Pyridoxal phosphate (PLP) is an enzyme cofactor required for the chemical transformation of biological amines in numerous essential cellular processes. PLP-dependent enzymes (PLP-DEs) are ubiquitous and evolutionarily diverse, making their classification based on sequence homology challenging. Here we present a chemical proteomic method for reporting on PLP-DEs using functionalized cofactor probes. We synthesized pyridoxal (PL)-analogues modified at the 2’-position which are taken up by cells and metabolized in situ. These PL-analogues are phosphorylated to functional cofactor surrogates by cellular PL kinases and bind to PLP-DEs via an aldimine bond which can be rendered irreversible by NaBH 4 reduction. Conjugation to a reporter tag enables the subsequent identification of PLP-DEs using quantitative, label-free mass spectrometry. Using these probes we accessed a significant portion of the Staphylococcus aureus PLP-DE proteome (73%) and annotate uncharacterized proteins as novel PLP-DEs. We also show that this approach can be used to study structural tolerance within PLP-DE active sites and to screen for off-targets of the PLP-DE inhibitor D-cycloserine.
Unprecedented bacterial targets are urgently needed to overcome the resistance crisis. Herein we systematically mine pyridoxal phosphate‐dependent enzymes (PLP‐DEs) in bacteria to focus on a target class which is involved in crucial metabolic processes. For this, we tailored eight pyridoxal (PL) probes bearing modifications at various positions. Overall, the probes exceeded the performance of a previous generation and provided a detailed map of PLP‐DEs in clinically relevant pathogens including challenging Gram‐negative strains. Putative PLP‐DEs with unknown function were exemplarily characterized via in‐depth enzymatic assays. Finally, we screened a panel of PLP binders for antibiotic activity and unravelled the targets of hit molecules. Here, an uncharacterized enzyme, essential for bacterial growth, was assigned as PLP‐dependent cysteine desulfurase and confirmed to be inhibited by the marketed drug phenelzine. Our approach provides a basis for deciphering novel PLP‐DEs as essential antibiotic targets along with corresponding ways to decipher small molecule inhibitors.
Highlights d Enrichment of human vitamin B 6-binding proteins with cofactor-derived probes d In situ target screening of vitamin B 6 antagonists d Comparison of human cell lines suggests cell-typedependent cofactor loading states
Cofactors are required for almost half of all enzyme reactions, but their functions and binding partners are not fully understood even after decades of research. Functionalised cofactor mimics that bind in place of the unmodified cofactor can provide answers, as well as expand the scope of cofactor activity. Through chemical proteomics approaches such as activity-based protein profiling, the interactome and localisation of the native cofactor in its physiological environment can be deciphered and previously uncharacterised proteins annotated. Furthermore, cofactors that supply functional groups to substrate biomolecules can be hijacked by mimics to site-specifically label targets and unravel the complex biology of post-translational protein modification. The diverse activity of cofactors has inspired the design of mimics for use as inhibitors, antibiotic therapeutics, and chemo-and biosensors, and cofactor conjugates have enabled the generation of novel enzymes and artificial DNAzymes.
Bacterial hibernating 100S ribosomes (the 70S dimers) are excluded from translation and are protected from ribonucleolytic degradation, thereby promoting long-term viability and increased regrowth. No extraribosomal target of any hibernation factor has been reported. Here, we discovered a previously unrecognized binding partner (YwlG) of hibernation-promoting factor (HPF) in the human pathogen Staphylococcus aureus . YwlG is an uncharacterized virulence factor in S. aureus . We show that the HPF–YwlG interaction is direct, independent of ribosome binding, and functionally linked to cold adaptation and glucose metabolism. Consistent with the distant resemblance of YwlG to the hexameric structures of nicotinamide adenine dinucleotide (NAD)–specific glutamate dehydrogenases (GDHs), YwlG overexpression can compensate for a loss of cellular GDH activity. The reduced abundance of 100S complexes and the suppression of YwlG-dependent GDH activity provide evidence for a two-way sequestration between YwlG and HPF. These findings reveal an unexpected layer of regulation linking the biogenesis of 100S ribosomes to glutamate metabolism.
Zur Überwindung der Antibiotika‐Resistenzkrise werden dringend neuartige bakterielle Angriffsziele benötigt. In diesem Artikel untersuchen wir systematisch Pyridoxalphosphat‐abhängige Enzyme (PLP‐DEs) in Bakterien, um uns auf Ziele zu konzentrieren, die an entscheidenden Stoffwechselprozessen beteiligt sind. Zu diesem Zweck haben wir acht Pyridoxal (PL)‐Sonden mit Modifikationen an verschiedenen Positionen entwickelt. Insgesamt übertrafen die Sonden die Leistung einer früheren Generation und lieferten detaillierte Informationen der PLP‐DEs in klinisch relevanten Krankheitserregern, einschließlich schwierig zu adressierender Gram‐negativer Stämme. Ausgewählte PLP‐DEs mit unbekannter Funktion wurden durch enzymatische Aktivitätsassays charakterisiert. Schließlich untersuchten wir eine Reihe von mutmaßlichen PLP‐Inhibitoren auf ihre antibiotische Aktivität und entschlüsselten die Zielenzyme aktiver Trefferverbindungen. Hier wurde ein kaum charakterisiertes Enzym, das für das bakterielle Wachstum unerlässlich ist, als PLP‐abhängige Cystein‐Desulfurase identifiziert und bestätigt, dass es durch das zugelassene Medikament Phenelzin gehemmt wird. Unser Ansatz bietet eine Grundlage für die Entschlüsselung neuartiger PLP‐DEs als essenzielle Antibiotikaziele sowie entsprechende Möglichkeiten zur Entschlüsselung kleiner Molekül‐Inhibitoren.
Cofaktoren werden für beinahe die Hälfte aller Enzymreaktionen benötigt. Ihre Funktionen und Bindungspartner sind jedoch auch nach jahrzehntelanger Forschung noch nicht vollständig verstanden. Funktionalisierte Cofaktoren (Analoga), die anstelle des natürlichen Cofaktors binden, können darauf Antworten liefern und den Aktivitätsbereich des jeweiligen Cofaktors aufklären. Mithilfe chemischer Proteomik‐Ansätze wie des aktivitätsbasierten Protein‐Profilings können das Interaktom und die Lokalisierung des nativen Cofaktors in seiner physiologischen Umgebung entschlüsselt und bisher uncharakterisierte Proteine annotiert werden. Darüber hinaus können Cofaktoren, die funktionelle Gruppen an Substrat‐Biomoleküle übertragen, genutzt werden, um als Analoge Enzyme ortsspezifisch zu markieren und die komplexe Biologie der posttranslationalen Proteinmodifikation zu untersuchen. Die vielfältige Aktivität von Cofaktoren hat die Entwicklung von deren Analoga für den Einsatz als Inhibitoren, Antibiotika sowie Chemo‐ und Biosensoren inspiriert. Darüber hinaus haben Cofaktor‐Konjugate die Herstellung neuartiger Enzyme und künstlicher DNA‐Enzyme ermöglicht.
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