The Journal of Antibiotics (2016) 69, 524–533 & 2016 Japan Antibiotics Research Association All rights reserved 0021-8820/16 www.nature.com/ja ORIGINAL ARTICLE Biosynthetic potential of sesquiterpene synthases: product profiles of Egyptian Henbane premnaspirodiene synthase and related mutants Hyun Jo Koo1,3, Christopher R Vickery1,2,3,YiXu1, Gordon V Louie1, Paul E O'Maille1, Marianne Bowman1, Charisse M Nartey1, Michael D Burkart2 and Joseph P Noel1 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. The Journal of Antibiotics (2016) 69, 524–533; doi:10.1038/ja.2016.68; published online 22 June 2016 INTRODUCTION cationic intermediates en route to the major product 5-epi-aristolo- Terpenoids are one of the largest groups of naturally produced chene (1).13 Egyptian henbane (Hyoscyamus muticus) premnaspiro- chemicals, widespread across all three domains of life.1,2 Plants in diene synthase (HPS) (also known as vetispirodiene synthase),14 aTPS particular encode an incredible diversity of terpenoid biosynthetic sharing 75.5% identity with TEAS, produces premnaspirodiene (2) pathways that produce molecules conferring flavor, fragrance, (also known as vetispiradiene) as a major product. In addition to some antimicrobial, attractive and repellent properties to plants.3,4 Terpe- overlap in product profiles, the source plants of HPS and TEAS share a noids from plants also possess a wide variety of antibiotic,5 common evolutionary lineage in the Solanaceae family of plants.15 antiparasitic6 and antifungal7 activities, as well as chemotherapeutic Therefore, HPS and TEAS serve as reliable extant models for catalytic properties for the treatment of various human maladies.8 Class I diversification from a shared ancestor. However, the minor product terpene synthases (TPSs) are classified as mono-, sesqui- and profile of HPS has not been previously characterized. In order to di-terpene synthases according to their catalytically preferred mechanistically interrogate the diverging enzymatic properties linking substrates geranyl diphosphate, farnesyl diphosphate (FPP) and HPS and TEAS structurally and functionally, we characterized the full geranylgeranyl diphosphate, respectively.1 Products are formed via product profile of HPS. rearrangement of a cationic intermediate arising from cleavage of the The product profiles of TEAS and HPS are sensitive to the C-O bond between the diphosphate moiety and the carbon chain.9 identities of nine amino acid residues on and surrounding the This allylic carbocation intermediate can undergo a variety of active site surface.15,16 Mutating these nine positions in TEAS to structural alterations before arriving at a final product (Figure 1).10 the corresponding HPS residues yields TEAS-M9 that produces Generally, TPSs produce several minor products in addition to one or premnaspirodiene (2)asitsmajorproduct.Conversely,HPS-M9 more abundant products.11 produces 5-epi-aristolochene (1)asitsmajorproduct.16 We also Previously, a detailed product profile for the TPS 5-epi-aristolo- investigated the product profiles of these two interconverted chene synthase (TEAS) from common tobacco (Nicotiana tabacum) mutants. Comparison of HPS, TEAS, HPS-M9 and TEAS-M9 was described.12 The observed minor products likely reflected key product profiles reveals intriguing information regarding active 1Howard Hughes Medical Institute, The Salk Institute for Biological Studies, Jack H Skirball Center for Chemical Biology and Proteomics, La Jolla, CA,USAand2Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, USA 3Co-first authors. Correspondence: Dr HJ Koo or Professor JP Noel, Howard Hughes Medical Institute, The Salk Institute for Biological Studies, Jack H Skirball Center for Chemical Biology and Proteomics, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA. E-mail: [email protected] or [email protected] Received 19 April 2016; revised 3 May 2016; accepted 7 May 2016; published online 22 June 2016 Biosynthetic potential of sesquiterpene synthases HJ Koo et al 525 Figure 1 Chemical pathways leading to products formed by Nicotiana tabacum 5-epi-aristolochene synthase (TEAS) and Hyoscyamus muticus premnaspirodiene synthase (HPS) from farnesyl diphosphate (FPP). Class A (gray) arises from early quenching of the carbocation; class B (yellow) derives from the eudesmyl cation without an alkyl migration; classes C1, C2 and C3 are from the eudesmyl cation with three different alkyl migrations; and classD products are from the (Z,E)-farnesyl cation. Products at the bottom of the figure are separated into those arising