A Single Residue Change Leads to a Hydroxylated Product from the Class II Diterpene Cyclization Catalyzed by Abietadiene Synthase

Criswell, Jared; Potter, Kevin C.; Shephard, Freya; Beale, Michael H.; Peters, Reuben J.

doi:10.1021/ol3026022

Cited by 52 publications

(55 citation statements)

References 24 publications

(45 reference statements)

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“…Here, we have shown that a single-point mutation that causes an amino acid substitution close to the active site dramatically alters product specificity in both SAD1 and LUP1, so uncovering hidden functional diversity in these triterpene synthases. It is conceivable that nature explores alternate modes of cyclization through such single mutational steps, as has been suggested for diterpene synthases (36)(37)(38)(39)(40)(41)(42)(43). A dedicated cyclase that makes epDM as its major product has not been reported before our work to our knowledge.…”

Section: Discussionmentioning

confidence: 86%

A conserved amino acid residue critical for product and substrate specificity in plant triterpene synthases

Salmon

Thimmappa

Minto

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Triterpenes are structurally complex plant natural products with numerous medicinal applications. They are synthesized through an origami-like process that involves cyclization of the linear 30 carbon precursor 2,3-oxidosqualene into different triterpene scaffolds. Here, through a forward genetic screen in planta, we identify a conserved amino acid residue that determines product specificity in triterpene synthases from diverse plant species. Mutation of this residue results in a major change in triterpene cyclization, with production of tetracyclic rather than pentacyclic products. The mutated enzymes also use the more highly oxygenated substrate dioxidosqualene in preference to 2,3-oxidosqualene when expressed in yeast. Our discoveries provide new insights into triterpene cyclization, revealing hidden functional diversity within triterpene synthases. They further open up opportunities to engineer novel oxygenated triterpene scaffolds by manipulating the precursor supply.terpenes | natural products | plant defense | cyclization | mutants

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Section: Discussionmentioning

confidence: 86%

A conserved amino acid residue critical for product and substrate specificity in plant triterpene synthases

Salmon

Thimmappa

Minto

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…For example, with diTPSs, change of function as a result of small changes in the active site composition has been illustrated (e.g. Wilderman and Peters, 2007;Xu et al, 2007b;Keeling et al, 2008;Morrone et al, 2008;Leonard et al, 2010;Zhou and Peters, 2011;Criswell et al, 2012;Gao et al, 2012;Zerbe et al, 2012b). However, emerging patterns of functional evolution, such as changes in domain architecture and presence or absence of active side signature motifs, can now guide functional annotation and characterization of new diTPSs.…”

Section: General Applications and Conclusionmentioning

confidence: 99%

Gene Discovery of Modular Diterpene Metabolism in Nonmodel Systems

et al. 2013

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(Bj.H., Br.H., S.S.B.)Plants produce over 10,000 different diterpenes of specialized (secondary) metabolism, and fewer diterpenes of general (primary) metabolism. Specialized diterpenes may have functions in ecological interactions of plants with other organisms and also benefit humanity as pharmaceuticals, fragrances, resins, and other industrial bioproducts. Examples of high-value diterpenes are taxol and forskolin pharmaceuticals or ambroxide fragrances. Yields and purity of diterpenes obtained from natural sources or by chemical synthesis are often insufficient for large-volume or high-end applications. Improvement of agricultural or biotechnological diterpene production requires knowledge of biosynthetic genes and enzymes. However, specialized diterpene pathways are extremely diverse across the plant kingdom, and most specialized diterpenes are taxonomically restricted to a few plant species, genera, or families. Consequently, there is no single reference system to guide gene discovery and rapid annotation of specialized diterpene pathways. Functional diversification of genes and plasticity of enzyme functions of these pathways further complicate correct annotation. To address this challenge, we used a set of 10 different plant species to develop a general strategy for diterpene gene discovery in nonmodel systems. The approach combines metabolite-guided transcriptome resources, custom diterpene synthase (diTPS) and cytochrome P450 reference gene databases, phylogenies, and, as shown for select diTPSs, single and coupled enzyme assays using microbial and plant expression systems. In the 10 species, we identified 46 new diTPS candidates and over 400 putatively terpenoid-related P450s in a resource of nearly 1 million predicted transcripts of diterpene-accumulating tissues. Phylogenetic patterns of lineage-specific blooms of genes guided functional characterization.

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“…Given the ease with which diTPSs can be diverted to alternative activity (Wilderman and Peters, 2007; Xu al., 2007;Keeling et al, 2008;Morrone et al, 2008;Criswell et al, 2012;Potter et al, 2014), it is not clear what underlies the expanded nature and divergent activity observed in the S. miltiorrhiza diTPS family (i.e. selective pressure or genetic drift).…”

Section: Positive Selection For Divergent Cps Activitymentioning

confidence: 99%

Functional divergence of diterpene syntheses in the medicinal plant Salvia miltiorrhiza Bunge

et al. 2015

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A Single Residue Change Leads to a Hydroxylated Product from the Class II Diterpene Cyclization Catalyzed by Abietadiene Synthase

Cited by 52 publications

References 24 publications

A conserved amino acid residue critical for product and substrate specificity in plant triterpene synthases

A conserved amino acid residue critical for product and substrate specificity in plant triterpene synthases

Gene Discovery of Modular Diterpene Metabolism in Nonmodel Systems

Functional divergence of diterpene syntheses in the medicinal plant Salvia miltiorrhiza Bunge

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