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Research Article Volume 6 Issue 3
Role of N166 residue in β–glucosidase catalysis and glucose tolerance
Sneha Sawant, Arvind Lali1 Lali, Syed Shams Yazdani, Sanjeev Kumar Chandrayan, Annamma Anil Odaneth, Nidhi Adlakha
DBT-ICT Centre for Energy Biosciences, Institute of Chemical Technology, Nathalal Parekh Marg, India
DBT-ICGEB Advanced Bioenergy Research, International Centre for Genetic Engineering and Biotechnology (ICGEB), India
Received: May 12, 2019 | Published: June 18, 2019
Citation: Sawant S, Adlakha N, Odaneth AA, et al. Role of N166 residue in ? –glucosidase catalysis and glucose tolerance. J Appl Biotechnol Bioeng. 2019;6(3):142-148. DOI: 10.15406/jabb.2019.06.00186
Abstract
Product inhibition of β-glucosidase is one of the limiting factors in complete saccharification of lignocellulosic biomass to fermentable sugars. Relieving the detrimental effects of glucose on β-glucosidase will, therefore, be of great economic interest for the cost-effective deconstruction of recalcitrant biomass. The indispensable role of β-glucosidase in saccharification of cellulose led to the identification of GH1 family of CAZymes which have increased glucose tolerance and are stimulated in the presence of glucose, however, the exact mechanism is still elusive. The present study aims at unravelling interaction of glucose with active site residues in β-glucosidase (Gluc1C) using the combinatorial approach. Computational analysis revealed that amongst the catalytic centre residues, N166 interacts exclusively with glucose and no interaction was observed with substrate analogue thiocellobiose. In order to further understand the putative role of this residue in catalysis and product inhibition, we generated six single site-directed mutants. Biochemical characterization of the mutants indicated that of the six mutants only N166D mutant was active against para-nitrophenol-β-D-glucopyranoside (pNPG) as substrate. The N166D mutant was highly stimulated at 100mM glucose and was having a marginal increase in affinity for substrate pNPG, whereas replacement of N166 with amino acids with bulkier side chains resulted in a complete loss in activity. Thus, signifying the role of N166 in positioning substrate accurately between +1 and -1 subsite for hydrolysis as well as in substrate/ inhibitor binding and stimulation. The present study, highlight that apart from the regions around the active site entrance, the residue adjacent to an active site plays an important role in modulating the binding preference for cellobiose and glucose. The aforesaid study highlights the mechanistic details for glucose binding preferences in the enzyme active site. The study will also aid in rational engineering and selection of β-glucosidase for diverse biotechnological applications.
Keywords: ?-glucosidase, glucose tolerance, stimulation, mutagenesis, biotechnological applications, hydrolysis, structural basis
©2019 Sawant, et al. This is an open access article distributed under the terms of the, which permits unrestricted use, distribution, and build upon your work non-commercially.