Computational studies on the catalytic potential of the double active site for enzyme engineering
| Autor: | Naveen Banchallihundi Krishna, Lalitha Roopa, R. Pravin Kumar, Gopenath T S |
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| Jazyk: | angličtina |
| Rok vydání: | 2024 |
| Předmět: | |
| Zdroj: | Scientific Reports, Vol 14, Iss 1, Pp 1-15 (2024) |
| Druh dokumentu: | article |
| ISSN: | 2045-2322 37504231 |
| DOI: | 10.1038/s41598-024-60824-x |
| Popis: | Abstract Proteins possessing double active sites have the potential to revolutionise enzyme design strategies. This study extensively explored an enzyme that contains both a natural active site (NAS) and an engineered active site (EAS), focusing on understanding its structural and functional properties. Metadynamics simulations were employed to investigate how substrates interacted with their respective active sites. The results revealed that both the NAS and EAS exhibited similar minimum energy states, indicating comparable binding affinities. However, it became apparent that the EAS had a weaker binding site for the substrate due to its smaller pocket and constrained conformation. Interestingly, the EAS also displayed dynamic behaviour, with the substrate observed to move outside the pocket, suggesting the possibility of substrate translocation. To gain further insights, steered molecular dynamics (SMD) simulations were conducted to study the conformational changes of the substrate and its interactions with catalytic residues. Notably, the substrate adopted distinct conformations, including near-attack conformations, in both the EAS and NAS. Nevertheless, the NAS demonstrated superior binding minima for the substrate compared to the EAS, reinforcing the observation that the engineered active site was less favourable for substrate binding due to its limitations. The QM/MM (Quantum mechanics and molecular mechanics) analyses highlight the energy disparity between NAS and EAS. Specifically, EAS exhibited elevated energy levels due to its engineered active site being located on the surface. This positioning exposes the substrate to solvents and water molecules, adding to the energy challenge. Consequently, the engineered enzyme did not provide a significant advantage in substrate binding over the single active site protein. Further, the investigation of internal channels and tunnels within the protein shed light on the pathways facilitating transport between the two active sites. By unravelling the complex dynamics and functional characteristics of this double-active site protein, this study offers valuable insights into novel strategies of enzyme engineering. These findings establish a solid foundation for future research endeavours aimed at harnessing the potential of double-active site proteins in diverse biotechnological applications. |
| Databáze: | Directory of Open Access Journals |
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