Functional analysis of (4S)-limonene synthase mutants reveals determinants of catalytic outcome in a model monoterpene synthase
Crystal structural data for (4S)-limonene synthase [(4S)-LS] of spearmint (Mentha spicata L.) were used to infer which amino acid residues are in close proximity to the substrate and carbocation intermediates of the enzymatic reaction. Alanine-scanning mutagenesis of 48 amino acids combined with enz...
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| Τόπος έκδοσης: | Proc Natl Acad Sci U S A |
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National Academy of Sciences
2015
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| Διαθέσιμο Online: | https://ncbi.nlm.nih.gov/pmc/articles/PMC4371936/ https://ncbi.nlm.nih.gov/pubmed/25733883 https://ncbi.nlm.nih.govhttp://dx.doi.org/10.1073/pnas.1501203112 |
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pubmed-43719362015-09-17 Functional analysis of (4S)-limonene synthase mutants reveals determinants of catalytic outcome in a model monoterpene synthase Srividya, Narayanan Davis, Edward M. Croteau, Rodney B. Lange, B. Markus Proc Natl Acad Sci U S A Biological Sciences Crystal structural data for (4S)-limonene synthase [(4S)-LS] of spearmint (Mentha spicata L.) were used to infer which amino acid residues are in close proximity to the substrate and carbocation intermediates of the enzymatic reaction. Alanine-scanning mutagenesis of 48 amino acids combined with enzyme fidelity analysis [percentage of (−)-limonene produced] indicated which residues are most likely to constitute the active site. Mutation of residues W324 and H579 caused a significant drop in enzyme activity and formation of products (myrcene, linalool, and terpineol) characteristic of a premature termination of the reaction. A double mutant (W324A/H579A) had no detectable enzyme activity, indicating that either substrate binding or the terminating reaction was impaired. Exchanges to other aromatic residues (W324H, W324F, W324Y, H579F, H579Y, and H579W) resulted in enzyme catalysts with significantly reduced activity. Sequence comparisons across the angiosperm lineage provided evidence that W324 is a conserved residue, whereas the position equivalent to H579 is occupied by aromatic residues (H, F, or Y). These results are consistent with a critical role of W324 and H579 in the stabilization of carbocation intermediates. The potential of these residues to serve as the catalytic base facilitating the terminal deprotonation reaction is discussed. National Academy of Sciences 2015-03-17 2015-03-02 /pmc/articles/PMC4371936/ /pubmed/25733883 http://dx.doi.org/10.1073/pnas.1501203112 Text en |
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Biological Sciences Srividya, Narayanan Davis, Edward M. Croteau, Rodney B. Lange, B. Markus Functional analysis of (4S)-limonene synthase mutants reveals determinants of catalytic outcome in a model monoterpene synthase |
| description |
Crystal structural data for (4S)-limonene synthase [(4S)-LS] of spearmint (Mentha spicata L.) were used to infer which amino acid residues are in close proximity to the substrate and carbocation intermediates of the enzymatic reaction. Alanine-scanning mutagenesis of 48 amino acids combined with enzyme fidelity analysis [percentage of (−)-limonene produced] indicated which residues are most likely to constitute the active site. Mutation of residues W324 and H579 caused a significant drop in enzyme activity and formation of products (myrcene, linalool, and terpineol) characteristic of a premature termination of the reaction. A double mutant (W324A/H579A) had no detectable enzyme activity, indicating that either substrate binding or the terminating reaction was impaired. Exchanges to other aromatic residues (W324H, W324F, W324Y, H579F, H579Y, and H579W) resulted in enzyme catalysts with significantly reduced activity. Sequence comparisons across the angiosperm lineage provided evidence that W324 is a conserved residue, whereas the position equivalent to H579 is occupied by aromatic residues (H, F, or Y). These results are consistent with a critical role of W324 and H579 in the stabilization of carbocation intermediates. The potential of these residues to serve as the catalytic base facilitating the terminal deprotonation reaction is discussed. |
| author |
Srividya, Narayanan Davis, Edward M. Croteau, Rodney B. Lange, B. Markus |
| author_facet |
Srividya, Narayanan Davis, Edward M. Croteau, Rodney B. Lange, B. Markus |
| author_sort |
Srividya, Narayanan |
| title |
Functional analysis of (4S)-limonene synthase mutants reveals determinants of catalytic outcome in a model monoterpene synthase |
| title_short |
Functional analysis of (4S)-limonene synthase mutants reveals determinants of catalytic outcome in a model monoterpene synthase |
| title_full |
Functional analysis of (4S)-limonene synthase mutants reveals determinants of catalytic outcome in a model monoterpene synthase |
| title_fullStr |
Functional analysis of (4S)-limonene synthase mutants reveals determinants of catalytic outcome in a model monoterpene synthase |
| title_full_unstemmed |
Functional analysis of (4S)-limonene synthase mutants reveals determinants of catalytic outcome in a model monoterpene synthase |
| title_sort |
functional analysis of (4s)-limonene synthase mutants reveals determinants of catalytic outcome in a model monoterpene synthase |
| publisher |
National Academy of Sciences |
| container_title |
Proc Natl Acad Sci U S A |
| publishDate |
2015 |
| url |
https://ncbi.nlm.nih.gov/pmc/articles/PMC4371936/ https://ncbi.nlm.nih.gov/pubmed/25733883 https://ncbi.nlm.nih.govhttp://dx.doi.org/10.1073/pnas.1501203112 |
| _version_ |
1803640793038061568 |