Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Jedrzejewski M

PROVIDER: S-EPMC10462857 | biostudies-literature | 2023

REPOSITORIES: biostudies-literature

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Publications

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

Jedrzejewski Mateusz M Belza Barbara B Lewandowska Iwona I Sadlej Marta M Perlinska Agata P AP Augustyniak Rafal R Christian Thomas T Hou Ya-Ming YM Kalek Marcin M Sulkowska Joanna I JI

Computational and structural biotechnology journal 20230809

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 3<sub>1</sub> 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 ...[more]

PMID: 37649713

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Project description:Reliable studies of enzymatic reactions by combined quantum mechanics/molecular mechanics (QM/MM) approaches, with an ab initio description of the quantum region, presents a major challenge to computational chemists. The main problem is the need for a very large computer time for the evaluation of the QM energy, which in turn makes it extremely challenging to perform proper configurational sampling. A seemingly reasonable alternative is to perform energy minimization studies of the type used in gas-phase ab initio studies. However, it is hard to see why such an approach should give reliable results in protein active sites. To examine the problems with energy minimization QM/MM approaches, we chose the hypothetical reaction of a metaphosphate ion with water in the Ras.GAP complex. This hypothetical reaction served as a simple benchmark reaction. The possible problems with the QM/MM minimization were explored by generating several protein configurations from long MD simulations and using energy minimization and scanning of the reaction coordinates to evaluate the corresponding potential energy surfaces of the reaction for each of these different protein configurations. Comparing these potential energy surfaces, we found major variations of the corresponding minima. Furthermore, the reaction energies and activation energies also varied significantly even for similar protein configurations. The specific coordination of a magnesium ion, present in the active center of the protein complex, turned out to influence the energetics of the reaction in a major way, where a direct coordination to the reactant leads to an increase of the activation energy by 17 kcal/mol. Apparently, using energy minimization to generate potential surfaces for an enzymatic reaction, while starting from a single protein structure, could lead to major errors in calculations of activation free energies and binding free energies. Thus we believe that extensive samplings of the configurational space of the protein are essential for meaningful determination of the energetics of enzymatic reactions. The possible relevance of our conclusion with regard to a recent study of the RasGAP reaction is discussed.

Dataset Information

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

Publications

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

Similar Datasets

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