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 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.
Institute of Biological Chemistry and M. J. Murdock Metabolomics Laboratory, Washington State University, Pullman, WA 99163.
Narayanan
N
Srividya
Institute of Biological Chemistry and M. J. Murdock Metabolomics Laboratory, Washington State University, Pullman, WA 99163.
Edward M
EM
Davis
Institute of Biological Chemistry and M. J. Murdock Metabolomics Laboratory, Washington State University, Pullman, WA 99163 croteau@wsu.edu lange-m@wsu.edu.
Rodney B
RB
Croteau
Institute of Biological Chemistry and M. J. Murdock Metabolomics Laboratory, Washington State University, Pullman, WA 99163 croteau@wsu.edu lange-m@wsu.edu.
B Markus
BM
Lange
D000409:Alanine / Q000235:genetics; D055162:Biocatalysis; D019753:Intramolecular Lyases / Q000235:genetics*; D031337:Mentha spicata / Q000201:enzymology; D008954:Models, Biological; D008958:Models, Molecular; D016296:Mutagenesis / Q000235:genetics; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics*; D013379:Substrate Specificity; D013729:Terpenes / Q000737:chemistry / Q000378:metabolism
D050505:Mutant Proteins; D013729:Terpenes; D019753:Intramolecular Lyases; C040265:pinene cyclase I; D000409:Alanine
carbocation; enzyme catalysis; mechanism; monoterpene synthase; structure–function relationship