from the two possible stereoisomers of the cationic intermediate stemming from germacrene A. Highlighted in red boxes are key intermediate germacrene A and the two major products of TEAS and HPS, 5-epi-aristolochene and premnaspirodiene, respectively. Compounds are numbered according to their appearance in the main text. site properties that confer product profile diversity in otherwise architectural details linking active site organization with product orthologous enzymes. formation and chemical diversity. Biosynthesis of minor products by TPSs increases at high temperature12,17 or extreme pH,17,18,19 accompanied by an overall MATERIALS AND METHODS decrease in total terpenoid products formed. In addition, widely Protein expression and purification varying temperatures may also modulate TPS activity in planta.20 In TEAS, TEAS-M9, HPS and HPS-M9 were cloned into the pHIS9GW vector and β some cases, previously formed minor products are produced at higher expressed in BL21 (DE3) at 18 °C for 16 h with 0.5 mM isopropyl -D-1- thiogalactopyranoside (IPTG) induction, as previously reported.12 Harvested levels than the major products with changes in enzyme reaction cells were resuspended in lysis buffer (50 mM Tris-HCl,pH8.0,500mM NaCl, conditions.17 However, it is not clear whether formation of minor 20 mM imidazole, 10% (v/v) glycerol, 1% (v/v) Tween-20 and 20 mM products is more, less or equally dependent on active site dynamics of β-mercaptoethanol), lysed by sonication and centrifuged to recover soluble the TPS or the altered chemical reactivity of substrates and inter- proteins. Soluble proteins were mixed with Ni-NTA resin (Qiagen, Hilden, mediates at elevated temperatures and pH. Temperature and pH Germany) for 1 h, then washed twice with 10 bed volumes of wash buffer screens of all four HPS and TEAS constructs were carried out to assess (50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 20 mM imidazole, 10% (v/v) glycerol β these environmental perturbations on terpenoid product formation. and 20 mM -mercaptoethanol) and eluted with 3 bed volumes of elution buffer Finally, in order to provide structural underpinnings for the initial (50 mM Tris-HCl,pH8.0,500mM NaCl, 250 mM imidazole, 10% (v/v) glycerol and 20 mM β-mercaptoethanol). Purified proteins were dialyzed in dialysis buffer steps of product formation in the TEAS–HPS family, we determined I(50mM Tris-HCl,pH8.0,500mM NaCl and 10 mM β-mercaptoethanol) with X-ray crystal structures of TEAS containing several small molecule thrombin at 4 °C for 18 h and passed over Ni-NTA and Benzamidine Sepharose fi ligands. These TPS structures, along with product pro ling of these (GE Healthcare, Little Chalfont, UK) columns to remove uncleaved His-tagged four closely related TPSs using a variety of conditions, provide protein and thrombin, respectively. His-tag removed proteins were further The Journal of Antibiotics Biosynthetic potential of sesquiterpene synthases HJ Koo et al 526 purified to homogeneity by size exclusion chromatography. Purified fractions default ‘Tm Calling’ analysis found in the LightCycler 480 Software (release 21 were combined and dialyzed in dialysis buffer II (12.5 mM Tris-HCl, pH 8.0, 1.5.0) (Roche). 50 mM NaCl and 2 mM DTT) followed by concentration using Amicon Ultra 30 000 molecular weight cutoff centrifugal filters (Millipore, Billerica, MA, USA) Enzyme assays in different pHs − before storage at 9.8–25.5 mg ml 1 at − 80 °C. TEAS and HPS were assayed at varying pH range (4.5–10) in BTP, sodium maleate, 2-(N-morpholino)ethanesulfonic acid (MES), BIS-TRIS methane Enzyme assays (BT), piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES), 3-(N-morpholino) (E,E)-Farnesyl diphosphate (FPP) was purchased from Isoprenoids.com propanesulfonic acid (MOPS), MOPSO and sodium phosphate buffers. (Tampa, FL, USA). In general, for comparing product profiles, enzyme assays Enzyme assays were carried out in a total volume of 500 μl(0.1μM enzyme, were carried out in 500 μl, including 100 μl of Protein Extraction Buffer 5mM MgCl2,10mM pH buffer and 25 μM FPP) overlaid with 500 μlhexane. (50 mM 3-Morpholino-2-hydroxypropanesulfonic acid (MOPSO), pH 7.0, 10% Next, 20 μlof625μM FPP was added to initiate enzyme reactions. Assays were (v/v) glycerol, 5 mM MgCl2,5mM DTT, 5 mM sodium ascorbate and 0.5 mM allowed to continue overnight and products were recovered as detailed above. phenylmethylsulfonyl fluoride), and using 0.1–0.5 μM enzyme and 25–50 μM For TEAS, HPS, TEAS-M9 and HPS-M9 in BTP (pH range 8–9.5), a 500 μl FPP overlaid with 500 μl hexane.