RESUMEN
Biofilm formation and surface attachment in multiple Alphaproteobacteria is driven by unipolar polysaccharide (UPP) adhesins. The pathogen Agrobacterium tumefaciens produces a UPP adhesin, which is regulated by the intracellular second messenger cyclic diguanylate monophosphate (c-di-GMP). Prior studies revealed that DcpA, a diguanylate cyclase-phosphodiesterase, is crucial in control of UPP production and surface attachment. DcpA is regulated by PruR, a protein with distant similarity to enzymatic domains known to coordinate the molybdopterin cofactor (MoCo). Pterins are bicyclic nitrogen-rich compounds, several of which are produced via a nonessential branch of the folate biosynthesis pathway, distinct from MoCo. The pterin-binding protein PruR controls DcpA activity, fostering c-di-GMP breakdown and dampening its synthesis. Pterins are excreted, and we report here that PruR associates with these metabolites in the periplasm, promoting interaction with the DcpA periplasmic domain. The pteridine reductase PruA, which reduces specific dihydro-pterin molecules to their tetrahydro forms, imparts control over DcpA activity through PruR. Tetrahydromonapterin preferentially associates with PruR relative to other related pterins, and the PruR-DcpA interaction is decreased in a pruA mutant. PruR and DcpA are encoded in an operon with wide conservation among diverse Proteobacteria including mammalian pathogens. Crystal structures reveal that PruR and several orthologs adopt a conserved fold, with a pterin-specific binding cleft that coordinates the bicyclic pterin ring. These findings define a pterin-responsive regulatory mechanism that controls biofilm formation and related c-di-GMP-dependent phenotypes in A. tumefaciens and potentially acts more widely in multiple proteobacterial lineages.
Asunto(s)
Agrobacterium tumefaciens , Proteínas Bacterianas , Biopelículas , GMP Cíclico , Pterinas , Biopelículas/crecimiento & desarrollo , Agrobacterium tumefaciens/metabolismo , Agrobacterium tumefaciens/genética , Pterinas/metabolismo , GMP Cíclico/metabolismo , GMP Cíclico/análogos & derivados , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteobacteria/metabolismo , Proteobacteria/genética , Cofactores de Molibdeno , Periplasma/metabolismo , Proteínas Periplasmáticas/metabolismo , Proteínas Periplasmáticas/genética , Proteínas de Unión Periplasmáticas/metabolismo , Proteínas de Unión Periplasmáticas/genética , Regulación Bacteriana de la Expresión GénicaRESUMEN
SARS-CoV-2 nsp13 helicase is an essential enzyme for viral replication and a promising target for antiviral drug development. This study compares the double-stranded RNA (dsRNA) unwinding activity of nsp13 and the Omicron nsp13R392C variant, which is predominant in currently circulating lineages. Using in vitro gel- and fluorescence-based assays, we found that both nsp13 and nsp13R392C have dsRNA unwinding activity with equivalent kinetics. Furthermore, the R392C mutation had no effect on the efficiency of the nsp13-specific helicase inhibitor SSYA10-001. We additionally confirmed the activity of several other helicase inhibitors against nsp13, including punicalagin that inhibited dsRNA unwinding at nanomolar concentrations. Overall, this study reveals the utility of using dsRNA unwinding assays to screen small molecules for antiviral activity against nsp13 and the Omicron nsp13R392C variant. Continual monitoring of newly emergent variants will be essential for considering resistance profiles of lead compounds as they are advanced towards next-generation therapeutic development.
Asunto(s)
Antivirales , Metiltransferasas , SARS-CoV-2 , Proteínas no Estructurales Virales , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/genética , Antivirales/farmacología , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas no Estructurales Virales/metabolismo , Humanos , ARN Bicatenario/genética , ARN Bicatenario/metabolismo , Mutación/genética , ARN Viral/genética , ARN Helicasas/antagonistas & inhibidores , ARN Helicasas/genética , ARN Helicasas/metabolismo , Replicación Viral/efectos de los fármacos , Replicación Viral/genética , COVID-19/virologíaRESUMEN
There is an urgent need for new antibiotics given the rise of antibiotic resistance, and succinyl-diaminopimelate desuccinylase (DapE, E.C. 3.5.1.18) has emerged as a promising bacterial enzyme target. DapE from Haemophilus influenzae (HiDapE) has been studied and inhibitors identified, but it is essential to explore DapE from different species to assess selective versus broad-spectrum therapeutics. We have determined the structure of DapE from the ESKAPE pathogen Acinetobacter baumannii (AbDapE) and studied inhibition by known inhibitors of HiDapE. AbDapE is inhibited by captopril and sulfate comparable to HiDapE, but AbDapE was not significantly inhibited by a known indoline sulfonamide HiDapE inhibitor. Captopril and sulfate both stabilize HiDapE by increasing the thermal melting temperature (Tm) in thermal shift assays. By contrast, sulfate decreases the stability of the AbDapE enzyme, whereas captopril increases the stability. Further, we report two crystal structures of selenomethionine-substituted AbDapE in the closed conformation, one with AbDapE in complex with succinate derived from enzymatic hydrolysis of N6-methyl-l,l-SDAP substrate and acetate (PDB code 7T1Q, 2.25 Å resolution), and a crystal structure of AbDapE with bound succinate along with l-(S)-lactate, a product of degradation of citric acid from the crystallization buffer during X-ray irradiation (PDB code 8F8O, 2.10 Å resolution).