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Identification of amino acid residues involved in substrate specificity of plant acyl-ACP thioesterases using a bioinformatics-guided approach
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 i...
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Formato: | Texto |
Lenguaje: | English |
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BioMed Central
2007
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1770913/ https://www.ncbi.nlm.nih.gov/pubmed/17201914 http://dx.doi.org/10.1186/1471-2229-7-1 |
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author | Mayer, Kimberly M Shanklin, John |
author_facet | Mayer, Kimberly M Shanklin, John |
author_sort | Mayer, Kimberly M |
collection | PubMed |
description | 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. |
format | Text |
id | pubmed-1770913 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2007 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-17709132007-01-17 Identification of amino acid residues involved in substrate specificity of plant acyl-ACP thioesterases using a bioinformatics-guided approach Mayer, Kimberly M Shanklin, John BMC Plant Biol Research Article 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. BioMed Central 2007-01-03 /pmc/articles/PMC1770913/ /pubmed/17201914 http://dx.doi.org/10.1186/1471-2229-7-1 Text en Copyright © 2007 Mayer and Shanklin; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( (http://creativecommons.org/licenses/by/2.0) ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Research Article Mayer, Kimberly M Shanklin, John Identification of amino acid residues involved in substrate specificity of plant acyl-ACP thioesterases using a bioinformatics-guided approach |
title | Identification of amino acid residues involved in substrate specificity of plant acyl-ACP thioesterases using a bioinformatics-guided approach |
title_full | Identification of amino acid residues involved in substrate specificity of plant acyl-ACP thioesterases using a bioinformatics-guided approach |
title_fullStr | Identification of amino acid residues involved in substrate specificity of plant acyl-ACP thioesterases using a bioinformatics-guided approach |
title_full_unstemmed | Identification of amino acid residues involved in substrate specificity of plant acyl-ACP thioesterases using a bioinformatics-guided approach |
title_short | Identification of amino acid residues involved in substrate specificity of plant acyl-ACP thioesterases using a bioinformatics-guided approach |
title_sort | identification of amino acid residues involved in substrate specificity of plant acyl-acp thioesterases using a bioinformatics-guided approach |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1770913/ https://www.ncbi.nlm.nih.gov/pubmed/17201914 http://dx.doi.org/10.1186/1471-2229-7-1 |
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