The Role of Critical Ion Pairs in the Evolution of a Novel Enzyme in Tomato

Although much research has been done on how new genes arising from gene duplications has been done, little is still known on how these genes arose and by what mechanisms they evolved their new function. Here I address a case study on how the evolution of a novel defensive gene in the Solanaceae family arose by examining the defensive copy of threonine deaminase (TD2) in tomato (Solanum lycopersicum). This gene evolved by gene duplication from an ancestor involved in biochemical gene synthesis to become an exceptionally stable anti-nutritive defensive gene in insect guts. This stability is hypothesized to have evolved due to three critical ion pairs, ionically bonded amino acids that stabilize proteins. Here, I test whether these three critical ion pairs stabilize the protein and what effects it has the activity of TD2. The removal of the second critical ion pair reduces the Kcat of the enzyme, indicating that it affects activity in a positive manner. Removal of the first and third critical ion pairs increase activity during the temperature and pH stability assays. The results suggest that the interactions and effects of each critical ion pair is complex within the enzyme, but are likely to be stabilizing without sacrificing much activity and in some cases increasing activity as well.