López-Villamizar, IralisCabezas, AliciaPinto, Rosa MaríaCanales, JoséRibeiro, João MeirelesRodrigues, Joaquim RuiCostas, María JesúsCameselle, José Carlos2026-03-302026-03-302021-02-17López-Villamizar, I.; Cabezas, A.; Pinto, R.M.; Canales, J.; Ribeiro, J.M.; Rodrigues, J.R.; Costas, M.J.; Cameselle, J.C. Molecular Dissection of Escherichia coli CpdB: Roles of the N Domain in Catalysis and Phosphate Inhibition, and of the C Domain in Substrate Specificity and Adenosine Inhibition. Int. J. Mol. Sci. 2021, 22, 1977. https://doi.org/10.3390/ijms22041977.1661-6596http://hdl.handle.net/10400.8/16053CpdB is a 3′-nucleotidase/2′ 3′-cyclic nucleotide phosphodiesterase, active also with rea-sonable efficiency on cyclic dinucleotides like c-di-AMP (3′,5′-cyclic diadenosine monophosphate) and c-di-GMP (3′,5′-cyclic diadenosine monophosphate). These are regulators of bacterial physi-ology, but are also pathogen-associated molecular patterns recognized by STING to induce IFN-β response in infected hosts. The cpdB gene of Gram-negative and its homologs of gram-positive bacteria are virulence factors. Their protein products are extracytoplasmic enzymes (either periplas-mic or cell–wall anchored) and can hydrolyze extracellular cyclic dinucleotides, thus reducing the innate immune responses of infected hosts. This makes CpdB(-like) enzymes potential targets for novel therapeutic strategies in infectious diseases, bringing about the necessity to gain insight into the molecular bases of their catalytic behavior. We have dissected the two-domain structure of Escherichia coli CpdB to study the role of its N-terminal and C-terminal domains (CpdB_Ndom and CpdB_Cdom). The specificity, kinetics and inhibitor sensitivity of point mutants of CpdB, and truncated proteins CpdB_Ndom and CpdB_Cdom were investigated. CpdB_Ndom contains the catalytic site, is inhibited by phosphate but not by adenosine, while CpdB_Cdom is inactive but contains a substrate-binding site that determines substrate specificity and adenosine inhibition of CpdB. Among CpdB substrates, 3′-AMP, cyclic dinucleotides and linear dinucleotides are strongly dependent on the CpdB_Cdom binding site for activity, as the isolated CpdB_Ndom showed much-diminished activity on them. In contrast, 2′,3′-cyclic mononucleotides and bis-4-nitrophenylphosphate were actively hydrolyzed by CpdB_Ndom, indicating that they are rather independent of the CpdB_Cdom binding site.engpathogen–host interactioncyclic dinucleotideextracytoplasmic phosphodiesteraseprotein domaintruncated proteinpoint mutantsubstrate-binding sitecatalytic sitesubstrate specificityinhibitor sensitivityjournal article10.3390/ijms220419771422-0067