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Precise Probing of Residue Roles by Post-Translational β,γ-C,N Aza-Michael Mutagenesis in Enzyme Active Sites.

Dadová, J and Wu, K-J and Isenegger, PG and Errey, JC and Bernardes, GJL and Chalker, JM and Raich, L and Rovira, C and Davis, BG (2017) Precise Probing of Residue Roles by Post-Translational β,γ-C,N Aza-Michael Mutagenesis in Enzyme Active Sites. ACS Cent Sci, 3. pp. 1168-1173. ISSN 2374-7943

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Abstract

Biomimicry valuably allows the understanding of the essential chemical components required to recapitulate biological function, yet direct strategies for evaluating the roles of amino acids in proteins can be limited by access to suitable, subtly-altered unnatural variants. Here we describe a strategy for dissecting the role of histidine residues in enzyme active sites using unprecedented, chemical, post-translational side-chain-β,γ C-N bond formation. Installation of dehydroalanine (as a "tag") allowed the testing of nitrogen conjugate nucleophiles in "aza-Michael"-1,4-additions (to "modify"). This allowed the creation of a regioisomer of His (iso-His, Hisiso) linked instead through its pros-Nπ atom rather than naturally linked via C4, as well as an aza-altered variant aza-Hisiso. The site-selective generation of these unnatural amino acids was successfully applied to probe the contributing roles (e.g., size, H-bonding) of His residues toward activity in the model enzymes subtilisin protease from Bacillus lentus and Mycobacterium tuberculosis pantothenate synthetase.

Item Type: Article
Subjects: UNSPECIFIED
Divisions: UNSPECIFIED
Depositing User: Cron Job
Date Deposited: 30 Jul 2019 01:57
Last Modified: 10 Apr 2021 22:35
DOI: 10.1021/acscentsci.7b00341