Structural and functional evolution of the trichomonad lactate and malate dehydrogenases

The biochemical and biophysical properties of proteins evolve over time, yet their implications for the evolution of functional and structural novelty are still poorly understood. When proteins evolve by gene duplication, are the ancestors promiscuous or specific? Do changes in function happen primarily due to mutations of small or large effect? How do interactions between mutations (epistasis) influence evolution? What changes can happen to a protein's structure over the course of evolution, and how do they affect function? To answer these questions in a historically relevant setting, we reconstructed ancestors of trichomonad lactate and malate dehydrogenases (LDHs and MDHs), which catalyze the reduction of pyruvate and oxaloacetate, respectively, by NADH. These essential metabolic enzymes diverged by a recent gene duplication. We have found that the last common ancestor of these enzymes was a highly specific MDH (106-fold preference for oxaloacetate over pyruvate), a specificity conserved in modern MDH. Meanwhile, modern-day LDHs evolved promiscuous 2-ketoacid reductase activity (101- to 103-fold preference for pyruvate over oxaloacetate, with significant activity for other substrates) in a slow process of neofunctionalization. Site-directed mutagenesis of ancestral enzymes shows that promiscuous function evolved through one mutation of highly deleterious effect (R91L) and many mutations of small effect. R91L had greatly different effects in LDHs and MDHs, indicating a large role for epistasis. Additionally, we have solved modern-day and ancestral MDH and LDH crystal structures. By comparing these structures with a novel superpositioning technique, we have shown that changes in activity and specificity were paralleled by changes in dimerization and a helical shift in the LDHs.