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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）是真核生物和原核生物中多功能酶促反应的重要辅因子。PLP催化的反应包括关键的过程，如转氨作用、脱羧作用和外消旋作用，这些过程对细胞功能很重要，并且与多种疾病的发生和治疗有关。PLP通过吡哆醛激酶（PLK）磷酸化吡哆醛（PL）而生物活化。这些酶在其活性部位利用保守的碱性残基（通常是半胱氨酸），促进5 ′-羟基对ATP的γ-磷酸的亲核攻击。最近，我们鉴定了一种新的酶亚类，其需要在柔性盖区域中的额外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。对几种细菌基因组序列的仔细检查显示，双半胱氨酸PLK（CC-PLK）的新亚类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变化，阻碍了基于序列的预测。因此，我们在这里设计了一种化学蛋白质组学策略，用于设计基于PL的抑制剂，该抑制剂通过在4 ′-醛位置引入的亲电子部分捕获推定的半硫代缩醛形成半胱氨酸残基。抑制剂将进一步配备有标记物，从而促进CC-PLK的蛋白质组范围的发现。我们预计，除了新的CC-PLK成员，我们也将解开其他蛋白质，使用半胱氨酸作为半硫缩醛中间体。其机械和功能特性是本提案的主要目标。此外，我们的目标是监测蛋白质组范围内纳入PLP依赖性酶的功能化探针通过特洛伊木马策略。探针被细胞吸收，被PLK磷酸化，然后通过共价醛亚胺结合在酶活性位点用作辅因子。我们的方法将这种不稳定的键转化为一个稳定的系链，随后将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致病菌，因为PLK和PLP依赖性酶代表抗生素治疗的主要药物靶点。因此，我们的功能化化合物不仅可以作为新型抑制剂，而且还可以代表用于鉴定有希望的靶标的发现工具，特别是在耐药菌株中。