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

Dyzma, Alicja; Wielgus-Kutrowska, Beata; Girstun, Agnieszka; Matošević, Zoe Jelić; Staroń, Krzysztof; Bertoša, Branimir; Trylska, Joanna; Bzowska, Agnieszka

doi:10.3390/ijms24032157

Open AccessArticle

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

by

Alicja Dyzma

¹,

Beata Wielgus-Kutrowska

^1,*

,

Agnieszka Girstun

²

,

Zoe Jelić Matošević

³

,

Krzysztof Staroń

²,

Branimir Bertoša

³

,

Joanna Trylska

⁴

and

Agnieszka Bzowska

^1,*

¹

Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, Pasteura 5, 02-093 Warsaw, Poland

²

Department of Molecular Biology, Institute of Biochemistry, Faculty of Biology University of Warsaw, Miecznikowa 1, 02-096 Warsaw, Poland

³

Department of Chemistry, Faculty of Science, University of Zagreb, Horvatovac 102a, HR-10000 Zagreb, Croatia

⁴

Centre of New Technologies, University of Warsaw, Banacha 2c, 02-097 Warsaw, Poland

^*

Authors to whom correspondence should be addressed.

Int. J. Mol. Sci. 2023, 24(3), 2157; https://doi.org/10.3390/ijms24032157

Submission received: 28 October 2022 / Revised: 13 January 2023 / Accepted: 16 January 2023 / Published: 21 January 2023

(This article belongs to the Collection Feature Papers in Molecular Biophysics)

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Abstract

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.

Keywords: purine nucleoside phosphorylase; homooligomeric proteins; obligate (obligatory) oligomer; subunit–subunit interface; rearrangement of the active site; tertiary/quaternary structure; in silico/in vivo/in vitro

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MDPI and ACS Style

Dyzma, A.; Wielgus-Kutrowska, B.; Girstun, A.; Matošević, Z.J.; Staroń, K.; Bertoša, B.; Trylska, J.; Bzowska, A. 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. Int. J. Mol. Sci. 2023, 24, 2157. https://doi.org/10.3390/ijms24032157

AMA Style

Dyzma A, Wielgus-Kutrowska B, Girstun A, Matošević ZJ, Staroń K, Bertoša B, Trylska J, Bzowska A. 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. 2023; 24(3):2157. https://doi.org/10.3390/ijms24032157

Chicago/Turabian Style

Dyzma, Alicja, Beata Wielgus-Kutrowska, Agnieszka Girstun, Zoe Jelić Matošević, Krzysztof Staroń, Branimir Bertoša, Joanna Trylska, and Agnieszka Bzowska. 2023. "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 24, no. 3: 2157. https://doi.org/10.3390/ijms24032157

APA Style

Dyzma, A., Wielgus-Kutrowska, B., Girstun, A., Matošević, Z. J., Staroń, K., Bertoša, B., Trylska, J., & Bzowska, A. (2023). 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, 24(3), 2157. https://doi.org/10.3390/ijms24032157

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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

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