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Tailored Cofactor Traps for the in Situ Detection of Hemithioacetal-Forming Pyridoxal Kinases
ACS Chemical Biology ( IF 3.5 ) Pub Date : 2020-12-03 , DOI: 10.1021/acschembio.0c00787
Ines Hübner 1 , Jan-Niklas Dienemann 1 , Julia Friederich 1, 2 , Sabine Schneider 3 , Stephan A Sieber 1
Affiliation  

Pyridoxal kinases (PLK) are crucial enzymes for the biosynthesis of pyridoxal phosphate, an important cofactor in a plethora of enzymatic reactions. The evolution of these enzymes resulted in different catalytic designs. In addition to the active site, the importance of a cysteine, embedded within a distant flexible lid region, was recently demonstrated. This cysteine forms a hemithioacetal with the pyridoxal aldehyde and is essential for catalysis. Despite the prevalence of these enzymes in various organisms, no tools were yet available to study the relevance of this lid residue. Here, we introduce pyridoxal probes, each equipped with an electrophilic trapping group in place of the aldehyde to target PLK reactive lid cysteines as a mimic of hemithioacetal formation. The addition of alkyne handles placed at two different positions within the pyridoxal structure facilitates enrichment of PLKs from living cells. Interestingly, depending on the position, the probes displayed a preference for either Gram-positive or Gram-negative PLK enrichment. By applying the cofactor traps, we were able to validate not only previously investigated Staphylococcus aureus and Enterococcus faecalis PLKs but also Escherichia coli and Pseudomonas aeruginosa PLKs, unravelling a crucial role of the lid cysteine for catalysis. Overall, our tailored probes facilitated a reliable readout of lid cysteine containing PLKs, qualifying them as chemical tools for mining further diverse proteomes for this important enzyme class.

中文翻译:

量身定制的辅助因子陷阱,用于原位检测形成半硫缩醛的吡rid醛激酶。

吡rid醛激酶(PLK)是生物合成吡ido醛磷酸盐的重要酶,吡ido醛磷酸盐是多种酶促反应中的重要辅助因子。这些酶的进化导致了不同的催化设计。除了活性部位,最近还证明了嵌入在遥远的柔性盖区域内的半胱氨酸的重要性。该半胱氨酸与吡ido醛形成半硫缩醛,对于催化是必不可少的。尽管这些酶在各种生物中普遍存在,但尚无工具可用于研究这种盖残渣的相关性。在这里,我们介绍了吡ido醛探针,每个探针都配备了一个亲电捕获基团来代替醛,以模拟半硫缩醛的形成,靶向PLK反应性盖半胱氨酸。放置在吡ido醛结构内两个不同位置的炔烃手柄有助于从活细胞中富集PLK。有趣的是,根据位置,探针显示出对革兰氏阳性或革兰氏阴性PLK富集的偏好。通过应用辅助因子陷阱,我们不仅能够验证先前进行的研究金黄色葡萄球菌粪肠球菌PLK,还有大肠杆菌铜绿假单胞菌PLK,揭示了半胱氨酸盖在催化中的关键作用。总体而言,我们量身定制的探针有助于可靠地读出含有盖半胱氨酸的PLK,使它们有资格作为化学工具来为该重要酶类别开发更多蛋白质组。
更新日期:2020-12-18
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