The structures of the C146A variant of the amidase from Pyrococcus horikoshii bound to glutaramide and acetamide suggest the basis of amide recognition.

The nitrilase superfamily enzymes from Pyrococcus abyssi and Pyrococcus horikoshii hydrolyze several different amides. No nitriles that we tested were hydrolyzed by either enzyme. Propionamide and acetamide were the most rapidly hydrolyzed of all the substrates tested. Amide substrate docking studies on the wild-type and C146A variant P. horikoshii enzymes suggest a sequence in which the incoming amide substrate initially hydrogen bonds to the amino group of Lys-113 and the backbone carbonyl of Asn-171. When steric hindrance is relieved by replacing the cysteine with alanine, the amide then docks such that the amino group of Lys-113 and the backbone amide of Phe-147 are hydrogen-bonded to the substrate carbonyl oxygen, while the backbone carbonyl oxygen of Asn-171 and the carboxyl oxygen of Glu-42 are hydrogen-bonded to the amino group of the substrate. Here, we confirm the location of the acetamide and glutaramide ligands experimentally in well-resolved crystal structures of the C146A mutant of the enzyme from P. horikoshii. This ligand location suggests that there is no direct interaction between the substrate amide and the other active site glutamate, Glu-120, and supports an active-site geometry leading to the formation of the thioester intermediate via an attack on the si-face of the amide by the sulfhydryl of the active site cysteine.

Discovery of Novel Prebiotic Carbohydrates and Sugar Mimics of BlMsmE, a Solute-Binding Protein of the ABC Transporter from Bacillus licheniformis.

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 Kd 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.

A cotton gene encoding novel MADS-box protein is preferentially expressed in fibers and functions in cell elongation.

Cotton fibers, as natural fibers, are widely used in the textile industry in the world. In order to find genes involved in fiber development, a cDNA (designated as GhMADS11) encoding a novel MADS protein with 151 amino acid residues was isolated from cotton fiber cDNA library. The deduced protein shares high similarity with Arabidopsis AP1 and AGL8 in MADS domain. However, the GhMADS11 protein (being absent of the partial K-domain and normal C-terminus) is shorter than AP1 and AGL8 by the reason of gene frameshift mutation during evolution. The experimental results revealed that GhMADS11 was not a transcriptional activator, and it did not form homodimer. GhMADS11 transcripts were specifically accumulated in elongating fibers, but no or very low signals of its expression were detected in other tissues of cotton. Overexpression of GhMADS11 in fission yeast promotes atypical cell elongation by 1.4-2.0-fold. Furthermore, morphological analysis indicated that the transformed cells expressing GhMADS11m, a MIKC-type derivative of GhMADS11 by the site-directed mutation, displayed the same phenotype as that of the transformed cells with GhMADS11. The concurrence of these data sets suggested that GhMADS11 protein may function in fiber cell elongation, and its MADS domain and partial K-domain are sufficient for this function.