The α-terpineol to 1,8-cineole cyclization reaction of tobacco … › ... › 2016 › 10 › 11...

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Short title: Cyclization reactions of monoterpene synthases 1

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Birgit Piechulla 3

Institute of Biological Sciences, Biochemistry, University of Rostock, Albert-Einstein-Straße 4

3, 18059 Rostock, Germany, Phone 0049 381 498 6130, Fax 0049 381 498 6132 5

e-mail [email protected] 6

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The α-terpineol to 1,8-cineole cyclization reaction of tobacco terpene synthases 8

Birgit Piechulla1, Richard Bartelt, Anne Brosemann, Uta Effmert, Harro Bouwmeester, Frank 9

Hippauf, Wolfgang Brandt 10

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Birgit Piechulla 12


3, 18059 Rostock, Germany, Phone 0049 381 498 6130, Fax 0049 381 498 6132 14


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Richard Bartelt 17

Leibniz Institute of Plant Biochemistry, Weinberg 3 18

06120 Halle (Saale), Germany 19

e-mail richard.bartelt @ipb-halle.de 20

21

Anne Brosemann 22


3, 18059 Rostock, Germany 24


Plant Physiology Preview. Published on October 11, 2016, as DOI:10.1104/pp.16.01378

Copyright 2016 by the American Society of Plant Biologists

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Frank Hippauf 27




31

Wolfgang Brandt 32

Leibniz Institute of Plant Biochemistry, Weinberg 3 33

06120 Halle (Saale), Germany 34

e-mail Wolfgang.Brandt @ipb-halle.de 35

36

Uta Effmert 37




41

Harro Bouwmeester 42

Plant Sciences, University of Wageningen, Droevendaalsesteeg 1, 6708PB Wageningen, The 43

Netherlands 44

e-mail: harro [email protected] 45

1 to whom correspondance may be addressed 46

Author contributions: B.P., W.B. designed the research; F.H., A.B., R.B., U.E., H.B. 47

performed the research; F.H., A.B., R.B., U.E., B.P., H.B. analyzed the data, R.B. and F.H 48

prepared the manuscript drafts; B.P. finalized the manuscript. Equal contribution of R.B., 49

A.B. and F.H. 50

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Funding: Financial support was given by the University of Rostock, the Institute for Plant 52

Biochemistry (Halle) and the Deutsche Forschungsgemeinschaft (Pi 153/22). 53

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One sentence summary: The conversion of α-terpineol to 1,8-cineole by monoterpene synthases 55

is initiated by a catalytic dyad, followed by a reaction via (S)-(-)-α-terpineol, and influenced by amino 56

acids more than 10 Å away from the active site. 57

58

59

keywords: α-terpineol, 1,8-cineole, monoterpene synthases, ‘cineole cassette’, cineole 60

synthase, terpineol synthase, Nicotiana 61

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63

Abstract 64

Flowers of Nicotiana species emit a characteristic blend including the ‘cineole cassette’ 65

monoterpenes. This set of terpenes is synthesized by multiproduct enzymes, with either 1,8-66

cineole or α-terpineol contributing most to the volatile spectrum, thus referring to cineole or 67

terpineol synthases (CIN, TER), respectively. To understand the molecular and structural 68

requirements of the enzymes that favor the biochemical formation of α-terpineol and 1,8-69

cineole, site-directed mutagenesis, in silico modelling and semi empiric calculations were 70

performed. Our results indicate the formation of α-terpineol by a nucleophilic attack of water. 71

During this attack the α-terpinyl cation is stabilized by π-stacking with a tryptophan side chain 72

(W253). The hypothesized catalytic mechanism of α-terpineol to 1,8-cineole conversion is 73

initiated by a catalytic dyad (H502 and E249), acting as a base, and a threonine (T278) 74

providing the subsequent re-arrangement from terpineol to cineol by catalyzing the auto-75

protonation of (S)-(-)-α-terpineol, which is the favored enantiomer product of the recombinant 76

enzymes. Furthermore, by site-directed mutagenesis we were able to identify amino acids at 77

position #147, #148 and #266 that determine the different terpineol-cineole ratios in N. 78

suaveolens CIN and N. langsdorffii TER. Since amino acid #266 is more than 10 Å away 79

from the active site, an indirect effect of this amino acid exchange on the catalysis is 80

discussed. 81



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82

Introduction 83

Monoterpenes are a large group of natural products often found in essential oils and defensive 84

oleoresins of plants (Bohlmann and Keeling, 2008). They consist of a 10-carbon skeleton and 85

can be divided into acyclic, monocyclic and bicyclic monoterpenes (Degenhardt et al., 2009; 86

