UNLABELLED: The ability to predict the transglycosylation activity of glycosidases by in silico analysis was investigated. The transglycosylation abilities of 7 different β-d-galactosidases from GH family 2 were tested experimentally using 7 different acceptors and p-nitrophenyl-β-d-galactopyranoside as a donor of galactosyl moiety. Similar transglycosylation abilities were confirmed for all enzymes originating from bacteria belonging to Enterobacteriaceae, which were able to use all tested acceptor molecules. Higher acceptor selectivity was observed for all others used bacterial strains. Structure models of all enzymes were constructed using homology modeling. Ligand-docking method was used for enzymes-transglycosylation products models construction and evaluation. Results obtained by in silico analysis were compared with results arisen out of experimental testing. The experiments confirmed that significant differences in transglycosylation abilities are caused by small differences in active sites composition of analyzed enzymes. According to obtained result, it is possible to conclude that homology modeling may serve as a quick starting point for detection or exclusion of enzymes with defined transglycosylation abilities, which can be used for subsequent synthesis of e.g., pharmaceutically interesting glycosides. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02715-w.

• Keywords
• Carbohydrate family, Catalysis, Homology modeling, Hydrolases, Ligand-docking,
• Publication type
• Journal Article MeSH

The heme-based oxygen sensor histidine kinase AfGcHK is part of a two-component signal transduction system in bacteria. O2 binding to the Fe(II) heme complex of its N-terminal globin domain strongly stimulates autophosphorylation at His183 in its C-terminal kinase domain. The 6-coordinate heme Fe(III)-OH- and -CN- complexes of AfGcHK are also active, but the 5-coordinate heme Fe(II) complex and the heme-free apo-form are inactive. Here, we determined the crystal structures of the isolated dimeric globin domains of the active Fe(III)-CN- and inactive 5-coordinate Fe(II) forms, revealing striking structural differences on the heme-proximal side of the globin domain. Using hydrogen/deuterium exchange coupled with mass spectrometry to characterize the conformations of the active and inactive forms of full-length AfGcHK in solution, we investigated the intramolecular signal transduction mechanisms. Major differences between the active and inactive forms were observed on the heme-proximal side (helix H5), at the dimerization interface (helices H6 and H7 and loop L7) of the globin domain and in the ATP-binding site (helices H9 and H11) of the kinase domain. Moreover, separation of the sensor and kinase domains, which deactivates catalysis, increased the solvent exposure of the globin domain-dimerization interface (helix H6) as well as the flexibility and solvent exposure of helix H11. Together, these results suggest that structural changes at the heme-proximal side, the globin domain-dimerization interface, and the ATP-binding site are important in the signal transduction mechanism of AfGcHK. We conclude that AfGcHK functions as an ensemble of molecules sampling at least two conformational states.

• Keywords
• bacterial protein kinase, crystal structure, globin, heme-containing oxygen sensor, histidine kinase, hydrogen-deuterium exchange, signal transduction, two component signal transduction system,
• MeSH
• Bacterial Proteins chemistry   metabolism   MeSH
• Phosphorylation MeSH
• Heme chemistry   MeSH
• Histidine Kinase chemistry   metabolism   MeSH
• Mass Spectrometry MeSH
• Crystallography, X-Ray MeSH
• Protein Structure, Quaternary MeSH
• Oxygen metabolism   MeSH
• Models, Molecular MeSH
• Myxococcales metabolism   MeSH
• Oxidation-Reduction MeSH
• Protein Domains MeSH
• Signal Transduction MeSH
• Deuterium Exchange Measurement MeSH
• Ferric Compounds chemistry   MeSH
• Ferrous Compounds chemistry   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Bacterial Proteins MeSH
• Heme MeSH
• Histidine Kinase MeSH
• Oxygen MeSH
• Ferric Compounds MeSH
• Ferrous Compounds MeSH

Many carbohydrate-binding proteins contain aromatic amino acid residues in their binding sites. These residues interact with carbohydrates in a stacking geometry via CH/π interactions. These interactions can be found in carbohydrate-binding proteins, including lectins, enzymes and carbohydrate transporters. Besides this, many non-protein aromatic molecules (natural as well as artificial) can bind saccharides using these interactions. Recent computational and experimental studies have shown that carbohydrate-aromatic CH/π interactions are dispersion interactions, tuned by electrostatics and partially stabilized by a hydrophobic effect in solvated systems.

