Submission Type
Poster
Abstract
Malate dehydrogenase (MDH) plays an essential role in metabolism by catalyzing the reversible conversion of malate to oxaloacetate in the tricarboxylic acid cycle. This reaction, which also generates NADH, is critical to metabolic homeostasis across species. Although the general structure and function of MDH are well-characterized, the contribution of its conserved “flexible loop” region to substrate binding and catalysis remains insufficiently understood. In this project, we investigated how point mutations within this loop affect enzymatic activity in Citrullus lanatus (watermelon) glycosomal MDH (wgMDH), a model system with a well-characterized expression protocol and sequence. We introduced four site-directed mutations—R130A, R130E, R124K, and R124A—into the wgMDH gene via Q5-based mutagenesis in sister laboratories. These constructs were transformed into E. coli TOP10 cells for plasmid propagation and later into BL21(DE3) cells for expression and purification. Wild-type and mutant proteins were expressed under IPTG induction and purified by Ni-NTA chromatography. Protein levels were determined using the Bradford assay, and purity was assessed by SDS-PAGE. We are now optimizing protein expression and purification methods, aiming to perform kinetic assays with varied concentrations of key MDH reactants; which will further elucidate how flexible loop mutations affect overall enzymatic activity. By analyzing the effects of these mutations on catalytic efficiency and substrate interaction, we aim to deepen our understanding of MDH’s structural dynamics. Our findings may inform the broader significance of loop-mediated regulation in enzymes and support future therapeutic exploration, particularly as MDH is indeed a potential target in parasitic diseases like African trypanosomiasis.
Recommended Citation
Glass, Jay; McFadden, Andrew; and Singh, Harshdeep, "181 - Investigating the Role of Flexible Loop Mutations in Malate Dehydrogenase: Structural and Kinetic Impacts on Enzyme Catalysis" (2025). GREAT Day Posters. 47.
https://knightscholar.geneseo.edu/great-day-symposium/great-day-2025/posters-2025/47
181 - Investigating the Role of Flexible Loop Mutations in Malate Dehydrogenase: Structural and Kinetic Impacts on Enzyme Catalysis
Malate dehydrogenase (MDH) plays an essential role in metabolism by catalyzing the reversible conversion of malate to oxaloacetate in the tricarboxylic acid cycle. This reaction, which also generates NADH, is critical to metabolic homeostasis across species. Although the general structure and function of MDH are well-characterized, the contribution of its conserved “flexible loop” region to substrate binding and catalysis remains insufficiently understood. In this project, we investigated how point mutations within this loop affect enzymatic activity in Citrullus lanatus (watermelon) glycosomal MDH (wgMDH), a model system with a well-characterized expression protocol and sequence. We introduced four site-directed mutations—R130A, R130E, R124K, and R124A—into the wgMDH gene via Q5-based mutagenesis in sister laboratories. These constructs were transformed into E. coli TOP10 cells for plasmid propagation and later into BL21(DE3) cells for expression and purification. Wild-type and mutant proteins were expressed under IPTG induction and purified by Ni-NTA chromatography. Protein levels were determined using the Bradford assay, and purity was assessed by SDS-PAGE. We are now optimizing protein expression and purification methods, aiming to perform kinetic assays with varied concentrations of key MDH reactants; which will further elucidate how flexible loop mutations affect overall enzymatic activity. By analyzing the effects of these mutations on catalytic efficiency and substrate interaction, we aim to deepen our understanding of MDH’s structural dynamics. Our findings may inform the broader significance of loop-mediated regulation in enzymes and support future therapeutic exploration, particularly as MDH is indeed a potential target in parasitic diseases like African trypanosomiasis.
Comments
Sponsored by Varuni Jamburuthugoda