Fähnrich et al., 2011). They often represent the majority in floral scent bouquets of seed 87

plants but other plant organs, e.g., leaves and roots also release them into the atmosphere or 88

rhizosphere. Monoterpenes have different biological functions, e.g., they serve as attractants 89

and guides for pollinators, as defence compounds to protect plants against herbivores, and act 90

as intra- and interspecific plant communication molecules (Gershenzon and Dudareva, 2007). 91

The biosynthesis of monoterpenes starts with the cleavage of the phosphoester bond of the 92

common substrate geranyl diphosphate (GPP). The fate of the resulting geranyl cation 93

determines the nature of the final product (Degenhardt et al., 2009) (Fig. 1). While acyclic 94

monoterpenes are direct products of the acyclic geranyl or linalyl cation, all cyclic 95

monoterpenes share one additional precursor, the α-terpinyl cation, which is formed by an 96

intramolecular electrophilic addition of the cation to the double bond at C6. Recent evidence 97

indicates that the direct isomerization of a geranyl cation to the cisoid isomer, which was so 98

far considered unlikely, is feasible (Zhang and Tiefenbacher, 2015). The α-terpinyl cation can 99

now undergo further intramolecular additions, re-arrangements and hydride-shifts leading to a 100

broad structural variety of carbocations. Quenching of these cations by either attack of a 101

nucleophile or deprotonation then leads to the final monoterpene products. The synthesis of 102

1,8-cineole is reported via α-terpineol (Degenhardt et al., 2009), but the molecular details 103

underlying the reaction mechanism to 1,8-cineole remain elusive so far. It is known that the 104

double bond of α-terpineol is protonated and this results in an intermediate, which is the 105

precursor for the cyclization to 1,8-cineole (Wise et al., 2002). However, α-terpineol cannot 106

be converted in cell free extracts (Croteau et al., 1994) and it is also hypothesized that a 107

pathway independent from α-terpineol might exist. 108

Within the genus Nicotiana several species emit a characteristic floral monoterpene volatile 109

pattern consisting of 1,8-cineole, β-myrcene, limonene, sabinene, α-/β-pinene and α-terpineol. 110

As 1,8-cineole represents the main component of the scent mixture this set of compounds was 111

named ʻcineole cassetteʼ (Raguso et al., 2006). Up to now ‘cineole cassette’ monoterpene 112

synthases were isolated from seven Nicotiana species. It was shown that for N. bonariensis, 113



5

N. forgetiana, N. longiflora, N. suaveolens and N. noctiflora the ‘cineole cassette’ 114

monoterpenes were indeed synthesized by just one enzyme, which was called cineole 115

synthase (CIN) (Fähnrich et al., 2011; 2012; 2014). The corresponding enzymes of N. alata 116

and N. langsdorffii, however, released α-terpineol as the main compound, and were 117

subsequently named terpineol synthases (TER) (Fähnrich et al., 2012). All Nicotiana ‘cineole 118

cassette’ monoterpene synthases possess sequence similarities and share conserved motifs 119

(Fig. 2 and Fig. S1). At the N-terminal end of the protein sequence is a transit peptide, which 120

is essential for the transport into the plastids (Turner et al., 1999). The first motif of the 121

mature protein sequence is RR(X)8W. It plays a role in isomerization of the substrate 122

(Williams et al., 1998). The motifs RWW and CYMNE have been identified in all CINs and 123

