Nucleolar Essential Protein 1 (Nep1): Elucidation of enzymatic catalysis mechanism by molecular dynamics simulation and quantum mechanics study
The Nep1 protein is essential for the formation of eukaryotic and archaeal small ribosomal subunits, and it catalyzes the site-directed SAM-dependent methylation of pseudouridine (Ψ) during pre-rRNA processing. It possesses a non–trivial topology, namely, a 31 knot in the active site. Here, we addre...
| Main Authors: | , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Elsevier
2023-01-01
|
| Series: | Computational and Structural Biotechnology Journal |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2001037023002763 |
| _version_ | 1797384108486164480 |
|---|---|
| author | Mateusz Jedrzejewski Barbara Belza Iwona Lewandowska Marta Sadlej Agata P. Perlinska Rafal Augustyniak Thomas Christian Ya-Ming Hou Marcin Kalek Joanna I. Sulkowska |
| author_facet | Mateusz Jedrzejewski Barbara Belza Iwona Lewandowska Marta Sadlej Agata P. Perlinska Rafal Augustyniak Thomas Christian Ya-Ming Hou Marcin Kalek Joanna I. Sulkowska |
| author_sort | Mateusz Jedrzejewski |
| collection | DOAJ |
| description | The Nep1 protein is essential for the formation of eukaryotic and archaeal small ribosomal subunits, and it catalyzes the site-directed SAM-dependent methylation of pseudouridine (Ψ) during pre-rRNA processing. It possesses a non–trivial topology, namely, a 31 knot in the active site. Here, we address the issue of seemingly unfeasible deprotonation of Ψ in Nep1 active site by a distant aspartate residue (D101 in S. cerevisiae), using a combination of bioinformatics, computational, and experimental methods. We identified a conserved hydroxyl-containing amino acid (S233 in S. cerevisiae, T198 in A. fulgidus) that may act as a proton-transfer mediator. Molecular dynamics simulations, based on the crystal structure of S. cerevisiae, and on a complex generated by molecular docking in A. fulgidus, confirmed that this amino acid can shuttle protons, however, a water molecule in the active site may also serve this role. Quantum-chemical calculations based on density functional theory and the cluster approach showed that the water-mediated pathway is the most favorable for catalysis. Experimental kinetic and mutational studies reinforce the requirement for the aspartate D101, but not S233. These findings provide insight into the catalytic mechanisms underlying proton transfer over extended distances and comprehensively elucidate the mode of action of Nep1. |
| first_indexed | 2024-03-08T21:30:44Z |
| format | Article |
| id | doaj.art-fce9cd31689d48fc924328112827005f |
| institution | Directory Open Access Journal |
| issn | 2001-0370 |
| language | English |
| last_indexed | 2024-03-08T21:30:44Z |
| publishDate | 2023-01-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Computational and Structural Biotechnology Journal |
| spelling | doaj.art-fce9cd31689d48fc924328112827005f2023-12-21T07:31:54ZengElsevierComputational and Structural Biotechnology Journal2001-03702023-01-012139994008Nucleolar Essential Protein 1 (Nep1): Elucidation of enzymatic catalysis mechanism by molecular dynamics simulation and quantum mechanics studyMateusz Jedrzejewski0Barbara Belza1Iwona Lewandowska2Marta Sadlej3Agata P. Perlinska4Rafal Augustyniak5Thomas Christian6Ya-Ming Hou7Marcin Kalek8Joanna I. Sulkowska9Centre of New Technologies, University of Warsaw, Banacha 2c, 02-097, Warsaw, PolandCentre of New Technologies, University of Warsaw, Banacha 2c, 02-097, Warsaw, PolandCentre of New Technologies, University of Warsaw, Banacha 2c, 02-097, Warsaw, PolandCentre of New Technologies, University of Warsaw, Banacha 2c, 02-097, Warsaw, PolandCentre of New Technologies, University of Warsaw, Banacha 2c, 02-097, Warsaw, PolandDepartment of Chemistry, University of Warsaw, Ludwika Pasteura 1, 02-093, Warsaw, PolandDepartment of Biochemistry and Molecular Biology, Thomas Jefferson University, 4201 Henry Ave, Philadelphia, PA 19144, USADepartment of Biochemistry and Molecular Biology, Thomas Jefferson University, 4201 Henry Ave, Philadelphia, PA 19144, USACentre of New Technologies, University of Warsaw, Banacha 2c, 02-097, Warsaw, Poland; Corresponding authors.Centre of New Technologies, University of Warsaw, Banacha 2c, 02-097, Warsaw, Poland; Corresponding authors.The Nep1 protein is essential for the formation of eukaryotic and archaeal small ribosomal subunits, and