Biology subjects

Suryawanshi, A. B.

Publications and source records attributed to Suryawanshi, A. B..

3 recordsLinked to original sources

bioRxiv2025

Rewiring Catalytic Craters: A Path for Engineering β-Glucosidases to Improve Glucose Tolerance

Bioethanol, a sustainable alternative to fossil fuels, is produced from cellulose via enzymatic saccharification. {beta}-Glucosidase, a key enzyme in this process, hydrolyses disaccharides into glucose but is limited by feedback inhibition. This study investigated GH1 {beta}-glucosidase from a soil metagenome (UnBGl1) to understand and overcome this limitation. We solved the near-atomic resolution crystal structure of UnBGl1 at 1.15 [A] as an apo enzyme and report the first high-resolution crystal structures of the enzyme in its pre-hydrolytic state as a cellobiose complex and covalent intermediate-bound state, capturing key stages of its catalytic mechanism. Structural analysis revealed three crucial glucose-binding subsites in UnBGl1s crater. Glucose binding at the -1 subsite induced a 1.4 [A] shift in E370, increasing the distance between catalytic glutamates and reducing enzymatic activity. The C188V variant, generated by rational engineering, significantly improved glucose tolerance to 2.5 M. Additionally, the H261W mutation at the +2 subsite enhanced kinetic properties by improving cellobiose affinity (Km = 22.87 {+/-} 1.1 mM) and shifted the optimal pH to 5.5 from 6.0. Comparative structural analysis with other glucose-tolerant GH1 {beta}-glucosidases revealed conserved residues at the -1 subsite crucial for substrate stabilization and +1 subsite residues interacting with glucose, offering targets for further optimization. Engineered UnBGl1 variants retained high stability and activity on sugarcane bagasse, demonstrating their potential for industrial cellulase cocktails. These findings provide a robust framework for engineering {beta}-glucosidases with enhanced glucose tolerance and catalytic efficiency, paving the way for improved bioethanol production and contributing to sustainable energy solutions.

biochemistry↗

bioRxiv2024

Molecular insights into the oligosaccharide binding, recognition and hydrolysis by a fungal exo-beta-(1,3)-glucanase

Carbohydrate hydrolysing enzymes assume special industrial and commercial interest as a source for yielding fermentable glucose especially for the biofuel industry. Among these enzymes, the exo-{beta}-(1,3) glucanases are promising for industrial use as they hydrolyze sugars such as laminarin, a major constituent of the algal cell wall. Exploring the structure and function of these enzymes is of particular interest for the improvement of their functional properties for industrial use. We report the structural and biochemical characterizations of Aspergillus oryzae exo-{beta}-(1,3) glucanase (AoBgl). We have expressed, purified, and performed biochemical characterizations of the recombinant AoBgl. Purified AoBgl is found to hydrolyse {beta}-(1,3)-glycosidic linkages present in the oligosaccharide laminaritriose and the polysaccharide, laminarin effectively while retaining >50% activity at glucose concentrations of around 1.5M. We have determined three high-resolution structures of AoBgl: (a) apo form at 1.75 [A], (b) complexed form with bound disaccharide at 1.73 [A] and (c) glucose-bound form at 1.20 [A]. Sequence analysis and structural comparison indicate that AoBgl belongs to the GH5 sugar hydrolase family. The sugar-bound structures reveal the mode of substrate binding and interactions at the active site of AoBgl. Further, molecular dynamics simulation and mutational studies indicate that AoBgl can effectively bind trisaccharides and higher oligosaccharides. Our biochemical and structural data provide detailed molecular insights into the active site of this GH5 enzyme and would be helpful in the rational engineering of glycosyl hydrolases belonging to similar families for industrial use.

biochemistry↗

bioRxiv2021

Structures of plasmepsin X from P. falciparum reveal a novel inactivation mechanism of the zymogen and molecular basis for binding of inhibitors in mature enzyme

Plasmodium falciparum plasmepsin X (PfPMX), involved in the invasion and egress of this deadliest malarial parasite, is essential for its survival and hence considered as an important drug target. We report the first crystal structure of PfPMX zymogen containing a novel fold of its prosegment. A unique twisted loop from the prosegment and arginine 244 from the mature enzyme are involved in zymogen inactivation; such mechanism, not previously reported, might be common for apicomplexan proteases similar to PfPMX. The maturation of PfPMX zymogen occurs through cleavage of its prosegment at multiple sites. Our data provide thorough insights into the mode of binding of a substrate and a potent inhibitor 49c to PfPMX. We present molecular details of inactivation, maturation, and inhibition of PfPMX that should aid in the development of potent inhibitors against pepsin-like aspartic proteases from apicomplexan parasites.

biophysics↗