TERs of Nicotiana, but the functions of these motifs remain elusive. The RDR motif is 124

involved in the protection of the carbocation intermediate against nucleophilic attack (Starks 125

et al., 1997). Mutant analyses and crystallization of monoterpene synthases from Salvia 126

species have demonstrated that the NALV motif is responsible for the product outcome and 127

enzyme specificity. The aspartate-rich DDXXD motif and the NSE/DTE motif play important 128

roles in the coordination and binding of divalent metal ions to form the trinuclear magnesium 129

cluster, which supports substrate ionization, followed by the cyclization reaction to the α-130

terpinyl cation (Christianson, 2006). 131

Despite these conserved motifs and sequence similarities there are considerable differences in 132

the product composition regarding the α-terpineol to cineole ratio. Consequently, in our work 133

we wanted to unravel structural features of Nicotiana ‘cineole cassette’ monoterpene 134

synthases, which control this ratio of the main products. To reach this goal, we wanted to 135

establish and verify a putative mechanism of the enzymatic formation of α-terpineol and 136

cineole. 137

138

Results 139

Site-directed-mutagenesis based on sequence analysis of Nicotiana ‘cineole cassette’ 140

monoterpene synthases 141

Sequence comparison of all identified Nicotiana ‘cineole cassette’ monoterpene synthases 142

(Fähnrich et al. 2011, 2012, 2014) revealed sequence identities of 89-99 % corresponding to 143

one to 53 amino acid alterations (Table S1). To identify sequence features discriminating 144

between the identified five CIN and two TER, enzymes sequences were analysed in more 145



6

detail. Twenty amino acids were assumed to be involved in the formation of the active pocket, 146

R244, W253, N274, D281 – D285, I376, L417 - D426 (numbering according to sequence of 147

N. forgetiana, Fig. 2) (summarized in Fähnrich et al., 2012). These residues are conserved in 148

all sequences, only in the N. suaveolens CIN the D426 is replaced by glutamate (E). We 149

therefore concluded that amino acids outside of the active pocket must influence the terpineol 150

and cineole synthesis. Subsequently, amino acid differences of two TER and five CIN 151

enzymes were summarized and analyzed (Table S2, amino acid numbers correspond to the N. 152

forgetiana sequence, Fähnrich et al., 2014). Remarkable differences include the amino acids 153

R147 and N148, which are missing in the TERs of N. alata and N. langsdorffii and R352, 154

which is conserved in all CINs and changed into an isoleucine in the TERs. The amino acids 155

R147 and N148 were inserted into the N. langsdorffii TER, but both had only a small impact 156

on cineole synthesis (Fig. 3C). The residue R352 lies on the surface of the outermost helix of 157

the C-terminal domain, more than 10 Å away from the active pocket. Because of this location 158

it was concluded that it may not influence the terpineol/cineole ratio. In addition, some semi-159

conserved amino acids appear at positions 167, 222 and 472, where three or four of five CINs 160

share a common amino acid, while amino acids of both TERs are different, but these positions 161

were also not convincing to influence the terpineol/cineole ratio. Consequently, solely the 162

multiple sequence alignment approach did not highlight possible candidate amino acids. 163

Therefore a one to one comparison approach supported by in silico modelling was used. 164

165

Site-directed mutagenesis based on sequence comparison of N. suaveolens CIN and N. 166

langsdorffii TER 167

In order to unravel the origin of the structural differences, which discriminate between 1,8-168

cineole or α-terpineol synthesis, site-directed mutagenesis of N. suaveolens CIN was 169

performed. Sequence alignment of the CIN of N. suaveolens and TER of N. langsdorffii (Fig. 170

2, Table S1, sequence identity 89%) and 3D modelling (Fig. 4C) were used to select amino 171

acids (Table S3) for a mutational screen. We hypothesized that mutations close to the active 172

pocket or conserved motifs disturb the proposed functions of these motifs and the enzyme. 173

Additionally, we mutated amino acids, which we suspected to be involved in the enzymatic 174

catalysis. The amino acids at respective sites of the N. suaveolens CIN were altered and 175

changed into the residues found at the equivalent position in the N. langsdorffii TER sequence 176

(Fig. 2 highlighted in cyan; Table S2). The product profiles of the mutated enzymes were 177

obtained from crude extracts of E. coli (Fig. S1A-G) and the results are summarized in Table 178



7

S4. Although the distributions of the five detectable products sabinene, β-myrcene, limonene, 179

