Altering product outcome in Abies grandis (-)-limonene synthase and (-)-limonene/(-)-alpha-pinene synthase by domain swapping and directed mutagenesis |
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Authors: | Katoh Sadanobu Hyatt David Croteau Rodney |
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Affiliation: | Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164-6340, USA. |
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Abstract: | (-)-(4S)-limonene synthase (LS) and (-)-(4S)-limonene/(-)-(1S, 5S)-alpha-pinene synthase (LPS) from grand fir (Abies grandis) exhibit nearly 91% sequence identity (93% similarity) at the amino acid level, yet produce very different mixtures of monoterpene olefins. To elucidate critical amino acids involved in determining monoterpene product distribution, a combination of domain swapping and reciprocal site-directed mutagenesis was carried out between these two enzymes. Exchange of the predicted helix D through F region in LS gave rise to an LPS-like product outcome, whereas reciprocal substitutions of four amino acids in LPS (two in the predicted helix D and two in the predicted helix F) altered the product distribution to that intermediate between LS and LPS, and resulted in a 5-fold increase in relative velocity. These results, in conjunction with modeling of the two enzymes, suggest that amino acids in the predicted D through F helix regions are critical for product determination. |
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Keywords: | Monoterpene synthases Monoterpene cyclases Terpene cyclase structure-function Geranyl diphosphate cyclization Turpentine biosynthesis Limonene Pinene Abies grandis Grand fir |
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