Recombinant Production, Purification, and Biochemical Characterization of a Novel L-lactate Dehydrogenase from NRC1 and Inhibition Study of Mangiferin

Lactate dehydrogenase (LDH, EC 1.1.1.27) is one of the vital glycolytic conditions, especially during anaerobic conditions. It is a significant diagnostic, prognostic, and monitoring biomarker parameter. A 950-bp DNA fragment containing the gene (LDH) encoding LDH was amplified from NRC1. The deduced amino acid sequence reveals that LDH (-LDH) is highly homologous to the LDHs of organisms. All LDH enzymes have a significant degree of conservation in their active site and several additional domains with unidentified functions. The gene for LDH, which catalyzes lactate synthesis, was cloned, sequenced (accession number: LC706200.1), and expressed in BL21 (DE3). In this investigation, -LDH was purified to homogeneity with a specific activity of 22.7 units/mg protein and a molecular weight of 35 kDa. It works optimally at pH 8.0. The purified enzyme was inhibited by FeCl, CuCl, ZnCl, and NiCl, whereas CoCl was found to boost the activity of -LDH. The molecular docking of the 3D model of the -LDH structure with a natural inhibitor, mangiferin, demonstrated excellent LDH inhibition, with a free binding energy of -10.2 kcal/mol. Moreover, mangiferin is a potent -LDH inhibitor that inhibits -LDH competitively and has one binding site with a Ki value of 0.075 mM. The LDH-mangiferin interaction exhibits a low RMSF value (>1.5 Å), indicating a stable contact at the residues. This study will pave the way for more studies to improve the understanding of mangiferin, which could be considered an intriguing candidate for creating novel and improved LDH inhibitors.