1,8-cineole and α-terpineol changed slightly in all these mutants, none of them shifted the 180

product spectrum significantly towards α-terpineol. The only exception represented the 181

mutation at position 266 (F266S). Therefore this amino acid position was investigated in more 182

detail in the mutants, F266T, F266V, F266Y, and F266C. Respective amino acid alterations 183

were selected to test i) aromatic vs non-aromatic/bulky vs. less unwieldy amino acids with 184

hydroxyl groups, ii) amino acids with potential other hydrogen bond residues, or iii) amino 185

acids without bulky and hydrophobic side chains. The purified proteins of three mutants (Fig. 186

S2) showed a decrease of the KM value of F266T (0.04 µM) and F266V (0.04 µM) compared 187

to the wild type enzyme (0.19 µM) and F266S mutant (0.12 µM) (Table 1). The kcat of F266T 188

and F266V is one order of magnitude lower compared to wild type enzyme and to F266S (2.9 189

– 4.0 x 10-4 s-1) the catalytic efficiencies (kcat/KM) of the wild type and mutant enzymes were 190

approximately the same and in the range of other plant cineole synthases (Table S12). The 191

examination of the product composition of purified enzymes resembled that of the 192

corresponding crude extracts. These mutations produced similar amounts of α-terpineol 193

compared to the wild type enzyme in similar terpineol/cineole ratios close to 1:1, while the 194

wild type ratio was 1:2.4 (Table S5 – S7, Fig. 3B). Again, only N. suaveolens F266S 195

produced more α-terpineol than cineole, thus this mutated enzyme changed from a CIN to a 196

TER according to the classification. 197

Table 1: Biochemical characterization of the wild type and three 198 mutant enzymes F266S, F266T and F266V of Nicotiana suaveolens 199 200

KM [µM

]

vmax [µM/min]

E0 [M]

kcat [s-1]

kcat/KM [s-1 x M-1]

Wild type 0.19 0.008

3.4 x 10-7

4.1 x 10-4 2157

F266S 0.12 0.006 2.9 x 10-4 2416

F266T 0.04 0.0012 5.8 x 10-5 1450

F266V 0.04 0.0017 8 x 10-5 2000

The enzyme assay using 3H-GPP was performed with 1 µg purified enzyme in a total volume 201 of 50 µl. The results vmax [µM/min], E0 [M], kcat [s-1], KM [µM] and the catalytically 202 efficiency kcat/KM [s-1 x M-1] were taken from Lineweaver-Burk plots. Wild type: n=1, mutant 203 F266S, F266T, F266V: n=3. 204 205

Further support for the importance of the F266 position for the 1,8-cineol synthesis was 206

provided by a reverse mutation in the TER of N. langsdorffii where serine (S264) is present in 207

the wild type enzyme (Fig. 2, Table S2). This mutant S264F produced twice as much 1,8-208



8

cineole than α-terpineol (Fig. 3C), indicating that the TER enzyme was converted into a CIN. 209

Interestingly, other mutations based on sequence differences between N. langsdorffii TER and 210

N. suaveolens CIN enzymes such as A277T and Q220E did not alter the product profiles nor 211

the 1,8-cineole/α-terpineol ratio (Fig. S3), while the insertions of the R147/N148 in 212

combination with A277T increased 1,8-cineole levels compared to the wild type TER (Fig. 213

3C) indicating again, that distantly located amino acids influence the reaction mechanism in 214

the active site. 215

216

In silico investigations and site directed mutagenesis of N. forgetiana CIN 217

Despite a sequence identity of 98% between N. langsdorffii TER and N. forgetiana CIN 218

(Table S1), both enzymes differ considerably in their main products and terpineol/cineole 219

ratios (Fähnrich et al., 2011; 2012; Fig. 3). Therefore the mechanism of the product formation 220

of the N. forgetiana CIN was investigated by i) in silico investigations and ii) the putative 221

reaction mechanism was verified by site-directed mutagenesis and in vitro assays with 222

purified enzymes. The wild type enzyme of N. forgetiana CIN synthesized α-pinene, β-223

pinene, sabinene, β-myrcene, limonene, α-terpineol and 1,8-cineole (Fig. 3, Table S8-S10, 224

