Biosynthetic potential of sesquiterpene synthases: product profiles of Egyptian Henbane premnaspirodiene synthase and related mutants

Abstract

The plant terpene synthase (TPS) family is responsible for the biosynthesis of a variety of terpenoid natural products possessing diverse biological functions. TPSs catalyze the ionization and, most commonly, rearrangement and cyclization of prenyl diphosphate substrates, forming linear and cyclic hydrocarbons. Moreover, a single TPS often produces several minor products in addition to a dominant product. We characterized the catalytic profiles of Hyoscyamus muticus premnaspirodiene synthase (HPS) and compared it with the profile of a closely related TPS, Nicotiana tabacum 5-epi-aristolochene synthase (TEAS). The profiles of two previously studied HPS and TEAS mutants, each containing nine interconverting mutations, dubbed HPS-M9 and TEAS-M9, were also characterized. All four TPSs were compared under varying temperature and pH conditions. In addition, we solved the X-ray crystal structures of TEAS and a TEAS quadruple mutant complexed with substrate and products to gain insight into the enzymatic features modulating product formation. These informative structures, along with product profiles, provide new insight into plant TPS catalytic promiscuity.

Structural and Chemical Biology of Terpenoid Cyclases

“… FPP cyclization reactions leading to the formation of major products 5-epi-aristolochene and premnaspirodiene, as well as alternative cyclization products generated by cyclase mutants classified by Noel and colleagues as follows: 309 class A products (gray) result from quenching of early carbocation intermediates; class B products (yellow) derive from the eudesmane cation in the absence of any alkyl migrations; class C products (C1, blue; C2, red; C3, green) derive from three different alkyl migrations in the eudesmane cation; and class D products (purple) derive from the ( cis,trans )-farnesyl cation. Reprinted with permission from ref ( 315 ). Copyright 2016 Macmillan Publishers Ltd. …”

Germacrene A–A Central Intermediate in Sesquiterpene Biosynthesis

“…[233] Ad etailed analysis of the product profiles of TEAS and HPS has led to the characterisation of several side products and demonstrated that TEAS produces minor amountso f95, [234] while HPS generates small quantities of 93 from FPP. [235] Domain swapping experiments between TEAS and HPS resulted in enzymev ariants making mixtures of 93 and 95 and allowed the identification of domains that conferred specificity for these two products. [236] After the crystal structure of TEAS had become available, as ystematic and rational approach targeting nine selected residues within andn ear the active site in all 2 9 = 512 combinations forafunctional interconversion between TEAS and HPS wass urveyed.…”

“…A detailed analysis of the product profiles of TEAS and HPS has led to the characterisation of several side products and demonstrated that TEAS produces minor amounts of 95 , [234] while HPS generates small quantities of 93 from FPP [235] . Domain swapping experiments between TEAS and HPS resulted in enzyme variants making mixtures of 93 and 95 and allowed the identification of domains that conferred specificity for these two products [236] .…”

De novo Transcriptome Characterization of Rhodomyrtus tomentosa Leaves and Identification of Genes Involved in α/β-Pinene and β-Caryophyllene Biosynthesis

“…Interestingly, 138 potential genes encoding these enzymes were identified by blasting the known protein database, including acetyl-CoA-acetyltransferase (AACT); hydroxymethylglutaryl-CoA synthase (HMGS); hydroxymethylglutaryl-CoA reductase (HMGR); mevalonate kinase (MK); phosphomevalonate kinase (PMK); diphosphomevalonate decarboxylase (MPD); 1-deoxy- D -xylulose-5-phosphate synthase (DXS); 1-deoxy- D -xylulose-5-phosphate reductoisomerase (DXR); 2- C -methyl- D -erythritol 4-phosphate cytidylyltransferase (MCT); 4-diphosphocytidyl-2- C -methyl- D -erythritol kinase (CMK); 2- C -methyl- D -erythritol 2,4-cyclodiphosphate synthase (MCS); ( E )-4-hydroxy-3-methylbut-2-enyl-diphosphate synthase (HDS); 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR); isopentenyl-diphosphate delta-isomerase (IPPI) ( Table 2 , Figure 1 , and Supplementary Table S3 ). Finally, IPP and isomer DMAPP synthesize GPP and FPP by geranyl diphosphate synthase (GPPS) and farnesyl diphosphate synthase (FPPS), respectively, which are then diverted to different mono- (e.g., α-pinene) and sesqui-terpenes (e.g., β-caryophyllene), respectively, after a single step enzymatic reaction mediated by TPS (Keszei et al, 2010; Koo et al, 2016; Piechulla et al, 2016) ( Figure 1 ). Therefore, identifying the function of TPS enzymes in the last step are important for understanding the molecular mechanism of terpenoid synthesis in R. tomentosa .…”

“…GPP and FPP serve as substrates for terpene synthases (TPSs) for synthesizing mono- and sesqui-terpenes, respectively (Yahyaa et al, 2015; Despinasse et al, 2017), during which the synthesis of monoterpenes is initiated by GPP dephosphorylation and ionization to geranyl carbocation, while the synthesis of sesquiterpene starts with FPP ionization to a farnesyl cation (Degenhardt et al, 2009; Huang et al, 2010). This is then followed by a series of complex chemical mechanisms involving isomerizations, cyclizations, and rearrangements catalyzed by TPSs, which finally generate structurally diverse terpenoids (Keszei et al, 2010; Koo et al, 2016; Piechulla et al, 2016) ( Figure 1 ).…”