• Keywords
• CH/π interactions, carbohydrate-protein interactions, interaction energy, lectins, non-canonical hydrogen bond,
• MeSH
• Lectins chemistry   metabolism   MeSH
• Models, Molecular MeSH
• Carbohydrates chemistry   MeSH
• Protein Binding MeSH
• Hydrogen Bonding MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Lectins MeSH
• Carbohydrates MeSH

β-Mannosidase (EC 3.2.1.25) is an important exoglycosidase specific for the hydrolysis of terminal β-linked mannoside in various oligomeric saccharide structures. β-Mannosidase from Aspergillus niger was expressed in Pichia pastoris and purified to clear homogeneity. β-Mannosidase was crystallized in the presence of D-mannose and the crystal diffracted to 2.41 Å resolution. The crystal belonged to space group P1, with unit-cell parameters a=62.37, b=69.73, c=69.90 Å, α=108.20, β=101.51, γ=103.20°. The parameters derived from the data collection indicate the presence of one molecule in the asymmetric unit.

• Keywords
• Aspergillus niger, β-mannosidase,
• MeSH
• Aspergillus niger chemistry   enzymology   MeSH
• beta-Mannosidase chemistry   genetics   MeSH
• Fungal Proteins chemistry   genetics   MeSH
• Crystallization MeSH
• Crystallography, X-Ray MeSH
• Mannose chemistry   MeSH
• Pichia chemistry   genetics   MeSH
• Recombinant Proteins chemistry   genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• beta-Mannosidase MeSH
• Fungal Proteins MeSH
• Mannose MeSH
• Recombinant Proteins MeSH

Recently, the gene coding for a new beta-glucuronidase enzyme has been identified and cloned from Streptococcus equi subsp. zooepidemicus. This is another report of a beta-glucuronidase gene cloned from bacterial species. The ORF Finder analysis of a sequenced DNA (EMBL, AJ890474) revealed a presence of 1,785 bp large ORF potentially coding for a 594 aa protein. Three protein families in (Pfam) domains were identified using the Conserved Domain Database (CDD) analysis: Pfam 02836, glycosyl hydrolases family 2, triose phosphate isomerase (TIM) barrel domain; Pfam 02837, glycosyl hydrolases family 2, sugar binding domain; and Pfam 00703, glycosyl hydrolases family 2, immunoglobulin-like beta-sandwich domain. To gain more insight into the enzymatic activity, the domains were used to generate a bootstrapped unrooted distance tree using ClustalX. The calculated distances for two domains, TIM barrel domain, and sugar-binding domain were comparable and exhibited similarity pattern based on function and thus being in accordance with recently published works confirming beta-glucuronidase activity of the enzyme. The calculated distances and the tree arrangement in the case of centrally positioned immonoglobulin-like beta-sandwich domain were somewhat higher when compared to other two domains but clustering with other beta-glucuronidases was rather clear. Nine proteins, including beta-glucuronidases, beta-galactosidase, and mannosidase were selected for multiple alignment and subsequent distance tree creation.

• MeSH
• Glucuronidase genetics   MeSH
• Horses MeSH
• Models, Molecular MeSH
• Molecular Sequence Data MeSH
• Amino Acid Sequence MeSH
• Base Sequence MeSH
• Sequence Analysis, DNA MeSH
• Sequence Homology, Amino Acid MeSH
• Sequence Homology, Nucleic Acid MeSH
• Cluster Analysis MeSH
• Streptococcus equi genetics   MeSH
• Protein Structure, Tertiary genetics   MeSH
• Computational Biology * MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Glucuronidase MeSH