An atomic-resolution view of neofunctionalization in the evolution of apicomplexan lactate dehydrogenases

Abstract

Malate and lactate dehydrogenases (MDH and LDH) are homologous, core metabolic enzymes that share a fold and catalytic mechanism yet possess strict specificity for their substrates. In the Apicomplexa, convergent evolution of an unusual LDH from MDH produced a difference in specificity exceeding 12 orders of magnitude. The mechanisms responsible for this extraordinary functional shift are currently unknown. Using ancestral protein resurrection, we find that specificity evolved in apicomplexan LDHs by classic neofunctionalization characterized by long-range epistasis, a promiscuous intermediate, and few gain-of-function mutations of large effect. In canonical MDHs and LDHs, a single residue in the active-site loop governs substrate specificity: Arg102 in MDHs and Gln102 in LDHs. During the evolution of the apicomplexan LDH, however, specificity switched via an insertion that shifted the position and identity of this ‘specificity residue’ to Trp107f. Residues far from the active site also determine specificity, as shown by the crystal structures of three ancestral proteins bracketing the key duplication event. This work provides an unprecedented atomic-resolution view of evolutionary trajectories creating a nascent enzymatic function.How are new genes created? Most of the mutations in the genome of an organism place the organism at some sort of disadvantage, but a small number confer an advantage. The beneficial changes are usually retained by subsequent generations and can ultimately lead to the creation of new genes.An example is the gene that encodes an enzyme called lactate dehydrogenase (LDH). This enzyme is involved in anaerobic respiration, the process that allows organisms to produce energy without using oxygen. The LDH enzyme is found in many species of animals and parasites, including those that spread malaria and other diseases. However, there are important differences in the structures of the LDH enzyme in animals and some parasites, like the malarial Plasmodium, because the genes for the enzymes in these two groups evolved separately.The parasite version of the LDH enzyme evolved hundreds of millions of years ago from an enzyme with a similar structure called malate dehydrogenase, which was inherited from bacteria. To work out how the LDH enzyme developed, Boucher et al. predicted and built the ancestral proteins that would have formed as the bacterial enzyme evolved into LDH.Studying these structures revealed that two mutations were mainly responsible for this evolution: six amino acids were added to the active site of the enzyme, and one amino acid (at position 102) was replaced by a different amino acid. However, introducing the same mutations into a modern version of the bacterial enzyme did not produce a working form of the LDH enzyme. This suggests that other amino acids, further away from the active site, also influenced how LDH evolved.The structures found by Boucher et al. reveal that the enzymes evolved as a result of a gene duplicating, followed by one of the copies evolving a new function. However, some of the mutations responsible for the novel function occurred far from the active site, and it is still unknown how they exert their functional effects. Untangling the important mutations from the mundane will be necessary to fully understand how protein functions are created and how to control them—both of which will aid in developing effective drugs that target essential parasite proteins.