Protein Crystallography Lab

Omega transaminases (omega-TAs) catalyze the chiral amination of unnatural substrates without the requirement of an alpha-COOH group and are highly commercially valuable for the industrial production of several pharmaceutical intermediates. Development of better variants of omega-TAs is hence essential for the biotransformation of unnatural substrates. We determined multiple crystals structures and performed extensive biochemical studies on two omega-TAs. Our results revealed the molecular basis for the development of omega-TAs with high catalytic efficiency for asymmetric synthesis, and further applications in large-scale biotransformation processes.

Glutamate dehydrogenase (GDH) is a pivotal metabolic enzyme in all living organisms. However, the mode of allosteric communication during the homotropic cooperativity in GDHs remains poorly understood. In this study, we examined two homologous GDHs, Aspergillus niger GDH (AnGDH) and Aspergillus terreus GDH (AtGDH), with differing substrate utilization kinetics to uncover the factors driving their distinct behavior. We report the first-ever cryo-EM structures of apo- AtGDH and AnGDH that captured arrays of conformational ensembles. We have identified a distant substitution in Domain II, which can impact the mouth opening and converts non-cooperative AtGDH into a cooperative enzyme. Our study demonstrates that remote residues can influence structural and kinetic properties in homologous GDHs.

Cellulases hydrolyze cellulose chains into fermentable glucose and hence are widely used in bioethanol production. The last enzyme of the cellulose degradation pathway, β-glucosidase, is inhibited by its product, glucose. The product inhibition by glucose hinders cellulose hydrolysis limiting the saccharification during bioethanol production. Thus, engineered β-glucosidases with enhanced glucose tolerance and catalytic efficiency are essential for industrial bioethanol production. In our study, structure-based rational design improved the glucose tolerance of a β-glucosidase from Acetivibrio thermocellus (WT-AtGH1). The WT-AtGH1 mutants exhibited improved glucose tolerance and catalytic efficiency while maintaining thermal stability. Our study emphasizes the importance of rational design in enhancing the properties of β-glucosidases for industrial applications.

Aspergillus oryzae exo-β-(1,3)-glucanase (AoBgl), a GH5 enzyme, hydrolyses β-(1,3)-glycosidic linkages. We have determined high-resolution structures of AoBgl: (a) the apo form at 1.75 Å, (b) the complexed form with bound cellobiose at 1.73 Å and (c) the glucose-bound form at 1.20 Å. The crystal structures, molecular dynamics simulation studies and site-directed mutagenesis reveal the mode of substrate binding and interactions at the active site. Our results unravel the molecular basis of sugar recognition in its active site of AoBgl. This study is highlighted on the cover page of FEBS Letters (January 2025 issue).

Tetrameric streptavidin along with a biotinylated metal complex is a promising artificial metalloenzyme for its industrial application in diverse non-natural reactions. In this collaborative work with Prof. Debabrata Maiti group (Department of Chemistry, IIT Bombay), a streptavidin-biotin-Rh(III) platform has been utilized to synthesize chiral isoindolones. We have determined a high-resolution crystal structure of streptavidin with unique conformations of the biotinylated Rh(III) cofactor, and the information from the structural analysis has aided to develop better biocatalyst.

We have shown that FDA approved HIV-1 drugs ritonavir (RTV) and lopinavir (LPV) exhibit the highest inhibition activity against plasmepsin II (PMII) and plasmepsin X (PMX) of P. falciparum, the parasite that causes the deadliest form of malaria. Crystal structures of the complexes of PMII with both RTV and LTV have been determined. Our structural investigations and biochemical data emphasize PMs as crucial targets for repurposing anti-HIV drugs as antimalarials.

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. Our data provide thorough insights into the mode of binding of a substrate and a potent inhibitor 49c to PfPMX.

Our paper in Emerging Topics in Life Sciences presents advancements in the field of protein crystallography or macromolecular crystallography (MX). The paper provides a brief history about the developments in methods as well as technologies in MX. With many exciting current techniques like X-ray Free Electron Lasers (XFELs) and more developments awaiting in the upcoming years, MX has the potential to contribute significantly to the growth of modern biology