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Title: Functional analysis of (4 S)-limonene synthase mutants reveals determinants of catalytic outcome in a model monoterpene synthase

Abstract

We used crystal structural data for (4S)-limonene synthase [(4S)-LS] of spearmint (Mentha spicata L.) 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. Furthermore, the 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). Our results are consistent with a critical role of W324 and H579 in the stabilization of carbocation intermediates. Finally, the potential of these residues to serve as the catalytic base facilitating the terminal deprotonation reaction is discussed.

Authors:
 [1];  [1];  [1];  [1]
  1. Washington State Univ., Pullman, WA (United States). Inst. of Biological Chemistry and M.J. Murdock Metabolomics Lab.
Publication Date:
Research Org.:
Washington State Univ., Pullman, WA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22); National Institutes of Health (NIH)
OSTI Identifier:
1356187
Grant/Contract Number:
SC0001553; GM-31354
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Proceedings of the National Academy of Sciences of the United States of America
Additional Journal Information:
Journal Volume: 112; Journal Issue: 11; Journal ID: ISSN 0027-8424
Publisher:
National Academy of Sciences, Washington, DC (United States)
Country of Publication:
United States
Language:
English
Subject:
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; monoterpene synthase; enzyme catalysis; mechanism; carbocation; structure–function relationship

Citation Formats

Srividya, Narayanan, Davis, Edward M., Croteau, Rodney B., and Lange, B. Markus. Functional analysis of (4S)-limonene synthase mutants reveals determinants of catalytic outcome in a model monoterpene synthase. United States: N. p., 2015. Web. doi:10.1073/pnas.1501203112.
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. United States. doi:10.1073/pnas.1501203112.
Srividya, Narayanan, Davis, Edward M., Croteau, Rodney B., and Lange, B. Markus. Mon . "Functional analysis of (4S)-limonene synthase mutants reveals determinants of catalytic outcome in a model monoterpene synthase". United States. doi:10.1073/pnas.1501203112. https://www.osti.gov/servlets/purl/1356187.
@article{osti_1356187,
title = {Functional analysis of (4S)-limonene synthase mutants reveals determinants of catalytic outcome in a model monoterpene synthase},
author = {Srividya, Narayanan and Davis, Edward M. and Croteau, Rodney B. and Lange, B. Markus},
abstractNote = {We used crystal structural data for (4S)-limonene synthase [(4S)-LS] of spearmint (Mentha spicata L.) 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. Furthermore, the 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). Our results are consistent with a critical role of W324 and H579 in the stabilization of carbocation intermediates. Finally, the potential of these residues to serve as the catalytic base facilitating the terminal deprotonation reaction is discussed.},
doi = {10.1073/pnas.1501203112},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
number = 11,
volume = 112,
place = {United States},
year = {Mon Mar 02 00:00:00 EST 2015},
month = {Mon Mar 02 00:00:00 EST 2015}
}

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Cited by: 12 works
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