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

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Precise Probing of Residue Roles by Post-Translational β,γ-C,N Aza-Michael Mutagenesis in Enzyme Active Sites.
ACS Central Science ( IF 12.7 ) Pub Date : 2017-11-13 , DOI: 10.1021/acscentsci.7b00341
Jitka Dadová ₁ , Kuan-Jung Wu ₁ , Patrick G Isenegger ₁ , James C Errey ₁ , Gonçalo J L Bernardes ₁ , Justin M Chalker ₁ , Lluís Raich ₂ , Carme Rovira _{2,

3} , Benjamin G Davis ₁

Affiliation

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.

中文翻译：

通过酶活性位点的翻译后β，γ-C，N Aza-Michael诱变精确探测残基作用。

仿生学非常有价值地理解了概括生物学功能所必需的基本化学成分，但是通过获得合适的，微妙改变的非天然变体，可以限制用于评估氨基酸在蛋白质中的作用的直接策略。在这里，我们描述了一种使用史无前例的，化学的，翻译后的侧链-β，γCN键形成方法来解剖组氨酸残基在酶活性位点中的作用的策略。脱氢丙氨酸的安装（作为“标签”）允许测试“氮杂-迈克尔” -1，4-加成中的氮缀合亲核试剂（以“修饰”）。这允许创建通过其pros-Nπ原子而不是通过C4自然连接的His（异His，Hisiso）区域异构体，以及aza改变的aza-Hisiso变体。

更新日期：2017-11-14

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