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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.

Background: Families of homologous enzymes evolved from common progenitors. The availability of multiple sequences representing each activity presents an opportunity for extracting information specifying the functionality of individual homologs. We present a straightforward method for the identification of residues likely to determine class specific functionality in which multiple sequence alignments are converted to an annotated graphical form by the Conserved Property Difference Locator (CPDL) program., Results: Three test cases, each comprised of two groups of functionally-distinct homologs, are presented. Of the test cases, one is a membrane and two are soluble enzyme families. The desaturase/hydroxylase data was used to design and test the CPDL algorithm because a comparative sequence approach had been successfully applied to manipulate the specificity of these enzymes. The other two cases, ATP/GTP cyclases, and MurD/MurE synthases were chosen because they are well characterized structurally and biochemically. For the desaturase/hydroxylase enzymes, the ATP/GTP cyclases and the MurD/MurE synthases, groups of 8 (of approximately 400), 4 (of approximately 150) and 10 (of >400) residues, respectively, of interest were identified that contain empirically defined specificity determining positions., Conclusion: CPDL consistently identifies positions near enzyme active sites that include those predicted from structural and/or biochemical studies to be important for specificity and/or function. This suggests that CPDL will have broad utility for the identification of potential class determining residues based on multiple sequence analysis of groups of homologous proteins. Because the method is sequence, rather than structure, based it is equally well suited for designing structure-function experiments to investigate membrane and soluble proteins.