Stachyose is a typical prebiotic that can be utilized by the probiotic strain Bacillus licheniformis. Pioneering X-ray crystallography has determined the structure of stachyose in complex with the solute-binding protein MsmE in B. licheniformis (BlMsmE). The present work describes a combined strategy for the identification of putative BlMsmE-specific ligands, which can be used for the development of prebiotics. After a ligand-based virtual similarity screening of a large ZINC database containing ∼22 M compounds, we identified 3575 ligands. A total of 600 structures for which the Tanimoto coefficient's value was larger than a cutoff of 0.23 were selected for molecular docking. Based on the docking scores, we identified 100 top-scoring ligands, followed by molecular dynamics (MD) simulations. During simulations, 35 candidates were abandoned because of serious steric clashes in the complexes. Finally, the top 10 ligands with free energies below an energy threshold of −50.84 kcal/mol were selected. The top two ligands were stachyose and raffinose, which have proved their health benefits as prebiotics and their safety. The remaining eight ligands were further analyzed by the in silico ADME tool; only galactinol did not violate any of the criteria required for a lead compound. These three ligands were further analyzed for understanding their binding to BlMsmE. Isothermal titration calorimetry analysis suggested that stachyose, raffinose, and galactinol bound strongly to BlMsmE with K d values of 299, 170, and 134 nM, respectively. Microsecond MD simulations suggested significant conformational changes of BlMsmE upon ligand binding. Our results provide new insight into the thermodynamics of sugars and MsmE, which would promote the development of novel prebiotics.
Of considerable interest are the determinants of nitrilase activity, in which a nitrile is converted to ammonia and the corresponding carboxylic acid, versus amidase activity, in which an amide is converted to ammonia and the corresponding carboxylic acid. In both cases it appears that the main catalytic residues are a cysteine, two glutamates and a lysine. Three recombinantly expressed archaeal enzymes of the nitrilase superfamily have been purified and characterized. The PaNit from Pyrococcus abyssi has been reported as true nitrilase which prefers dinitrile substrates. Its X-ray structure (PDB ID 3IVZ) shows an almost identical active site to that of the known amidases. The AmiE from Pyrococcus yayanosii shares 78% sequence homology with PaNit has an amidase activity, hydrolyzing acetamide, L-glutamine, hexanamide, acrylamide, formamide and L-asparagine but no nitrilase activity against the corresponding nitriles. A similar enzyme found in Pyrococcus horikoshii was crystallized and its structure was determimed (PDB ID 1J31) to be extremely similar to PaNit (RMSD = 0.45A and the sequence identity is 86%). However, no functional characterization of this enzyme was reported. The genes from the archaeal enzymes from P. abyssi and P. horikoshii were synthesized and were inserted in expression plasmids that contain a C-terminal, thrombin cleavable his-tag. Abundant soluble expression of both proteins was obtained from the constructs. Amidase activity was observed for formamide, acetamide, propionamide, butyramide, hexanamideand malonamide but no nitrilase activity was observed for any substrate. The crystal structure of C146V variant of the enzyme from P. horikoshii has been determinied and will be used to observe substrate binding in the active site.
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