Lablog4-63:Bypassing evolutionary dead ends and switching the rate-limiting step of a human immunotherapeutic enzyme

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John Blazeck, Christos S. Karamitros, Kyle Ford, Catrina Somody, Ahlam Qerqez, Kyle Murray, Nathaniel T. Burkholder, Nicholas Marshall, Anirudh Sivakumar, Wei-Cheng Lu, Bing Tan, Candice Lamb, Yuri Tanno, Menna Y. Siddiqui, Norah Ashoura, Silvia Coma, Xiaoyan M. Zhang, Karen McGovern, Yoichi Kumada, Yan Jessie Zhang, Mark Manfredi, Kenneth A. Johnson, Sheena D’Arcy, Everett Stone, George Georgiou

First author

John Blazeck, Christos S. Karamitros

Corresponding author

George Georgiou

Publication Style

Journal name Nature Catalysis


Volume, issue, pages

5(10) 952-967


The Trp metabolite L-kynurenine (KYN) accumulates in numerous solid tumours and mediates potent immunosuppression. Bacterial kynureninases (KYNases), which preferentially degrade KYN, can relieve immunosuppression in multiple cancer models, but immunogenicity concerns preclude their clinical use, while the human enzyme (HsKYNase) has very low activity for KYN and shows no therapeutic effect. Using fitness selections, we evolved a HsKYNase variant with 28-fold higher activity, beyond which exploration of >30 evolutionary trajectories involving the interrogation of >109 variants led to no further improvements. The 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 the 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.