Dataset Information

Moonlighting Metals: Insights into Regulation of Cyclization Pathways in Fungal ?(6) -Protoilludene Sesquiterpene Synthases.

ABSTRACT: Fungal 1,11 cyclizing sesquiterpene synthases are product specific under typical reaction conditions. However, in vivo expression of certain ?(6)-protoilludene synthases results in dual 1,11 and 1,10 cyclization. To determine the factors regulating this mechanistic variation, in-depth in vitro characterization of ?(6)-protoilludene synthases was conducted. Divalent metal ions determine cyclization specificity and this product variability. Promiscuity in metal binding is mediated by secondary metal-binding sites away from the conserved D(D/E)XX(D/E) motif in sesquiterpene synthases. Phylogenetic analysis revealed a divergent evolution of Basidiomycota trans-humulyl cation producing sesquiterpene synthases, results that indicate a wider diversity in function than previously predicted. This study provides key insights into the function and evolution of 1,11 cyclizing fungal sesquiterpene synthases.

SUBMITTER: Quin MB

PROVIDER: S-EPMC4742524 | biostudies-literature | 2015 Oct

REPOSITORIES: biostudies-literature

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Moonlighting Metals: Insights into Regulation of Cyclization Pathways in Fungal Δ(6) -Protoilludene Sesquiterpene Synthases.

Quin Maureen B MB Michel Stephen N SN Schmidt-Dannert Claudia C

Chembiochem : a European journal of chemical biology 20150901 15

Fungal 1,11 cyclizing sesquiterpene synthases are product specific under typical reaction conditions. However, in vivo expression of certain Δ(6)-protoilludene synthases results in dual 1,11 and 1,10 cyclization. To determine the factors regulating this mechanistic variation, in-depth in vitro characterization of Δ(6)-protoilludene synthases was conducted. Divalent metal ions determine cyclization specificity and this product variability. Promiscuity in metal binding is mediated by secondary met ...[more]

PMID: 26239156

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Project description:BackgroundSesquiterpene lactones are characteristic metabolites of Asteraceae (or Compositae) which often display potent bioactivities and are sequestered in specialized organs such as laticifers, resin ducts, and trichomes. For characterization of sunflower sesquiterpene synthases we employed a simple method to isolate pure trichomes from anther appendages which facilitated the identification of these genes and investigation of their enzymatic functions and expression patterns during trichome development.ResultsGlandular trichomes of sunflower (Helianthus annuus L.) were isolated, and their RNA was extracted to investigate the initial steps of sesquiterpene lactone biosynthesis. Reverse transcription-PCR experiments led to the identification of three sesquiterpene synthases. By combination of in vitro and in vivo characterization of sesquiterpene synthase gene products in Escherichia coli and Saccharomyces cerevisiae, respectively, two enzymes were identified as germacrene A synthases, the key enzymes of sesquiterpene lactone biosynthesis. Due to the very low in vitro activity, the third enzyme was expressed in vivo in yeast as a thioredoxin-fusion protein for functional characterization. In in vivo assays, it was identified as a multiproduct enzyme with the volatile sesquiterpene hydrocarbon delta-cadinene as one of the two main products with alpha-muuorlene, beta-caryophyllene, alpha-humulene and alpha-copaene as minor products. The second main compound remained unidentified. For expression studies, glandular trichomes from the anther appendages of sunflower florets were isolated in particular developmental stages from the pre- to the post-secretory phase. All three sesquiterpene synthases were solely upregulated during the biosynthetically active stages of the trichomes. Expression in different aerial plant parts coincided with occurrence and maturity of trichomes. Young roots with root hairs showed expression of the sesquiterpene synthase genes as well.ConclusionThis study functionally identified sesquiterpene synthase genes predominantly expressed in sunflower trichomes. Evidence for the transcriptional regulation of sesquiterpene synthase genes in trichome cells suggest a potential use for these specialized cells for the identification of further genes involved in the biosynthesis, transport, and regulation of sesquiterpene lactones.

Project description:BackgroundTerpene synthases are versatile catalysts in all domains of life, catalyzing the formation of an enormous variety of different terpenoid secondary metabolites. Due to their diverse bioactive properties, terpenoids are of great interest as innovative ingredients in pharmaceutical and cosmetic applications. Recent advances in genome sequencing have led to the discovery of numerous terpene synthases, in particular in Basidiomycota like the wood rotting fungus Coniophora puteana, which further enhances the scope for the manufacture of terpenes for industrial purposes.ResultsIn this study we describe the identification of two novel (+)-δ-cadinol synthases from C. puteana, Copu5 and Copu9. The sesquiterpene (+)-δ-cadinol was previously shown to exhibit cytotoxic activity therefore having an application as possible, new, and sustainably sourced anti-tumor agent. In an Escherichia coli strain, optimized for sesquiterpene production, titers of 225 mg l-1 and 395 mg l-1, respectively, could be achieved. Remarkably, both enzymes share the same product profile thereby representing the first two terpene synthases from Basidiomycota with identical product profiles. We solved the crystal structure of Copu9 in its closed conformation, for the first time providing molecular details of sesquiterpene synthase from Basidiomycota. Based on the Copu9 structure, we conducted structure-based mutagenesis of amino acid residues lining the active site, thereby altering the product profile. Interestingly, the mutagenesis study also revealed that despite the conserved product profiles of Copu5 and Copu9 different conformational changes may accompany the catalytic cycle of the two enzymes. This observation suggests that the involvement of tertiary structure elements in the reaction mechanism(s) employed by terpene synthases may be more complex than commonly expected.ConclusionThe presented product selectivity and titers of Copu5 and Copu9 may pave the way towards a sustainable, biotechnological production of the potentially new bioactive (+)-δ-cadinol. Furthermore, Copu5 and Copu9 may serve as model systems for further mechanistic studies of terpenoid catalysis.

Dataset Information

Moonlighting Metals: Insights into Regulation of Cyclization Pathways in Fungal ?(6) -Protoilludene Sesquiterpene Synthases.

Publications

Moonlighting Metals: Insights into Regulation of Cyclization Pathways in Fungal Δ(6) -Protoilludene Sesquiterpene Synthases.

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