Abstract: A Study of Mitochondrial Malate Dehydrogenase in Gallus gallus and Other Avian Species

Malate dehydrogenase (MDH) functions as a catalyst for the NAD+/NADH-dependent reversible reaction between malate and oxaloacetate. The mitochondrial form of the enzyme (MDH2) is important in the citric acid cycle, a key part of aerobic metabolism. Previous studies into avian MDH2 have focused on studying enzyme gel mobility between taxonomic families, activity differences between migratory and non-migratory species, and activity differences of a species at high versus low altitudes. Individual enzyme kinetics and structural data on the wild-type MDH2, however, are not documented. A cDNA library is utilized to obtain the gene for Gallus gallus (chicken). The Gibson Cloning assembly was used to insert the MDH2 gene into the pET28(a)+ expression vector for expression of the protein in E. coli cells. Initial experiments to test expression conditions indicated that codon optimization may be required. Once optimal expression of the enzyme is achieved, the Kcat, Vmax, and Km values of wild-type MDH2 can be determined. Additionally, protein modeling software was employed to predict the 3D structure of the Gallus gallus and other avian species mitochondrial MDH proteins. These comparisons not only give insight on how differences in the amino acid sequence affect the structure, but they provide clues as to what mutations to the Gallus gallus expression vector may mimic the MDH2 enzyme of another avian species providing future research directions.