Mechanistic conformational and substrate selectivity profiles emerging in the evolution of enzymes via parallel trajectories.

Autor: Karamitros CS; Department of Chemical Engineering, University of Texas at Austin (UT Austin), Austin, TX, USA.; Research and Clinical Development, Nestlé Health Science, Lausanne, 1000, Switzerland., Murray K; Department of Chemistry and Biochemistry, The University of Texas at Dallas, Richardson, TX, USA.; Trajan Scientific America's, Inc. Boston, Massachusetts, MA, USA., Kumada Y; Department of Molecular Chemistry and Engineering, Kyoto Institute of Technology, Kyoto, Japan., Johnson KA; Department of Molecular Biosciences, University of Texas at Austin (UT Austin), Austin, TX, USA., D'Arcy S; Department of Chemistry and Biochemistry, The University of Texas at Dallas, Richardson, TX, USA., Georgiou G; Department of Chemical Engineering, University of Texas at Austin (UT Austin), Austin, TX, USA. gg@che.utexas.edu.; Department of Molecular Biosciences, University of Texas at Austin (UT Austin), Austin, TX, USA. gg@che.utexas.edu.; Department of Biomedical Engineering, University of Texas at Austin (UT Austin), Austin, TX, USA. gg@che.utexas.edu.; Institute for Cellular and Molecular Biology, The University of Texas at Austin (UT Austin), Austin, TX, USA. gg@che.utexas.edu.
Jazyk: angličtina
Zdroj: Nature communications [Nat Commun] 2024 Aug 16; Vol. 15 (1), pp. 7068. Date of Electronic Publication: 2024 Aug 16.
DOI: 10.1038/s41467-024-51133-y
Abstrakt: Laboratory evolution studies have demonstrated that parallel evolutionary trajectories can lead to genetically distinct enzymes with high activity towards a non-preferred substrate. However, it is unknown whether such enzymes have convergent conformational dynamics and mechanistic features. To address this question, we use as a model the wild-type Homo sapiens kynureninase (HsKYNase), which is of great interest for cancer immunotherapy. Earlier, we isolated HsKYNase_66 through an unusual evolutionary trajectory, having a 410-fold increase in the k cat /K M for kynurenine (KYN) and reverse substrate selectivity relative to HsKYNase. Here, by following a different evolutionary trajectory we generate a genetically distinct variant, HsKYNase_93D9, that exhibits KYN catalytic activity comparable to that of HsKYNase_66, but instead it is a "generalist" that accepts 3'-hydroxykynurenine (OH-KYN) with the same proficiency. Pre-steady-state kinetic analysis reveals that while the evolution of HsKYNase_66 is accompanied by a change in the rate-determining step of the reactions, HsKYNase_93D9 retains the same catalytic mechanism as HsKYNase. HDX-MS shows that the conformational dynamics of the two enzymes are markedly different and distinct from ortholog prokaryotic enzymes with high KYN activity. Our work provides a mechanistic framework for understanding the relationship between evolutionary mechanisms and phenotypic traits of evolved generalist and specialist enzyme species.
(© 2024. The Author(s).)
Databáze: MEDLINE
Externí odkaz: