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Background: The large amount of available sequence information for the plant acyl-ACP thioesterases (TEs) made it possible to use a bioinformatics-guided approach to identify amino acid residues involved in substrate specificity. The Conserved Property Difference Locator (CPDL) program allowed the identification of putative specificity-determining residues that differ between the FatA and FatB TE classes. Six of the FatA residue differences identified by CPDL were incorporated into the FatB-like parent via site-directed mutagenesis and the effect of each on TE activity was determined. Variants were expressed in E. coli strain K27 that allows determination of enzyme activity by GCMS analysis of fatty acids released into the medium. Results: Substitutions at four of the positions (74, 86, 141, and 174) changed substrate specificity to varying degrees while changes at the remaining two positions, 110 and 221, essentially inactivated the thioesterase. The effects of substitutions at positions 74, 141, and 174 (3-MUT) or 74, 86, 141, 174 (4-MUT) were not additive with respect to specificity. Conclusion: Four of six putative specificity determining positions in plant TEs, identified with the use of CPDL, were validated experimentally; a novel colorimetric screen that discriminates between active and inactive TEs is also presented. [ABSTRACT FROM AUTHOR]

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 His229, localize to the same region of the model as catalytic residues found in other enzymes with helix/multistranded 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 β-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. [ABSTRACT FROM AUTHOR]