Germacrene A synthase in yarrow (Achillea millefolium) is an enzyme with mixed substrate specificity: gene cloning, functional characterization and expression analysis

Abstract

Terpenoid synthases constitute a highly diverse gene family producing a wide range of cyclic and acyclic molecules consisting of isoprene (C5) residues. Often a single terpene synthase produces a spectrum of molecules of given chain length, but some terpene synthases can use multiple substrates, producing products of different chain length. Only a few such enzymes has been characterized, but the capacity for multiple-substrate use can be more widespread than previously thought. Here we focused on germacrene A synthase (GAS) that is a key cytosolic enzyme in the sesquiterpene lactone biosynthesis pathway in the important medicinal plant Achillea millefolium (AmGAS). The full length encoding gene was heterologously expressed in Escherichia coli BL21 (DE3), functionally characterized, and its in vivo expression was analyzed. The recombinant protein catalyzed formation of germacrene A with the C15 substrate farnesyl diphosphate (FDP), while acyclic monoterpenes were formed with the C10 substrate geranyl diphosphate (GDP) and cyclic monoterpenes with the C10 substrate neryl diphosphate (NDP). Although monoterpene synthesis has been assumed to be confined exclusively to plastids, AmGAS can potentially synthesize monoterpenes in cytosol when GDP or NDP become available. AmGAS enzyme had high homology with GAS sequences from other Asteraceae species, suggesting that multi-substrate use can be more widespread among germacrene A synthases than previously thought. Expression studies indicated that AmGAS was expressed in both autotrophic and heterotrophic plant compartments with the highest expression levels in leaves and flowers. To our knowledge, this is the first report on the cloning and characterization of germacrene A synthase coding gene in A. millefolium, and multi-substrate use of GAS enzymes.

Chemotypic variation in terpenes emitted from storage pools influences early aphid colonisation on tansy

“…So far approximately 25,000 structures have been reported. Terpenes are initially produced by terpene synthases, which are often able to produce multiple compounds from single substrates1011. Subsequently these terpene backbones are modified in multiple ways, generating the large number of chemical structures112.…”

Substrate specificity of α‐humulene synthase from Zingiber zerumbet Smith and determination of kinetic constants by a spectrophotometric assay

“…Compounds were identified using mass spectrometry and comparisons with the NIST Mass Spectral Database and determination of Kovats indices. Structures of (2E)-GPP (1), (2Z)-NPP (2), β-myrcene (3), limonene (4), linalool (5), α-terpineol (6), geraniol (7), α-terpinolene (8), p-menth-2-en-1-ol (9), piperitol (10), nerol (11).…”

“…Out of eleven products seven could be identified. Enzymatic conversion of (2E)-GPP mainly yielded acyclic products, geraniol (7) being the main one. The biotransformation with (2Z)-NPP predominantly furnished cyclic products with limonene (4) being the main product.…”

“…Terpene synthases, the key enzymes responsible for the conversion of linear prenyl pyrophosphates to diverse terpene skeletons, have a variable product specificity that primarily depends on how well the reactive substrate carbocation can be stabilized in the enzyme´s active site. Although terpene synthesis in plant metabolism is highly regulated and compartmentalized, there is evidence that several terpene synthases are capable of forming terpenes with different chain lengths depending on the corresponding substrate available [5][6][7]. Some catalyze the synthesis of only a limited number of products and others yield a variety of products.…”

“…Some catalyze the synthesis of only a limited number of products and others yield a variety of products. Although terpene synthesis in plant metabolism is highly regulated and compartmentalized, there is evidence that several terpene synthases are capable of forming terpenes with different chain lengths depending on the corresponding substrate available [5][6][7]. Previous studies showed that sesquiterpene synthases possess the ability to convert geranyl and neryl pyrophosphates to monoterpene products [8].…”