Fig. 1

Two amino acid motifs consistently occur in ALTs that prefer β-keto and 3-hydroxy acyl-ACP substrates. A Motifs that assort with ALT oxidation state specificity (31-CQH[G/C]RH-36, 108-KXXA-111) are highlighted on a partial alignment of the thioesterase domain sequences of ALT-type thioesterases from diverse plant species, which have been previously characterized in E. coli using endogenous acyl-ACP pools [20, 23, 24]. Residues are numbered beginning from the N-terminus of the hot-dog fold thioesterase domain; a predicted N-terminal plastid targeting sequence has been excluded [20, 24]. Protein sequences were aligned using ClustalW and sorted by oxidation state specificity in E. coli, and motifs of interest were identified using the STREME program [37, 38]. At = Arabidopsis thaliana, Bd = Brachypodium distachyon, Cr = Chlamydomonas reinhardtii, Cs = Cannabis sativa, Gm = Glycine max, Mt = Medicago truncatula, Os = Oryza sativa subsp. japonica, Sh = Solanum hirsutum subsp. glabratum, Sm = Solanum melongena, Vv = Vitis vinifera, Zm = Zea mays. B The structural positions of aa31–36 and aa108–111 are indicated on a three-dimensional model of the predicted Arabidopsis thaliana ALT4 homotetramer, generated with AlphaFold 2.0 and HSYMDOCK [39,40,41]. Examination of this model at the amino acid level shows that the side-chains of certain residues within these motifs are within potential interacting distance of one another (≤5 Å) and a predicted catalytic site residue, N24. Models were visualized in ChimeraX 1.2.5 software [42]