Evolution and Adaptation in Malate Dehydrogenases

Abstract

What is the evolutionary relationship between structure, function and stability is a central concept in the molecular life sciences. Malate Dehydrogenase is an excellent model enzyme to ask questions about this concept since i) it exists throughout biology, playing a central role in energy metabolism and ii) has a number of isoforms including cytosolic, mitochondrial, and in plants, glyoxysomal: overall the organelle forms are thought to have evolved by symbiosis with bacterial forms and are quite different in sequence and properties than the cytosolic forms. Cytosolic forms tend to have higher affinity for NADH while organelle forms have higher affinity for oxaloacetate, broadly consistent with the metabolic roles of the isoforms. Initially, we compared sequence conservation in 22 mammaliam cytoplasmic forms with watermelon glyoxysomal malate dehydrogenase (WMgMDH). Of 40 conserved amino acids in the cytosolic forms, 36 were conserved in WMgMDH. From the structure of WMgMDH, we used HINT and PyMol to examine atomic interactions both within the protein and with substrate and cofactor of the four residues, L159, A221, I250 and K311. We changed each residue to that resembling the cytosolic form to explore which contributed most to cytosolic like properties. We hypothesized that by changing Leucine 159 to a Valine (L159V), the hydrophobic core and cofactor bonding network would be altered. Changing Alanine 221 to Serine (A221S) would create hydrogen bonding potential at this site. Changing Isoleucine 250 to Valine (I250V) would reduce the hydrophobic area and potential impact stability of the protein. Lastly, changing Lysine 311 to Proline (K311P) may affect an interaction with cofactor due to its close proximity. Each mutation was constructed using QuikChange mutagenesis. Sequence confirmed mutants were expressed and purified using NiNTA Affinity Chromatography. Purified proteins were screened using SDS PAGE to assess purity. Specific Activities and Michaelis Constants for NADH and Oxaloacetate were then determined. The K311P and L159V mutants had specific activities similar to wildtype. I250V and A221T had 2 fold and 8 fold lower specific activities, respectively. Within standard errors, the Km for NADH was similar to wildtype for K311P and I250V, while L159V had a two fold lower Km (at a greater than 95% confidence limit). For oxaloacetate binding (as judged by Km for Oxaloacetate), I250V had a significantly higher Km, while L159V had a significantly lower Km (both at greater than 95% confidence limits). The changes produced by the L159 and I250 mutations result in more “cytosolic like” properties, while K311 mutation produced no significant effect. These findings contribute to our understanding of the subtle changes that can contribute to evolution and adaptation of enzyme structure and function.