Abstract
Enzyme structures and functions evolve over time, but how changes occur is often unexplained. How do enzymes evolve new functions? How many mutations did it take to produce the new enzymatic activity? Are functions lost over time or can one activity simply be swapped for another? Questions pertaining to enzyme evolution are often difficult to address which leaves the process poorly understood. I used the homologous enzymes lactate and malate dehydrogenase (L/MDH) from Apicomplexa as a system to answer these questions. The modern enzymes are highly structurally similar but are specific for different substrates. LDH vs MDH specificity is canonically governed by the identity of a single sequence position, with charge balance being the primary source of substrate discrimination. However, LDH function has arisen multiple times and more than one molecular mechanism for specificity has developed. Several are examined in this work. Using Plasmodium falciparum LDH, I methodically investigated what was necessary to maintain LDH activity, ultimately asking what was needed to create the enzyme. While LDH activity originally evolved by way of a five-residue insertion, evolution of the LDH seems contingent on very little. Many insertions were likely possible to confer LDH function as activity is resilient to radical perturbations of both loop identity and length. Additionally, most apicomplexan LDHs are specific through charge balance but in Plasmodium LDH evolution, an active site glycine to alanine mutation added orders of magnitude stricter specificity. Specificity cannot be relaxed by reversing the mutation, suggesting strong specificity is functionally constrained in the Plasmodium lineage. The LDH from Cryptosporidium had no identified functional residues and lacked a thorough description of the evolution of the enzyme after a recent gene duplication. My results show that Cryptosporidium LDH evolved through the neofunctionalization of a specific MDH due to a few mutations of large effect. The ancestral LDH had a large shift in its rate limiting step, implying significant changes to the active site after the functional change. I was able to create several bifunctional enzymes using historic mutations, implying possible promiscuous intermediates. The modern enzyme now acts as a specific LDH with a network of epistatic interactions assisting an essential tryptophan in a novel position.