A structural model of the plant acyl-acyl carrier protein thioesterase FatB comprises two helix/4-stranded sheet domains, the N-terminal domain containing residues that affect specificity and the C-terminal domain containing catalytic residues.

Plant acyl-acyl carrier protein thioesterases (TEs) terminate the acyl-acyl carrier protein track of fatty acid biosynthesis and play an essential role in determining the amount and composition of fatty acids entering the storage lipid pool. A combination of bioinformatics tools was used to predict a three-dimensional model for Arabidopsis FatB (AtFatB), which comprises a fold similar to that of Escherichia coli TEII, an enzyme that is functionally similar to plant TEs but lacks significant sequence similarity and displays different inhibitor sensitivity. The catalytic residues in AtFatB, Cys-264 and His-229, localize to the same region of the model as catalytic residues found in other enzymes with helix/multi-stranded sheet motifs (hot dog folds). Based on the model, we identified Asn-227 as a possible third member of the proposed papain-like catalytic triad. The conversion of Asn-227 to Ala resulted in a loss of detectable activity (>200-fold reduction), similar to the result seen for the equivalent mutation in papain. Mapping of plant TE specificity-affecting mutations onto the structural model showed that these mutations all cluster around the catalytic triad. Also, superposition of the crystallographically determined structures of the complexes of 4-hydroxybenzoyl-CoA TE with substrate and beta-hydroxydecanoyl thiol ester dehydrase with inhibitor onto the AtFatB model showed that the substrate and inhibitor localize to the same region as the AtFatB catalytic triad in their respective structures. Together these data corroborate the structural model and show that the hot dog fold is common to enzymes from both prokaryotes and eukaryotes and that this fold supports at least three different catalytic mechanisms.