU - Chronopoulou, Evangelia G.

AU - Mutabdzija, Lana

AU - Poudel, Nirmal

AU - Papageorgiou, Anastassios C.

AU - Labrou, Nikolaos E.

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N2 - 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.

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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KW - site-directed mutagenesis

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KW - alpha class glutathione transferase

KW - thermostability

KW - site-directed mutagenesis

KW - motif-based protein engineering

KW - conserved amino acids

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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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