Additional file 2 of Determinants of substrate specificity in a catalytically diverse family of acyl-ACP thioesterases from plants

Additional file 2: Fig. S1. Predicted catalytic and acyl-binding cavity residues in modelled ALTs align with those of E. coli YbgC and Umbellularia californica FatB [45, 46, 48]. A. Monomer models of the hot-dog fold domains of MtALT1 and the E. coli acyl-CoA thioesterase YbgC, with numbered α-helices and β-strands. ALT monomers were modelled by AlphaFold 2.0, and the crystal structure of E. coli YbgC was retrieved from the RCSB PDB (PDB ID: 5 T06) [39, 40, 45]. B. Superimposition of Arabidopsis thaliana ALT3/4, Medicago truncatula ALT1/2, and Zea mays ALT1/3 homotetramer models assembled with HSYMDOCK (grey) with the crystal structure of the E. coli acyl-CoA thioesterase YbgC (yellow) [39–41, 45]. C. Comparison of predicted catalytic residues in modelled ALTs and the crystal structure of EcYbgC. AtALT4 is used as a representative example. Catalytic triad residues belong to two neighbouring subunits. D. Comparison of predicted acyl-binding cavity structure in modelled ALTs, and the crystal structures of EcYbgC and UcFatB. MtALT1 is used as a representative example. Ribbon structure of α1-α2 of the hot-dog fold domain is hidden from models to increase visibility of key residues. The crystal structure of UcFatB was retrieved from the RCSB PDB (PDB ID: 5X04), and the N-terminal hot-dog fold domain (residues 100–247) were isolated in ChimeraX 1.2.5 software. Top: Predicted substrate-binding cavity residues of MtALT1 and EcYbgC are shown as stick models. Middle: Molecular surfaces formed by predicted acyl binding cavity residues are coloured according to hydrophobicity (yellow = hydrophobic, white = amphipathic, blue = hydrophilic). Bottom: Predicted substrate-binding cavity residues of MtALT1, and experimentally determined substrate-binding cavity residues of UcFatB are shown as stick models [48]. E. Alignment of the MtALT1, EcYbgC, and N-terminal UcFatB hot-dog fold domain sequences [55]. Acyl-binding cavity residues belonging to each protein are highlighted in red [46]. Asterisks (*) indicate predicted acyl-binding cavity-forming residues of MtALT1 that align with those of both EcYbgC and UcFatB. Dots (●) indicate cavity-forming residues of MtALT1 that align with those of EcYbgC, but not UcFatB. Diamonds (◊) indicate cavity-forming residues of UcFatB that align structurally, but not on the sequence alignment, with those of MtALT1.

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