7S3V

Structure of HsKYNase_66, an evolved variant of human kynureninase with greatly increased activity towards kynurenine

7S3V の概要

• エントリーDOI: 10.2210/pdb7s3v/pdb
• 分子名称: Kynureninase (1 entity in total)
• 機能のキーワード: kynurenine, tryptophan, cancer, directed-evolution, antitumor protein, hydrolase
• 由来する生物種: Homo sapiens (Human)
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 106019.68

構造登録者

Burkholder, N.T.,Zhang, Y.J. (登録日: 2021-09-08, 公開日: 2022-12-21, 最終更新日: 2023-11-15)

主引用文献

Blazeck, J.,Karamitros, C.S.,Ford, K.,Somody, C.,Qerqez, A.,Murray, K.,Burkholder, N.T.,Marshall, N.,Sivakumar, A.,Lu, W.C.,Tan, B.,Lamb, C.,Tanno, Y.,Siddiqui, M.Y.,Ashoura, N.,Coma, S.,Zhang, X.M.,McGovern, K.,Kumada, Y.,Zhang, Y.J.,Manfredi, M.,Johnson, K.A.,D'Arcy, S.,Stone, E.,Georgiou, G.
Bypassing evolutionary dead ends and switching the rate-limiting step of a human immunotherapeutic enzyme.
Nat Catal, 5:952-967, 2022

PubMed 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 >10 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.

PubMed: 36465553
DOI: 10.1038/s41929-022-00856-6

実験手法

X-RAY DIFFRACTION (3.249 Å)