Defining the Potassium Binding Region in an Apple Terpene Synthase

Abstract

Terpene synthases are a family of enzymes largely responsible for synthesizing the vast array of terpenoid compounds known to exist in nature. Formation of terpenoids from their respective 10-, 15-, or 20-carbon atom prenyl diphosphate precursors is initiated by divalent (M 2؉ ) metal ion-assisted electrophilic attack. In addition to M 2؉ , monovalent cations (M ؉ ) have also been shown to be essential for the activity of certain terpene synthases most likely by facilitating substrate binding or catalysis. An apple ␣-farnesene synthase (MdAFS1), which has a dependence upon potassium (K ؉ ), was used to identify active site regions that may be important for M ؉ binding. Protein homology modeling revealed a surface-exposed loop (H-␣l loop) in MdAFS1 that fulfilled the necessary requirements for a K ؉ binding region. Site-directed mutagenesis analysis of specific residues within this loop then revealed their crucial importance to this K ؉ response and strongly implicated specific residues in direct K ؉ binding. The role of the H-␣l loop in terpene synthase K ؉ coordination was confirmed in a Conifer pinene synthase also using site-directed mutagenesis. These findings provide the first direct evidence for a specific M ؉ binding region in two functionally and phylogenetically divergent terpene synthases. They also provide a basis for understanding K ؉ activation in other terpene synthases and establish a new role for the H-␣l loop region in terpene synthase catalysis.Terpenoids comprise the largest and most diverse class of natural products known (1). Terpenoid structures range from simple acyclic molecules to complex heterocyclic ring systems. These compounds are synthesized by a large family of enzymes called terpene synthases (TPS). 2 In plants, monoterpenes (C10-based) are synthesized from the plastidderived geranyl diphosphate (GDP), sesquiterpenes (C15-based) from the cytoplasmically derived farnesyl diphosphate (FDP) and diterpenes (C20-based) from geranylgeranyl diphosphate, which is also plastid-derived. Although these compounds provide biosynthetic building blocks to various primary metabolites including hormones and photosynthetic pigments, the majority function as secondary metabolites. These improve the ecological fitness of the organism by enabling communication to pollinators and seed dispersal agents and the provision for defense against pathogens and herbivores (2-5). Terpenes can also affect quality traits, including susceptibility to storage disorders, in crops such as apples (6) and pears (7).Divergent TPS share various structural features. These include a highly conserved C-terminal domain, which contains their catalytic site (8) and an aspartate-rich DDXXD motif (9) essential for the divalent metal ion (typically Mg 2ϩ or Mn 2ϩ ) assisted substrate binding in these enzymes (10 -14). This conservation is also shared in their reaction chemistry, which is characterized by electrophilically initiated carbocation formation, carbocation stabilization, and rearrangement, incorporating exquisite regio-a...

Experimental design-aided systematic pathway optimization of glucose uptake and deoxyxylulose phosphate pathway for improved amorphadiene production

“…The enzymes ispD and ispF were reported to be more soluble than the enzymes dxs and idi and may have higher activities (Zhou et al 2012); hence, the overexpression of ispDF may be unnecessary when the pathway was optimized, and further study is required to address this issue. The fourth module was required to be expressed at higher levels as the activity of heterologous ADS from Artemisia annua is known to be poor in E. coli Green et al 2009). Consistent with the prediction of the a The strains were named based on their sequence of promoters; number B0,^B1,^B2,^and B3^stood for T7, TM1, TM2, and TM3 promoters, respectively (e.g., P1000 referred to a construct in which galP-glk, dxs, idi, and ispA-ADS were controlled by TM1, T7, T7, and T7 promoters, respectively) regression model, the combination of a relatively lower expression of galP-glk and higher expression of dxs and ispA-ADS enhanced AD production.…”

Selectivity of Fungal Sesquiterpene Synthases: Role of the Active Site's H-1α Loop in Catalysis

“…5). Green et al (15) recently reported that this residue is also conserved as Asn, Lys, or Ser in the H-1␣-loops of a large subgroup of plant terpene synthases. A conserved Ser was found to participate in K ϩ binding in ␣-farnesene synthase from apple and pinene synthase from spruce; replacement of Ser with Lys abolished the dependence of these enzymes on K ϩ for activity (15).…”

“…Despite the identification of several loops and helices involved in this conformational rearrangement, only one study has specifically targeted residues of the H-␣1 loop that caps the active site upon ligand binding (15). Here we used structural modeling and sitedirected mutagenesis to investigate the role of the H-␣1 loop in the cyclization mechanism of three fungal sesquiterpene synthases (Cop3, Cop4, and Cop6) that have different product profiles and fidelities.…”

“…However, despite the structural importance of the H-␣1 loop in this group of enzymes, very little is known about its influence on the cyclization mechanism of terpene synthases. In one recent study, homology modeling and site-directed mutagenesis of ␣-farnesene synthase from apple (Malus x domestica) identified residues in the H-␣1 loop as important for monovalent metal binding (15).…”

“…Sesquiterpene synthases catalyze the cyclization of farnesyl pyrophosphate (FPP) to structurally diverse C 15 -hydrocarbons. These enzymes belong to the large group of terpene synthases that convert isoprene pyrophosphate substrates into hundreds of described terpenoid compounds by employing some of the most complex carbon-carbon forming reactions known (8).…”