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

磷酸吡哆醛（Pyridoxal phosphate， PLP）是真核生物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，这些过程对细胞功能至关重要，并与多种疾病的发病和治疗有关。PLP通过吡哆醛激酶（PLK）磷酸化吡哆醛（PL）而被生物激活。这些酶在活性位点利用保守的碱性残基（通常是半胱氨酸），促进5′-羟基对ATP -磷酸的亲核攻击。最近，我们发现了一种新的酶亚类，它需要一个额外的亲核半胱氨酸残基在一个灵活的盖子区域，与PL的4′-醛形成半缩醛中间体，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，半胱氨酸在柔性眼睑的位置不同，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′-醛位置结合的亲电性片段捕获推定的半硫缩醛形成的半胱氨酸残基。抑制剂将进一步配备一个标记，从而促进蛋白质组范围内发现CC-PLKs。我们预计，除了新的CC-PLK成员外，我们还将揭示其他使用半胱氨酸作为半硫缩醛中间体的蛋白质。它们的机制和功能特征是本建议的主要目标。此外，我们的目标是通过特洛伊木马策略监测功能化探针在蛋白质组范围内与plp依赖性酶的结合。这些探针被细胞吸收，被plk磷酸化，然后通过共价醛胺结合作为酶活性位点的辅助因子。我们的方法将这种不稳定的键转化为稳定的系绳，随后将用于质谱实验和蛋白质鉴定的定量分析。这些研究的重点将放在致病菌上，因为plk和plp依赖性酶是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的抑制剂，而且还可以作为鉴定有希望的靶点的发现工具，特别是在耐药菌株中。

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.