Precise Probing of Residue Roles by Post-Translational ß,y-C,N Aza-Michael Mutagenesis in Enzyme Active Sites

Jitka Dadová, Kuan-Jung Wu, Patrick Isenegger, James Errey, Goncalo Bernardes, Justin Chalker, Lluís Raich, Carme Rovira, Benjamin Davis

    Research output: Contribution to journalArticle

    10 Citations (Scopus)

    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.

    Original languageEnglish
    Pages (from-to)1168-1173
    Number of pages6
    JournalACS Central Science
    Volume3
    Issue number11
    DOIs
    Publication statusE-pub ahead of print - 2017

    Fingerprint Dive into the research topics of 'Precise Probing of Residue Roles by Post-Translational ß,y-C,N Aza-Michael Mutagenesis in Enzyme Active Sites'. Together they form a unique fingerprint.

  • Cite this

    Dadová, J., Wu, K-J., Isenegger, P., Errey, J., Bernardes, G., Chalker, J., Raich, L., Rovira, C., & Davis, B. (2017). Precise Probing of Residue Roles by Post-Translational ß,y-C,N Aza-Michael Mutagenesis in Enzyme Active Sites. ACS Central Science, 3(11), 1168-1173. https://doi.org/10.1021/acscentsci.7b00341