Fig. 3

Sequence fragments exchanged between ALT pairs to create chimeric thioesterases and their contributions to substrate specificity. Regions exchanged between ALT enzymes are annotated on pairwise alignments of the hot-dog fold domain sequences of AtALT3/4 from Arabidopsis thaliana, MtALT1/2 from Medicago truncatula, and ZmALT1/3 from Zea mays, and highlighted on a three-dimensional model of the predicted ALT homotetramer. These are annotated as follows: A = aa31–36, B = aa108–111, C = aa78–93, D = aa94–96, E = aa64–67. A brief summary of how each region influences ALT chain-length and oxidation specificity, as could be deduced from the behaviour of mutant ALT enzymes in E. coli, is also provided. Residues predicted to be of particular importance in dictating ALT substrate specificity based on experimental results or computational modelling of protein structure are marked with asterisks. Sequence pairs were aligned using the Needleman-Wünsch algorithm, and structural models of the ALT catalytic domain were generated with AlphaFold 2.0 and HSYMDOCK [39, 40, 47]