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Modulating activati...
Modulating activation entropyand enthalpy of human oxidosqualene cyclase reaction by tunnel mutagenesis
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- Schriever, Karen (författare)
- KTH,Science for Life Laboratory, SciLifeLab,Ytbehandlingsteknik
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- Hueting, David A. (författare)
- KTH,Ytbehandlingsteknik,Science for Life Laboratory, SciLifeLab
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- Biundo, Antonino (författare)
- KTH,Ytbehandlingsteknik,Science for Life Laboratory, SciLifeLab
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visa fler...
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- Braun, Tatjana (författare)
- Schrödinger GmbH
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- Govindarajan, Sridhar (författare)
- ATUM
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- Gustafsson, Claes (författare)
- ATUM
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- Syrén, Per-Olof (författare)
- KTH,Science for Life Laboratory, SciLifeLab,Ytbehandlingsteknik
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visa färre...
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(creator_code:org_t)
- Engelska.
- Relaterad länk:
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https://urn.kb.se/re...
Abstract
Ämnesord
Stäng
- The formation of tetracyclic lanosterol from (S)-2,3-oxidosqualene is catalyzed by oxidosqualenecyclase (OSC). Lanosterol is of high interest due to its essential role in steroid metabolism. Therefore,understanding how the inherent high entropic cost of forming a multicyclic core from a flexible linearsubstrate is energetically driven is of high interest. Enzyme mechanisms can involve a reducedhydration state of rearranging transient charges in intermediates and transition states. Often thesereactions have an unusually low entropy barrier. We studied the activation enthalpy and entropy inrelation to solvent tunnels accessing the active site in the carbocationic polycyclization cascadecatalyzed by human OSC (hOSC). We applied Eyring transition state analysis of lanosterol formationby hOSC at different temperatures, alongside Molecular Dynamics simulations and CAVER analysis.hOSC showed a high favorable entropy of activation (+6.4 kcal mol-1 at 310 K) at ambienttemperatures. The introduction of bulky residues at the interface of several water tunnels, resulted inenzyme variants with altered thermodynamic properties. One of the variants was enthalpy-driven andshowed an inversed temperature dependence of cyclization. We further biochemically characterizeddifferent enzyme libraries, in which rational tunnel-mutations combined with mutations suggested byphylogeny-guided protein design were introduced in different combinations. This approach yieldedseveral highly active hOSC variants (5-6x increased activity at 37 °C), as well as a highly active variantat colder temperature with inversed temperature dependence. In summary, the present workhighlights the importance of activation entropy in enzymes, which is often considered negligible, aswell as the challenges associated with rational protein design aiming to modify activationthermodynamic parameters.
Ämnesord
- NATURVETENSKAP -- Biologi -- Biokemi och molekylärbiologi (hsv//swe)
- NATURAL SCIENCES -- Biological Sciences -- Biochemistry and Molecular Biology (hsv//eng)
Publikations- och innehållstyp
- vet (ämneskategori)
- ovr (ämneskategori)