it catalyzes the site-directed SAM-dependent methylation of pseudouridine (Ψ) during pre-rRNA processing. It possesses a non–trivial topology, namely, a 31 knot in the active site. Here, we address the issue of seemingly unfeasible deprotonation of Ψ in Nep1 active site by a distant aspartate residue (D101 in S. cerevisiae), using a combination of bioinformatics, computational, and experimental methods. We identified a conserved hydroxyl-containing amino acid (S233 in S. cerevisiae, T198 in A. fulgidus) that may act as a proton-transfer mediator. Molecular dynamics simulations, based on the crystal structure of S. cerevisiae, and on a complex generated by molecular docking in A. fulgidus, confirmed that this amino acid can shuttle protons, however, a water molecule in the active site may also serve this role. Quantum-chemical calculations based on density functional theory and the cluster approach showed that the water-mediated pathway is the most favorable for catalysis. Experimental kinetic and mutational studies reinforce the requirement for the aspartate D101, but not S233. These findings provide insight into the catalytic mechanisms underlying proton transfer over extended distances and comprehensively elucidate the mode of action of Nep1.http://www.sciencedirect.com/science/article/pii/S2001037023002763Nep1Trefoil knotRNA processingMethylationProton transferEnzymatic catalysis |
| spellingShingle | Mateusz Jedrzejewski Barbara Belza Iwona Lewandowska Marta Sadlej Agata P. Perlinska Rafal Augustyniak Thomas Christian Ya-Ming Hou Marcin Kalek Joanna I. Sulkowska Nucleolar Essential Protein 1 (Nep1): Elucidation of enzymatic catalysis mechanism by molecular dynamics simulation and quantum mechanics study Computational and Structural Biotechnology Journal Nep1 Trefoil knot RNA processing Methylation Proton transfer Enzymatic catalysis |
| title | Nucleolar Essential Protein 1 (Nep1): Elucidation of enzymatic catalysis mechanism by molecular dynamics simulation and quantum mechanics study |
| title_full | Nucleolar Essential Protein 1 (Nep1): Elucidation of enzymatic catalysis mechanism by molecular dynamics simulation and quantum mechanics study |
| title_fullStr | Nucleolar Essential Protein 1 (Nep1): Elucidation of enzymatic catalysis mechanism by molecular dynamics simulation and quantum mechanics study |
| title_full_unstemmed | Nucleolar Essential Protein 1 (Nep1): Elucidation of enzymatic catalysis mechanism by molecular dynamics simulation and quantum mechanics study |
| title_short | Nucleolar Essential Protein 1 (Nep1): Elucidation of enzymatic catalysis mechanism by molecular dynamics simulation and quantum mechanics study |
| title_sort | nucleolar essential protein 1 nep1 elucidation of enzymatic catalysis mechanism by molecular dynamics simulation and quantum mechanics study |
| topic | Nep1 Trefoil knot RNA processing Methylation Proton transfer Enzymatic catalysis |
| url | http://www.sciencedirect.com/science/article/pii/S2001037023002763 |
| work_keys_str_mv | AT mateuszjedrzejewski nucleolaressentialprotein1nep1elucidationofenzymaticcatalysismechanismbymoleculardynamicssimulationandquantummechanicsstudy AT barbarabelza nucleolaressentialprotein1nep1elucidationofenzymaticcatalysismechanismbymoleculardynamicssimulationandquantummechanicsstudy AT iwonalewandowska nucleolaressentialprotein1nep1elucidationofenzymaticcatalysismechanismbymoleculardynamicssimulationandquantummechanicsstudy AT martasadlej nucleolaressentialprotein1nep1elucidationofenzymaticcatalysismechanismbymoleculardynamicssimulationandquantummechanicsstudy AT agatapperlinska nucleolaressentialprotein1nep1elucidationofenzymaticcatalysismechanismbymoleculardynamicssimulationandquantummechanicsstudy AT rafalaugustyniak nucleolaressentialprotein1nep1elucidationofenzymaticcatalysismechanismbymoleculardynamicssimulationandquantummechanicsstudy AT thomaschristian nucleolaressentialprotein1nep1elucidationofenzymaticcatalysismechanismbymoleculardynamicssimulationandquantummechanicsstudy AT yaminghou nucleolaressentialprotein1nep1elucidationofenzymaticcatalysismechanismbymoleculardynamicssimulationandquantummechanicsstudy AT marcinkalek nucleolaressentialprotein1nep1elucidationofenzymaticcatalysismechanismbymoleculardynamicssimulationandquantummechanicsstudy AT joannaisulkowska nucleolaressentialprotein1nep1elucidationofenzymaticcatalysismechanismbymoleculardynamicssimulationandquantummechanicsstudy |