Δημήτριος Χατζηνικολάου, Αναπληρωτής Καθηγητής Μικροβιακής Βιοτεχνολογίας, Τομέας Βοτανικής, Τμήμα Βιολογίας, Εθνικό και Καποδιστριακό Πανεπιστήμιο Αθηνών.
Many aerobic soil bacteria contain a phospho-enolpyruvate-dependent phosphotransferase system (PTS), which enables them to utilize oligomeric glycosides resulting from cellulose and/or other glucans degradation. Two key components of this system are (a) a transmembrane phosphotransferase, which phosphorylates the oligoglycans, while transporting them inside the cell and (b) an intracellular broad specificity GH1 β-P-glucosidase, which further hydrolyzes the resulted P-oligoglycans. This enzyme functions through a retaining mechanism having two glutamic acid residues as proton donor and as a nucleophilic group, respectively. In the present study, the results obtained from structural studies performed on a 6-phospho-β-glycosidase that belongs in family 1 glycosidase phosphate enzymes (GH1) (EC 126.96.36.199, 6-β-β-GH1) are presented. 6-β-β-GH1 is capable of hydrolyzing the β-1,4-glucosidic bond in a large number of β-P-bi-hexoses and was derived from a thermophilic microorganism, unlike the known structures investigated until that time. The ultimate aim of the study was to determine the 3D structure of the wild-type enzyme in its free form and in complex with substrate analogues. More specifically, the enzyme was isolated by the genome of the thermophilic bacterium Geobacillus sp. SP24, from the volcanic environment of Santorini island, Aegean Sea, Greece. Crystallisation trials were set with the vapor diffusion technique, using our in-house robotic crystallization facility at the Institute of Biology, Medicinal Chemistry and Biotechnology of the National Hellenic Research Foundation (NHRF). A selection of crystallization conditions was rationally designed based on literature research and commercially available crystallization screens. Selection of the suitable screens was performed after thorough investigation of the enzyme’s properties based on GH1 homologues. Initial hits were observed in a number of different conditions, either as thin plates or needles. Further optimization of protein: precipitant mixing ratio, resulted in single rhombohedral-shaped 6-β-β-GH1 crystals in the presence of PEG3350, sodium iodide and Bis-Tris propane pH 7. Meanwhile, another enzyme from a thermophilic Gram-positive soil bacterium was crystallized and its 3D structure was determined. For comparison purposes, modelling studies were performed using two different servers i-Tasser and Phyre2. The results showed that the predicited 3D structure obtained by Phyre 2 fitted best in one of the chains with glycosidase activity while the one from i-Tasser matched with another chain with galactosidase activity. Preliminary characterization of 6-P-β-GH1 crystals for the control of their diffraction capability was made using our in-house X-ray source at NHRF; however, the size of the crystals does not allow the collection of sufficient data. High resolution data, that will allow 6-P-β-GH1 structure determination, will be collected at Synchrotron Radiation sources in large scale European Facilities.
enzyme, 6-P-β-glycosidase, x-ray protein crystallography, structural studies