一种基因编码的同型半胱氨酸前体,可探测蛋白质活性位点并使大肠杆菌对直接参与催化的非规范氨基酸上瘾。

Clara Dunker, Henning D Mootz
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引用次数: 0

摘要

非规范氨基酸(non - canonical amino acids, ncAAs)通过终止密码子的抑制被整合到蛋白质中,是研究和扩展蛋白质结构和功能的有力工具。虽然同型半胱氨酸(Hcy)是一种普遍存在的天然氨基酸,但它被排除在普遍的遗传密码之外。Hcy在探测蛋白质活性位点方面非常有趣,但大部分尚未被开发,因为它与半胱氨酸和丝氨酸有着微妙的结构和电子差异,而半胱氨酸和丝氨酸是酶中广泛存在的催化残基。我们报道了一种新的受保护的Hcy前体HcyX的遗传编码,它可以被化学还原剂或生物正交试剂方便地去保护。我们发现不同纯化酶在催化位置具有不同的活性水平,有时是显著的。通过利用细胞内对Hcy的部分去保护,我们发现两种依赖于Hcy的蛋白质,一种是肠素,另一种是胸苷酸合成酶,可以通过催化细胞生存所必需的反应来挽救大肠杆菌的生长。据我们所知,这是细胞生长与直接参与催化作用的基因结合的ncAA联系在一起的第一个例子。我们进一步证明,hys基二硫键在化学上比半胱氨酸二硫键更稳定。总之,这些发现为遗传密码的实验进化开辟了新的途径。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
A Genetically Encoded Homocysteine Precursor to Probe Protein Active Sites and to Addict Escherichia coli to a Noncanonical Amino Acid Directly Involved in Catalysis.

Noncanonical amino acids (ncAAs) incorporated into proteins by stop codon suppression are powerful tools to probe and expand protein structure and function. Although homocysteine (Hcy) is a ubiquitous, naturally occurring amino acid, it was excluded from the universal genetic code. Hcy is very interesting, yet mostly unexplored, for probing protein active sites because of its subtle structural and electronic differences from cysteine and serine, which are widespread catalytic residues in enzymes. We report the genetic encoding of a new protected Hcy precursor, HcyX, that can be conveniently deprotected by chemical reductants or bioorthogonal reagents. We find varying and sometimes remarkable levels of activity for different purified enzymes with Hcy at catalytic positions. By exploiting partial intracellular deprotection to Hcy, we show that two proteins rendered Hcy-dependent, an intein and thymidylate synthase, can rescue growth of Escherichia coli by catalyzing a reaction essential for cell survival. To the best of our knowledge, these are the first examples in which cell growth is linked to a genetically incorporated ncAA directly involved in catalysis. We further demonstrate that Hcy-based disulfide bonds are chemically more stable than cysteine disulfides. Together, these findings open new paths for the experimental evolution of the genetic code.

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