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Abstract <p>This study examined the structures of <i>Sp</i>Cas9 endonuclease of <i>Streptococcus pyogenes</i> and their evolutionary variants using different computational biophysical models to investigate the behavior of hydration in these endonucleases. Although the mechanism of <i>Sp</i>Cas9 is well understood from an evolutionary perspective, its hydration has not been thoroughly explored. The study found that all endonucleases tended to compact together and expose less surface area to water as a solvent, resulting in a significant loss of water molecules from the hydration layer, as occurs in the folding of many globular proteins. A comparative analysis revealed that the distribution of water molecules in the hydration shell and PI domain, which is responsible for the biological recognition function of ligand, differed between each endonuclease. All endonucleases have a higher density in their hydration shell in relation to the density of water as a solvent, with <i>Sp</i>Cas9 having the highest density in the hydration shell (19%) and the lowest being the primitive endonuclease SCA (4%) in relation to the bulk water. The previously reported catalytic activity of these endonucleases toward the OCA2 and TYR genes increased nonlinearly with both maximum of probability density of the number of water molecules <InlineEquation ID="IEq1"> <EquationSource Format="TEX">\({{P}_{{\left( {{{n}_{w}}} \right)}}}\)</EquationSource> <!--BioChMsB2560096Delgado-m1--> </InlineEquation> and the degree of hydration <InlineEquation ID="IEq2"> <EquationSource Format="TEX">\(~{{P}_{{\left( {{{\delta }_{h}}} \right)}}}\)</EquationSource> <!--BioChMsB2560096Delgado-m2--> </InlineEquation> in the evolutionary direction from the oldest to the current. These findings suggest that water molecules in the hydration shell play an important role in the conformational changes, biological recognition, and activity of this endonuclease of great biotechnological interest.</p>

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Relationship between Hydration and Catalytic Activity of Endonucleases: The Case of Cas9 and Its Evolutionary Variants

  • 期刊论文

  • 发表日期：

    2025年12月1日

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  • Ariana Delgado,
  • Ysaías J. Alvarado,
  • Alejandro Vivas,
  • Anibal Méndez,
  • Patricia Rodríguez-Lugo,
  • María Elena Troconis,
  • Lenin González-Paz,
  • Yasmir Quiróz,
  • José Luis Paz,
  • Yovani Marrero-Ponce,
  • Felix Martinez-Rios,
  • Joan Vera-Villalobos

摘要

Abstract

This study examined the structures of SpCas9 endonuclease of Streptococcus pyogenes and their evolutionary variants using different computational biophysical models to investigate the behavior of hydration in these endonucleases. Although the mechanism of SpCas9 is well understood from an evolutionary perspective, its hydration has not been thoroughly explored. The study found that all endonucleases tended to compact together and expose less surface area to water as a solvent, resulting in a significant loss of water molecules from the hydration layer, as occurs in the folding of many globular proteins. A comparative analysis revealed that the distribution of water molecules in the hydration shell and PI domain, which is responsible for the biological recognition function of ligand, differed between each endonuclease. All endonucleases have a higher density in their hydration shell in relation to the density of water as a solvent, with SpCas9 having the highest density in the hydration shell (19%) and the lowest being the primitive endonuclease SCA (4%) in relation to the bulk water. The previously reported catalytic activity of these endonucleases toward the OCA2 and TYR genes increased nonlinearly with both maximum of probability density of the number of water molecules \({{P}_{{\left( {{{n}_{w}}} \right)}}}\) and the degree of hydration \(~{{P}_{{\left( {{{\delta }_{h}}} \right)}}}\) in the evolutionary direction from the oldest to the current. These findings suggest that water molecules in the hydration shell play an important role in the conformational changes, biological recognition, and activity of this endonuclease of great biotechnological interest.