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

• Applicant: Professor Dr. Stephan A. Sieber
• Subject Area: Biological and Biomimetic Chemistry
• Term: from 2016 to 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 314976069

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.

• DFG Programme: Research Grants