Interaction of the Diguanylate Cyclase YdeH of Escherichia coli with 2′,(3′)-Substituted Purine and Pyrimidine Nucleotides

Abstract

Diguanylate cyclases (DGCs) synthesize the bacterial second messenger cyclic 3Ј,5Ј-diguanosine monophosphate (c-di-GMP), which is degraded by specific phosphodiesterases. c-di-GMP levels control the transition of bacteria from a motile to a biofilm-forming lifestyle. These bacterial communities are highly resistant to antibiotic treatment and represent the predominant lifestyle in most chronic infections. Hence, DGCs serve as starting point for the development of novel therapeutics interfering with the second messenger-signaling network in bacteria. In previous studies, we showed that 2Ј(3Ј)-O-(Nmethylanthraniloyl) (MANT)-and 2Ј,3Ј-O-(2,4,6-trinitrophenyl) (TNP)-substituted nucleotides are potent adenylyl and guanylyl cyclase inhibitors. The catalytic domain of DGCs is homologous to the mammalian adenylyl cyclase catalytic domain. Therefore, we investigated the interaction of various MANT purine and pyrimidine nucleotides with the model DGC YdeH from Escherichia coli. We observed strong fluorescence resonance energy transfer between tryptophan and tyrosine residues of YdeH and the MANT group of MANT-NTPs (MANT-ATP, -CTP, -GTP, -ITP, -UTP, and -XTP) and an enhanced direct MANT fluorescence upon interaction with YdeH. We assessed the affinity of MANT-NTPs to YdeH by performing competition assays with NTPs. We conducted an amino acid alignment of YdeH with the earlier crystallized Caulobacter crescentus DGC PleD and found high similarities in the nucleotide-binding site of PleD. In vitro mass-spectrometric activity assays with YdeH resulted in the identification of new MANT/ TNP nucleotide-based inhibitors of DGC activity. Together, the analysis of interactions between MANT/TNP nucleotides and YdeH provides a new basis for the identification and development of DGC inhibitors and allows insights into nucleotideprotein interactions.

The YmdB Phosphodiesterase Is a Global Regulator of Late Adaptive Responses in Bacillus subtilis

“…To test whether YmdB might have an unexpected diguanylate cyclase activity, we incubated YmdB with GTP and assayed for c-di-GMP production. In parallel, the experiment was performed with the E. coli diguanylate cyclase YdeH (29). While YdeH exhibited the expected diguanylate cyclase activity, no activity was detectable for YmdB.…”

“…The supernatants were analyzed for cyclic di-GMP (c-di-GMP) by liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS), as described previously (29). In parallel, the assays were also performed with the E. coli diguanylate cyclase YdeH (29). In all kinetic experiments, K m and V max were determined by nonlinear curve fitting from Lineweaver-Burk plots.…”

“…The samples were centrifuged for 10 min at 14,000 rpm at 4°C. The supernatants were analyzed for cyclic di-GMP (c-di-GMP) by liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS), as described previously (29). In parallel, the assays were also performed with the E. coli diguanylate cyclase YdeH (29).…”

Identification of Anti-Inflammatory and Anti-Hypertensive Drugs as Inhibitors of Bacterial Diguanylate Cyclases

“…26,27 Alternatively, strategies based on c-di-GMP modification and GTP analogs also identified specific DGCs inhibitors targeting both active and the allosteric binding sites. 28,29 In this report, we identified novel inhibitors of bacterial DGCs by virtual screening methods (VS) from the DrugBank database. DrugBank database has a collection of FDA-approved drugs with approximately 1500 entries that are in therapeutic use or are being tested in a wide variety of diseases.…”

“…The highly conserved residues N 335 and D 344 were set as constraints given their pivotal role in the recognition and specificity to GTP over other nucleotides by DGCs. 29,41 Initial tests demonstrated that the FRED program was able to reproduce successfully the PleD-GTP-α-S complex with a docking energy of −40 kcal mol -1 , as calculated by the Chemgauss3 scoring function. We then docked each of the 1,500 DrugBank compounds in 500 orientations at the PleD A-site (Figure 2A).…”