Identification of amino acid residues involved in substrate specificity of plant acyl-ACP thioesterases using a bioinformatics-guided approach

Mayer, Kimberly M.; Shanklin, John

doi:10.1186/1471-2229-7-1

Identification of amino acid residues involved in substrate specificity of plant acyl-ACP thioesterases using a bioinformatics-guided approach

Journal Article · Wed Jan 03 00:00:00 EST 2007 · BMC Plant Biology

DOI:https://doi.org/10.1186/1471-2229-7-1· OSTI ID:1626509

Mayer, Kimberly M. ^[1]; Shanklin, John ^[2]

Brookhaven National Lab. (BNL), Upton, NY (United States); Univ. of North Carolina at Wilmington, NC (United States); DOE/OSTI
Brookhaven National Lab. (BNL), Upton, NY (United States)

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. 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. 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.

View Accepted Manuscript (DOE)

Research Organization:: Brookhaven National Laboratory (BNL), Upton, NY (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER). Biological Systems Science Division

Grant/Contract Number:: SC0012704

OSTI ID:: 1626509

Journal Information:: BMC Plant Biology, Journal Name: BMC Plant Biology Journal Issue: 1 Vol. 7; ISSN 1471-2229

Publisher:: BioMed CentralCopyright Statement

Country of Publication:: United States

Language:: English

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