Identification of amino acid residues involved in substrate specificity of plant acyl-ACP thioesterases using a bioinformatics-guided approach
<p>Abstract</p> <p>Background</p> <p>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 P...
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| Format: | Article |
| Language: | English |
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BMC
2007-01-01
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| Series: | BMC Plant Biology |
| Online Access: | http://www.biomedcentral.com/1471-2229/7/1 |
| _version_ | 1811286652462563328 |
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| author | Mayer Kimberly M Shanklin John |
| author_facet | Mayer Kimberly M Shanklin John |
| author_sort | Mayer Kimberly M |
| collection | DOAJ |
| description | <p>Abstract</p> <p>Background</p> <p>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 <it>E. coli </it>strain K27 that allows determination of enzyme activity by GCMS analysis of fatty acids released into the medium.</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>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.</p> |
| first_indexed | 2024-04-13T03:04:51Z |
| format | Article |
| id | doaj.art-fe15fb6d81ed41b0bd97777be51a4ff5 |
| institution | Directory Open Access Journal |
| issn | 1471-2229 |
| language | English |
| last_indexed | 2024-04-13T03:04:51Z |
| publishDate | 2007-01-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Plant Biology |
| spelling | doaj.art-fe15fb6d81ed41b0bd97777be51a4ff52022-12-22T03:05:19ZengBMCBMC Plant Biology1471-22292007-01-0171110.1186/1471-2229-7-1Identification of amino acid residues involved in substrate specificity of plant acyl-ACP thioesterases using a bioinformatics-guided approachMayer Kimberly MShanklin John<p>Abstract</p> <p>Background</p> <p>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 <it>E. coli </it>strain K27 that allows determination of enzyme activity by GCMS analysis of fatty acids released into the medium.</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>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.</p>http://www.biomedcentral.com/1471-2229/7/1 |
| spellingShingle | Mayer Kimberly M Shanklin John Identification of amino acid residues involved in substrate specificity of plant acyl-ACP thioesterases using a bioinformatics-guided approach BMC Plant Biology |
| 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 |
| url | http://www.biomedcentral.com/1471-2229/7/1 |
| work_keys_str_mv | AT mayerkimberlym identificationofaminoacidresiduesinvolvedinsubstratespecificityofplantacylacpthioesterasesusingabioinformaticsguidedapproach AT shanklinjohn identificationofaminoacidresiduesinvolvedinsubstratespecificityofplantacylacpthioesterasesusingabioinformaticsguidedapproach |