The santalene synthase from Cinnamomum camphora: Reconstruction of a sesquiterpene synthase from a monoterpene synthase

Plant terpene synthases (TPSs) can mediate formation of a large variety of terpenes, and their diversification contributes to the specific chemical profiles of different plant species and chemotypes. Plant genomes often encode a number of related terpene synthases, which can produce very different t...

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Published in:Archives of biochemistry and biophysics 2020-11, Vol.695, p.108647-108647, Article 108647
Main Authors: Di Girolamo, Alice, Durairaj, Janani, van Houwelingen, Adèle, Verstappen, Francel, Bosch, Dirk, Cankar, Katarina, Bouwmeester, Harro, de Ridder, Dick, van Dijk, Aalt D.J., Beekwilder, Jules
Format: Article
Language:English
Subjects:
Alkyl and Aryl Transferases - chemistry
Alkyl and Aryl Transferases - genetics
Alkyl and Aryl Transferases - metabolism
Cinnamomum camphora
Cinnamomum camphora - enzymology
Cinnamomum camphora - genetics
Escherichia coli - genetics
Escherichia coli - metabolism
Hybrid enzymes
Monoterpene synthase
Monoterpenes - chemistry
Monoterpenes - metabolism
Plant Proteins - chemistry
Plant Proteins - genetics
Plant Proteins - metabolism
Recombinant Proteins - chemistry
Recombinant Proteins - genetics
Recombinant Proteins - metabolism
Santalene synthase
Sesquiterpene synthase
Sesquiterpenes - chemistry
Sesquiterpenes - metabolism
Terpene
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Summary:Plant terpene synthases (TPSs) can mediate formation of a large variety of terpenes, and their diversification contributes to the specific chemical profiles of different plant species and chemotypes. Plant genomes often encode a number of related terpene synthases, which can produce very different terpenes. The relationship between TPS sequence and resulting terpene product is not completely understood. In this work we describe two TPSs from the Camphor tree Cinnamomum camphora (L.) Presl. One of these, CiCaMS, acts as a monoterpene synthase (monoTPS), and mediates the production of myrcene, while the other, CiCaSSy, acts as a sesquiterpene synthase (sesquiTPS), and catalyses the production of α-santalene, β-santalene and trans-α-bergamotene. Interestingly, these enzymes share 97% DNA sequence identity and differ only in 22 amino acid residues out of 553. To understand which residues are essential for the catalysis of monoterpenes resp. sesquiterpenes, a number of hybrid synthases were prepared, and supplemented by a set of single-residue variants. These were tested for their ability to produce monoterpenes and sesquiterpenes by in vivo production of sesquiterpenes in E. coli, and by in vitro enzyme assays. This analysis pinpointed three residues in the sequence which could mediate the change in product specificity from a monoterpene synthase to a sesquiterpene synthase. Another set of three residues defined the sesquiterpene product profile, including the ratios between sesquiterpene products. [Display omitted] •Two novel highly related terpene synthases for myrcene (CiCaMS) and santalene (CiCaSSy) from Cinnamomum camphora.•Hybrid enzymes of CiCaMS and CiCaSSy identify regions crucial for sesquiterpene synthase activity.•Six mutation of residues is sufficient for converting the myrcene synthase to the santalene synthase.
ISSN:0003-9861
1096-0384
DOI:10.1016/j.abb.2020.108647