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Nucleolar Essential Protein 1 (Nep1): Elucidation of enzymatic catalysis mechanism by molecular dynamics simulation and quantum mechanics study

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dc.abstract.enThe 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.
dc.affiliationUniwersytet Warszawski
dc.contributor.authorAugustyniak, Rafał
dc.contributor.authorSułkowska, Joanna
dc.contributor.authorKałek, Marcin
dc.contributor.authorPerlińska, Agata
dc.contributor.authorHou, Ya-Ming
dc.contributor.authorChristian, Thomas
dc.contributor.authorSadlej, Marta
dc.contributor.authorLewandowska, Iwona
dc.contributor.authorBelza, Barbara
dc.contributor.authorJedrzejewski, Mateusz
dc.date.accessioned2024-01-25T14:06:22Z
dc.date.available2024-01-25T14:06:22Z
dc.date.copyright2023-08-09
dc.date.issued2023
dc.description.accesstimeAT_PUBLICATION
dc.description.financePublikacja bezkosztowa
dc.description.versionFINAL_PUBLISHED
dc.description.volume21
dc.identifier.doi10.1016/J.CSBJ.2023.08.001
dc.identifier.urihttps://repozytorium.uw.edu.pl//handle/item/114069
dc.identifier.weblinkhttps://api.elsevier.com/content/article/PII:S2001037023002763?httpAccept=text/xml
dc.languageeng
dc.pbn.affiliationchemical sciences
dc.relation.ispartofComputational and Structural Biotechnology Journal
dc.relation.pages3999-4008
dc.rightsCC-BY-NC-ND
dc.sciencecloudnosend
dc.subject.enNep1
dc.subject.enTrefoil knot
dc.subject.enRNA processing
dc.subject.enMethylation
dc.subject.enProton transfer
dc.subject.enEnzymatic catalysis
dc.titleNucleolar Essential Protein 1 (Nep1): Elucidation of enzymatic catalysis mechanism by molecular dynamics simulation and quantum mechanics study
dc.typeJournalArticle
dspace.entity.typePublication