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Bypassing evolutionary dead ends and switching the rate-limiting step of a human immunotherapeutic enzyme.
Blazeck, John; Karamitros, Christos S; Ford, Kyle; Somody, Catrina; Qerqez, Ahlam; Murray, Kyle; Burkholder, Nathaniel T; Marshall, Nicholas; Sivakumar, Anirudh; Lu, Wei-Cheng; Tan, Bing; Lamb, Candice; Tanno, Yuri; Siddiqui, Menna Y; Ashoura, Norah; Coma, Silvia; Zhang, Xiaoyan M; McGovern, Karen; Kumada, Yoichi; Zhang, Yan Jessie; Manfredi, Mark; Johnson, Kenneth A; D'Arcy, Sheena; Stone, Everett; Georgiou, George.
Afiliación
  • Blazeck J; Department of Chemical Engineering, University of Texas at Austin (UT Austin), Austin, Texas, USA.
  • Karamitros CS; Department of Chemical Engineering, University of Texas at Austin (UT Austin), Austin, Texas, USA.
  • Ford K; Department of Chemical Engineering, University of Texas at Austin (UT Austin), Austin, Texas, USA.
  • Somody C; Department of Chemical Engineering, University of Texas at Austin (UT Austin), Austin, Texas, USA.
  • Qerqez A; Department of Chemical Engineering, University of Texas at Austin (UT Austin), Austin, Texas, USA.
  • Murray K; Department of Chemistry and Biochemistry, The University of Texas at Dallas, Richardson, Texas, USA.
  • Burkholder NT; Department of Molecular Biosciences, University of Texas at Austin (UT Austin), Austin, Texas, USA.
  • Marshall N; Department of Chemical Engineering, University of Texas at Austin (UT Austin), Austin, Texas, USA.
  • Sivakumar A; Department of Chemical Engineering, University of Texas at Austin (UT Austin), Austin, Texas, USA.
  • Lu WC; Department of Chemical Engineering, University of Texas at Austin (UT Austin), Austin, Texas, USA.
  • Tan B; Department of Chemical Engineering, University of Texas at Austin (UT Austin), Austin, Texas, USA.
  • Lamb C; Department of Chemical Engineering, University of Texas at Austin (UT Austin), Austin, Texas, USA.
  • Tanno Y; Department of Chemical Engineering, University of Texas at Austin (UT Austin), Austin, Texas, USA.
  • Siddiqui MY; Department of Chemical Engineering, University of Texas at Austin (UT Austin), Austin, Texas, USA.
  • Ashoura N; Department of Molecular Biosciences, University of Texas at Austin (UT Austin), Austin, Texas, USA.
  • Coma S; Ikena Oncology, Cambridge, Massachusetts, USA.
  • Zhang XM; Ikena Oncology, Cambridge, Massachusetts, USA.
  • McGovern K; Ikena Oncology, Cambridge, Massachusetts, USA.
  • Kumada Y; Department of Molecular Chemistry and Engineering, Kyoto Institute of Technology, Kyoto, Japan.
  • Zhang YJ; Department of Molecular Biosciences, University of Texas at Austin (UT Austin), Austin, Texas, USA.
  • Manfredi M; Institute for Cellular and Molecular Biology, The University of Texas at Austin (UT Austin), Austin, Texas, USA.
  • Johnson KA; Ikena Oncology, Cambridge, Massachusetts, USA.
  • D'Arcy S; Department of Molecular Biosciences, University of Texas at Austin (UT Austin), Austin, Texas, USA.
  • Stone E; Department of Chemistry and Biochemistry, The University of Texas at Dallas, Richardson, Texas, USA.
  • Georgiou G; Department of Molecular Biosciences, University of Texas at Austin (UT Austin), Austin, Texas, USA.
Nat Catal ; 5(10): 952-967, 2022 Oct.
Article en En | MEDLINE | ID: mdl-36465553
ABSTRACT
The Trp metabolite kynurenine (KYN) accumulates in numerous solid tumours and mediates potent immunosuppression. Bacterial kynureninases (KYNases), which preferentially degrade kynurenine, can relieve immunosuppression in multiple cancer models, but immunogenicity concerns preclude their clinical use, while the human enzyme (HsKYNase) has very low activity for kynurenine and shows no therapeutic effect. Using fitness selections, we evolved a HsKYNase variant with 27-fold higher activity, beyond which exploration of >30 evolutionary trajectories involving the interrogation of >109 variants led to no further improvements. Introduction of two amino acid substitutions conserved in bacterial KYNases reduced enzyme fitness but potentiated rapid evolution of variants with ~500-fold improved activity and reversed substrate specificity, resulting in an enzyme capable of mediating strong anti-tumour effects in mice. Pre-steady-state kinetics revealed a switch in rate-determining step attributable to changes in both enzyme structure and conformational dynamics. Apart from its clinical significance, our work highlights how rationally designed substitutions can potentiate trajectories that overcome barriers in protein evolution.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Idioma: En Revista: Nat Catal Año: 2022 Tipo del documento: Article País de afiliación: Estados Unidos
Texto completo
Añadir a Mi BVS
Imprimir
XML
PubMed Links
Buscar en Google

Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Idioma: En Revista: Nat Catal Año: 2022 Tipo del documento: Article País de afiliación: Estados Unidos