NE0047 from Nitrosomonas europaea has been annotated as a zinc-dependent deaminase; however, the substrate specificity is unknown because of the low level of structural similarity and sequence identity compared to other family members. In this study, the function of NE0047 was established as a guanine deaminase (catalytic efficiency of 1.2 × 10(5) M(-1) s(-1)), exhibiting secondary activity towards ammeline. The structure of NE0047 in the presence of the substrate analogue 8-azaguanine was also determined to a resolution of 1.9 Å. NE0047 crystallized as a homodimer in an asymmetric unit. It was found that the extreme nine-amino acid C-terminal loop forms an active site flap; in one monomer, the flap is in the closed conformation and in the other in the open conformation with this loop region exposed to the solvent. Calorimetric data obtained using the full-length version of the enzyme fit to a sequential binding model, thus supporting a cooperative mode of ligand occupancy. In contrast, the mutant form of the enzyme (ΔC) with the deletion of the extreme nine amino acids follows an independent model of ligand occupancy. In addition, the ΔC mutant also does not exhibit any enzyme activity. Therefore, we propose that the progress of the reaction is communicated via changes in the conformation of the C-terminal flap and the closed form of the enzyme is the catalytically active form, while the open form allows for product release. The catalytic mechanism of deamination was also investigated, and we found that the mutagenesis of the highly conserved active site residues Glu79 and Glu143 resulted in a complete loss of activity and concluded that they facilitate the reaction by serving as proton shuttles.
Antibiotic production and resistance pathways in Streptomyces are dictated by the interplay of transcriptional regulatory proteins that trigger downstream responses via binding to small diffusible molecules. To decipher the mode of DNA binding and the associated allosteric mechanism in the sub-class of transcription factors that are induced by γ-butyrolactones, we present the crystal structure of CprB in complex with the consensus DNA element to a resolution of 3.25 Å. Binding of the DNA results in the restructuring of the dimeric interface of CprB, inducing a pendulum-like motion of the helix-turn-helix motif that inserts into the major groove. The crystal structure revealed that, CprB is bound to DNA as a dimer of dimers with the mode of binding being analogous to the broad spectrum multidrug transporter protein QacR from the antibiotic resistant strain Staphylococcus aureus. It was demonstrated that the CprB displays a cooperative mode of DNA binding, following a clamp and click model. Experiments performed on a subset of DNA sequences from Streptomyces coelicolor A3(2) suggest that CprB is most likely a pleiotropic regulator. Apart from serving as an autoregulator, it is potentially a part of a network of proteins that modulates the γ-butyrolactone synthesis and antibiotic regulation pathways in S. coelicolor A3(2).
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Quorum sensing is a cell density dependent phenomenon that utilizes small molecule inducers like γ-butyrolactones (GBLs) and their receptor proteins for adaptation to the environment. The cognate GBLs that bind to several of this GBL receptor family of proteins remain elusive. Here, using CprB protein from Streptomyces coelicolor A3(2) as a model system, we devise a method suited for ligand screening that would be applicable to the entire family of GBL receptors. Docking studies were performed to confirm the identity of the ligand binding pocket, and it was ascertained that the common γ-butyrolactone moiety interacts with the conserved tryptophan residue (W127) residing in the ligand binding pocket. The presence of W127 in the cavity was exploited to monitor its fluorescence quenching on the addition of two chemically synthesized GBLs. Analysis of the data with both the native and W185L mutant versions of the protein confirmed that the compounds used as quenchers reside in the ligand binding pocket. Furthermore, fluorescence lifetime and potassium iodide (KI) quenching studies established that the quenching is static in nature and that the tryptophan residue is buried and inaccessible to surface quenchers. Additionally, a combination of concentration dependent fluorescence quenching and dynamic light scattering experiments revealed that the binding properties of the protein are concentration dependent and it was concluded that the most efficient binding of the ligand is evoked by working at the lowest concentration of protein, providing a sufficient signal, where the aggregation effects are negligible.
In this work, we present a rapid, metal‐free and efficient method for thiocyanation of aromatic amines under microwave irradiation for the synthesis of aryl thiocyanates using inexpensive and nontoxic NH4SCN as thiocyanato source and K2S2O8 as an oxidant. This protocol tolerates a wide range of substituted anilines to provide the corresponding thiocyanated products in good to excellent yields. The features of this strategy include a broad substrate scope, high functional group tolerance, mild reaction conditions, and short reaction time.
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