The goal of this research is to explore the dynamic structural mechanisms by which proteins have evolved novel functions in an important class of enzymes, the malate and lactate dehydrogenases (MDHs and LDHs, M/LDH family). We plan to resurrect entire evolutionary lineages of ancestral M/LDH enzymes, probe their biochemical functions, solve their structures by X-ray crystallography, and correlate the functional changes with structural changes observed along these evolutionary trajectories. Our model system is the malate and lactate dehydrogenase superfamily, which contains some of the most readily crystallizable proteins known. Both enzymes are widely distributed throughout life. MDH is found in the citric acid cycle and catalyzes the interconversion of malate to oxaloacetate, while LDH converts pyruvate, the final product of glycolysis, to lactate. The evolution of this family has been accompanied by many interesting and important functional innovations, including sharp changes in substrate specificity, acquisition of thermophilic and cryophilic stability, and gain of allosteric control by small effector molecules, and homo-multimerization via new protein- protein interfaces. The specific aims are to: 1) Investigate the evolutionary history of MDH and LDH proteins, reconstruct ancestral protein sequences along multiple trajectories, and resurrect them in the lab (using phylogenetic inference and artificial gene synthesis). Of exceptional interest are ancestral proteins bracketing significant evolutionary events, such as changes in specificity, catalytic rate, temperature stability, allostery, and oligomerization state. 2) Crystallize and determine the high-resolution structures of MDH and LDH lineages by X-ray crystallography. Analyze the evolution of observed structural changes. 3) Functionally characterize the resurrected proteins and correlate the functional changes along an evolutionary pathway with their concomitant structural perturbations. To determine which changes in the ancestors are functionally important, key residues will be mutated and biochemically characterized. By investigating these ancestral proteins and their mutants, we will empirically address many unanswered questions in molecular biology and in evolutionary theory. How do substitutions distal from the active site affect activity? Do historical amino acid substitutions have different effects on enzyme structure and function compared to the artificial mutations we engineer in modern enzymes? What is the importance of correlations among mutations (epistasis)? Does specificity increase during evolution? Was the common ancestor of MDH and LDH a promiscuous multifunctional protein? This study will provide the first dynamic, high-resolution picture of how novel biomolecular functions are generated during evolution by mutations and gene duplications. The resulting data will help answer several long-standing evolutionary questions and will bolster our knowledge of how enzymatic functions can be rationally engineered.