Trimeric Architecture Ensures the Stability and Biological Activity of the Calf Purine Nucleoside Phosphorylase: In Silico and In Vitro Studies of Monomeric and Trimeric Forms of the Enzyme
Mammalian purine nucleoside phosphorylase (PNP) is biologically active as a homotrimer, in which each monomer catalyzes a reaction independently of the others. To answer the question of why the native PNP forms a trimeric structure, we constructed, in silico and in vitro, the monomeric form of the e...
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2023-01-01
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| Series: | International Journal of Molecular Sciences |
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| author | Alicja Dyzma Beata Wielgus-Kutrowska Agnieszka Girstun Zoe Jelić Matošević Krzysztof Staroń Branimir Bertoša Joanna Trylska Agnieszka Bzowska |
| author_facet | Alicja Dyzma Beata Wielgus-Kutrowska Agnieszka Girstun Zoe Jelić Matošević Krzysztof Staroń Branimir Bertoša Joanna Trylska Agnieszka Bzowska |
| author_sort | Alicja Dyzma |
| collection | DOAJ |
| description | Mammalian purine nucleoside phosphorylase (PNP) is biologically active as a homotrimer, in which each monomer catalyzes a reaction independently of the others. To answer the question of why the native PNP forms a trimeric structure, we constructed, in silico and in vitro, the monomeric form of the enzyme. Molecular dynamics simulations showed different geometries of the active site in the non-mutated trimeric and monomeric PNP forms, which suggested that the active site in the isolated monomer could be non-functional. To confirm this hypothesis, six amino acids located at the interface of the subunits were selected and mutated to alanines to disrupt the trimer and obtain a monomer (6Ala PNP). The effects of these mutations on the enzyme structure, stability, conformational dynamics, and activity were examined. The solution experiments confirmed that the 6Ala PNP mutant occurs mainly as a monomer, with a secondary structure almost identical to the wild type, WT PNP, and importantly, it shows no enzymatic activity. Simulations confirmed that, although the secondary structure of the 6Ala monomer is similar to the WT PNP, the positions of the amino acids building the 6Ala PNP active site significantly differ. These data suggest that a trimeric structure is necessary to stabilize the geometry of the active site of this enzyme. |
| first_indexed | 2024-03-11T09:43:18Z |
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| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-11T09:43:18Z |
| publishDate | 2023-01-01 |
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| series | International Journal of Molecular Sciences |
| spelling | doaj.art-df058c79d3db4557a14ee93651d79c852023-11-16T16:53:08ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672023-01-01243215710.3390/ijms24032157Trimeric Architecture Ensures the Stability and Biological Activity of the Calf Purine Nucleoside Phosphorylase: In Silico and In Vitro Studies of Monomeric and Trimeric Forms of the EnzymeAlicja Dyzma0Beata Wielgus-Kutrowska1Agnieszka Girstun2Zoe Jelić Matošević3Krzysztof Staroń4Branimir Bertoša5Joanna Trylska6Agnieszka Bzowska7Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, Pasteura 5, 02-093 Warsaw, PolandDivision of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, Pasteura 5, 02-093 Warsaw, PolandDepartment of Molecular Biology, Institute of Biochemistry, Faculty of Biology University of Warsaw, Miecznikowa 1, 02-096 Warsaw, PolandDepartment of Chemistry, Faculty of Science, University of Zagreb, Horvatovac 102a, HR-10000 Zagreb, CroatiaDepartment of Molecular Biology, Institute of Biochemistry, Faculty of Biology University of Warsaw, Miecznikowa 1, 02-096 Warsaw, PolandDepartment of Chemistry, Faculty of Science, University of Zagreb, Horvatovac 102a, HR-10000 Zagreb, CroatiaCentre of New Technologies, University of Warsaw, Banacha 2c, 02-097 Warsaw, PolandDivision of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, Pasteura 5, 02-093 Warsaw, PolandMammalian purine nucleoside phosphorylase (PNP) is biologically active as a homotrimer, in which each monomer catalyzes a reaction independently of the others. To answer the question of why the native PNP forms a trimeric structure, we constructed, in silico and in vitro, the monomeric form of the enzyme. Molecular dynamics simulations showed different geometries of the active site in the non-mutated trimeric and monomeric PNP forms, which suggested that the active site in the isolated monomer could be non-functional. To confirm this hypothesis, six amino acids located at the interface of the subunits were selected and mutated to alanines to disrupt the trimer and obtain a monomer (6Ala PNP). The effects of these mutations on the enzyme structure, stability, conformational dynamics, and activity were examined. The solution experiments confirmed that the 6Ala PNP mutant occurs mainly as a monomer, with a secondary structure almost identical to the wild type, WT PNP, and importantly, it shows no enzymatic activity. Simulations confirmed that, although the secondary structure of the 6Ala monomer is similar to the WT PNP, the positions of the amino acids building the 6Ala PNP active site significantly differ. These data suggest that a trimeric structure is necessary to stabilize the geometry of the active site of this enzyme.https://www.mdpi.com/1422-0067/24/3/2157purine nucleoside phosphorylasehomooligomeric proteinsobligate (obligatory) oligomersubunit–subunit interfacerearrangement of the active sitetertiary/quaternary structure |
| spellingShingle | Alicja Dyzma Beata Wielgus-Kutrowska Agnieszka Girstun Zoe Jelić Matošević Krzysztof Staroń Branimir Bertoša Joanna Trylska Agnieszka Bzowska Trimeric Architecture Ensures the Stability and Biological Activity of the Calf Purine Nucleoside Phosphorylase: In Silico and In Vitro Studies of Monomeric and Trimeric Forms of the Enzyme International Journal of Molecular Sciences purine nucleoside phosphorylase homooligomeric proteins obligate (obligatory) oligomer subunit–subunit interface rearrangement of the active site tertiary/quaternary structure |
| title | Trimeric Architecture Ensures the Stability and Biological Activity of the Calf Purine Nucleoside Phosphorylase: In Silico and In Vitro Studies of Monomeric and Trimeric Forms of the Enzyme |
| title_full | Trimeric Architecture Ensures the Stability and Biological Activity of the Calf Purine Nucleoside Phosphorylase: In Silico and In Vitro Studies of Monomeric and Trimeric Forms of the Enzyme |
| title_fullStr | Trimeric Architecture Ensures the Stability and Biological Activity of the Calf Purine Nucleoside Phosphorylase: In Silico and In Vitro Studies of Monomeric and Trimeric Forms of the Enzyme |
| title_full_unstemmed | Trimeric Architecture Ensures the Stability and Biological Activity of the Calf Purine Nucleoside Phosphorylase: In Silico and In Vitro Studies of Monomeric and Trimeric Forms of the Enzyme |
| title_short | Trimeric Architecture Ensures the Stability and Biological Activity of the Calf Purine Nucleoside Phosphorylase: In Silico and In Vitro Studies of Monomeric and Trimeric Forms of the Enzyme |
| title_sort | trimeric architecture ensures the stability and biological activity of the calf purine nucleoside phosphorylase in silico and in vitro studies of monomeric and trimeric forms of the enzyme |
| topic | purine nucleoside phosphorylase homooligomeric proteins obligate (obligatory) oligomer subunit–subunit interface rearrangement of the active site tertiary/quaternary structure |
| url | https://www.mdpi.com/1422-0067/24/3/2157 |
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