2025/03/06 by Christoph Gruber, Εlad Noor, Marieke F. Buffing +1 · 1 voice
Biochemistry, Genetics and Molecular Biology · Computer Science · #Microbial Metabolic Engineering and Bioproduction #Computational Drug Discovery Methods #Metabolomics and Mass Spectrometry Studies
paper · pdf · doi:10.1073/pnas.2423767122
openalex publication_date 2025/03/06 · openalex created_date 2025/10/10 · openalex updated_date 2026/07/22
Recent physical binding screens suggest that protein–metabolite interactions are more extensive than previously recognized. To elucidate the functional relevance of these interactions, we developed a mass spectrometry–based screening method for higher throughput in vitro enzyme assays. By systematically quantifying the effects of 79 metabolites on the activity of 20 central Escherichia coli enzymes, we not only assess functional relevance but also gauge the depth of the current understanding of regulatory interactions within one of the best-characterized networks. Our identification of 50 inhibitors and 14 activators not only expands the range of known input signals but also uncovers novel regulatory logic. For instance, we observed that AMP inhibits malic enzyme to safeguard the cyclic operation of the tricarboxylic acid cycle, and erythrose-4-phosphate inhibits 6-phosphogluconate dehydrogenase to redirect flux from the pentose phosphate pathway into the Entner–Doudoroff pathway. Discrepancies between our standardized assays and existing database entries suggest that many previously reported interactions might occur only under specific, often nonphysiological conditions. Our dataset represents a systematically determined functional protein–metabolite interaction network, establishing a baseline for allosteric regulation in central metabolism. These results enhance our understanding of the regulatory logic governing metabolic processes and underscore its significance in cellular adaptation and growth.