Malate dehydrogenase (MDH) is an enzyme that is prevalent in many metabolic processes, such as the Krebs cycle and the citric acid cycle4. It reversibly forms oxaloacetate and NADH from (S)-malate and NAD+,1. Tryptophan fluorescence would be ideal to monitor conformational changes of MDH, but watermelon glycosomal MDH does not contain any tryptophan amino acids4. The research question being tested here is whether or not it is possible to mutate MDH to contain a tryptophan while still retaining native-like specific activity. This research question has not been addressed as a literature search has shown no research has been done to mutate Trp residues into wgMDH (watermelon). The mutant designed would be F353W. The factors that helped with the selection would be the similarity in shape to Trp as well as being distant from the active site, factors that are important towards maintance of native-like activity. The forward and reverse primer was first designed for 353W. The criteria that was provided for successful mutagenesis would be to have C or G at the end of both forward and reverse primer, to be 25-45 bases in length, and to have a melting temperature of over 78 degrees C using the QuickChange Site-Directed Mutagenesis Kit from Agilent Technologies. The reaction mixture was treated for DPN-1 and transformed into competent cells. Mutant colonies were picked and mutant protein was synthesized overnight in LB AMP. Five replicas of the mutant were produced and the plasmid was extracted. The five replicas were sequenced and will be used for further research, such as the subsequent protein production and purification of the mutant protein. The impact of this research would help with the further research towards binding for MDH including substrate binding, another enzyme binding, ligand binding and other research related to binding. Adding Trp would give the ability to track MDH and it would help track the conformational changes as conformational changes control diverse cellular processes, like signal transduction.
Yu, William, "Protein Engineering of a Fluorescent Watermelon Malate Dehydrogenase" (2023). Celebrating Scholarship and Creativity Day (2018-). 231.