Chemical-proteomic tools to monitor pyridoxal phosphorylation and its function as an enzyme cofactor in disease-related pathways

用于监测吡哆醛磷酸化及其作为疾病相关途径中酶辅因子的功能的化学蛋白质组学工具

基本信息

批准号：
314976069
负责人：
Professor Dr. Stephan A. Sieber
金额：
--
依托单位：
Lehrstuhl für Organische Chemie II
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2016
资助国家：
德国
起止时间：
2015-12-31 至 2019-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/314976069?language=en
关键词：
Chemical proteomic tools monitor pyridoxal

项目摘要

Pyridoxal phosphate (PLP) represents an important cofactor for versatile enzymatic reactions in eukaryotic and prokaryotic organisms. PLP catalyzed reactions include crucial processes such as transamination, decarboxylation and racemization, which are important for cellular function and relevant to the onset and treatment of several diseases. PLP is bioactivated by phosphorylation of pyridoxal (PL) via pyridoxal kinases (PLK). These enzymes utilize a conserved basic residue (often cysteine) in their active site that facilitates the nucleophilic attack of the 5´-hydroxy group onto gamma-phosphate of ATP. Recently we identified a novel enzyme subclass that requires an additional nucleophilic cysteine residue in a flexible lid region to form a hemithioacetal intermediate with the 4´-aldehyde of PL to promote phosphorylation. A closer inspection of several bacterial genome sequences revealed that the new subclass of dual cysteine PLKs (CC-PLK) is likely present in many other strains. In addition, the position of cysteine in the flexible lid varies, impeding sequence based predictions. We thus devise here a chemical proteomic strategy for the design of PL-based inhibitors that trap putative hemithioacetal-forming cysteine residues via electrophilic moieties incorporated at the 4´-aldehyde position. The inhibitors will be further equipped with a marker, thereby facilitating the proteome-wide discovery of CC-PLKs. We anticipate that in addition to novel CC-PLK members, we will also unravel other proteins that use cysteines as hemithioacetal intermediates. Their mechanistic and functional characterization is a major objective of this proposal. In addition, we aim to monitor the proteome-wide incorporation of functionalized probes into PLP-dependent enzymes via a Trojan horse strategy. The probes are taken up by the cells, phosphorylated by PLKs and then utilized as cofactors in enzyme active sites via covalent aldimine binding. Our approach converts this labile bond into a stable tether which will subsequently be utilized in mass-spectrometric experiments and quantitative analysis for protein identification. The main emphasis of these studies will be on pathogenic bacteria as PLKs and PLP-dependent enzymes represent major drug targets for antibiotic therapy. Our functionalized compounds will thus not only serve as novel inhibitors but also represent discovery tools for the identification of promising targets, especially in resistant strains.

磷酸吡哆醛 (PLP) 是真核和原核生物中多种酶促反应的重要辅助因子，PLP 催化的反应包括转氨、脱羧和外消旋等关键过程，这些过程对于细胞功能很重要，并且与多种疾病的发病和治疗相关。 PLP 通过吡哆醛激酶 (PLK) 磷酸化吡哆醛 (PL) 来生物激活。这些酶利用保守的酶。其活性位点中的碱性残基（通常是半胱氨酸）有助于 5'-羟基对 ATP 的 γ-磷酸盐进行亲核攻击。最近，我们发现了一种新的酶亚类，它需要在柔性盖区域中添加额外的亲核半胱氨酸残基。与 PL 的 4´-醛形成半硫缩醛中间体以促进磷酸化。对几个细菌基因组序列的仔细检查表明，双半胱氨酸 PLK 的新亚类。 (CC-PLK) 可能存在于许多其他菌株中。此外，半胱氨酸在柔性盖中的位置各不相同，这阻碍了基于序列的预测，因此我们在此设计了一种用于设计基于 PL 的抑制剂的化学蛋白质组策略。通过在 4'-醛位置掺入的亲电子部分捕获假定的半缩醛形成半胱氨酸残基。抑制剂将进一步配备标记，从而促进蛋白质组范围内的发现。 CC-PLK。我们预计，除了新的 CC-PLK 成员外，我们还将揭示使用半胱氨酸作为半硫缩醛中间体的其他蛋白质，其机制和功能表征是该提案的主要目标。通过特洛伊木马策略将功能化探针在蛋白质组范围内掺入 PLP 依赖性酶中。探针被细胞吸收，被 PLK 磷酸化，然后用作辅助因子。我们的方法通过共价醛亚胺结合将这种不稳定的键转化为稳定的键，随后将其用于质谱实验和蛋白质鉴定的定量分析，这些研究的主要重点将是针对 PLK 和 PLP 等病原菌。因此，依赖酶代表了抗生素治疗的主要药物靶标，因此我们的功能化化合物不仅可以作为新型抑制剂，而且还代表了识别有希望的靶标的发现工具，特别是在耐药菌株中。