Fig. S4). This volatile profile was identical to the in planta emission spectrum (Fähnrich et al., 225

2012). Additionally geraniol, linalool and in low amounts β-ocimene were detected in the in 226

vitro assay. These acyclic monoterpene alcohols do not belong to the ‘cineole cassette’ and 227

may occur spontaneously by hydrolysis of the substrate GPP, as shown in negative controls. 228

Therefore these compounds were not considered further. 229

A homology model (Fig. S5, Table S11) of the N. forgetiana CIN was created and the last 230

known common intermediate in the synthesis of terpineol and cineole, the α-terpinyl cation, 231

was placed in the active site (Fig. 4A,B). Since the stereospecificity of this intermediate is still 232

unknown, both enantiomers were treated equally during the following considerations. The 233

back of the α-terpinyl cation is located on a hydrophobic patch at the bottom of the active site 234

while the tertiary cation in the C7 of the α-terpinyl cation is stabilized by cation-π stacking 235

upon the indole ring of W253 (Fig. 5). In vitro tests of the mutant W253A revealed a strongly 236

decreased amount of cyclic monoterpenes (Table S8-S10). Therefore, we conclude cation-π 237

stacking to be crucial for the stabilization of the α-terpinyl cation and thus, for the formation 238

of all cyclic monoterpenes of the ‘cineole cassette’. An exchange to methionine (W253M) did 239

not seem to provide a comparable stabilization (Table S8-S10). 240



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By in silico modelling, a water molecule was identified, which is able to attack the α-terpinyl 241

cation at its C7. Within the active site of the protein, the water is fixed by hydrogen bonds to 242

the amino acid side chains of H502 and T278 (Fig. 6). During geometry optimization of the 243

active site of this protein, using the semiempirical method PM7 (Stewart, 2013), the water 244

nucleophilicity attacks the α-terpinyl cation without any energy barrier (Fig. S5). During this 245

attack a putative catalytic dyad comprising H502 and E249 could act as a base, accepting one 246

of the protons of the attacking water molecule. Interestingly, the mutation of this histidine 247

(H502A) did not only lead to a decreased amount of cyclic products, but also suppressed the 248

formation of 1,8-cineole thus producing α-terpineol as main product (Table S8). 249

Monoterpenes that require as a last step in biosynthesis the abstraction of proton need a 250

specific proton acceptor in the active site as well (Fig. 1). The mechanism of the following 251

cyclization of α-terpineol to 1,8-cineole strongly depends on the stereochemistry of the 252

intermediate α-terpinyl cation. For both stereoisomers semi-empirical grid calculations 253

suggest an auto-protonation of the α-terpineol and a subsequent ring closure to 1,8-cineole 254

(Fig. S6). The R-isomer transfers its proton to the hydroxyl group of Y496, which 255

simultaneously protonates the double bond of the α-terpineol (Fig. 6). Using the S-isomer as 256

starting point the same proton relay occurs via the hydroxyl group of T278 (Fig. 6). In our 257

calculations the energy barriers for these reaction paths are rather high (R-isomer = 19.8 258

kcal/mol, S-isomer = 19.7 kcal/mol) (Fig. S6, S7). 259

The mutation of the tyrosine (Y496F) caused a drastic decrease of cyclic products (Fig. 3, 260

Table S8- S10) suggesting a major role of this tyrosine at an earlier step in the biosynthetic 261

pathway. In addition, the hydroxyl group of this tyrosine might be necessary to control the 262

orientation of N419. This asparagine itself is proposed to be involved in binding and fixation 263

of the diphosphate moiety of the substrate. The mutation N419A resulted in a drastic drop of 264

enzyme activity and except for traces of α-terpineol no cyclic products were detected (Table 265

S8- S10). 266

The product composition of the threonine mutant (T278A) was far more characteristic. In 267

comparison to the wild type enzyme, the most striking change was the decreased amount of 268

1,8-cineole (Table S8). Thus, this mutation converted the wild type cineole synthase into a 269

α-terpineol synthase (Table S10). Assuming that α-terpineol is a distinct precursor in the 270

biosynthesis of 1,8-cineole, a decrease of cineole within the product profile indicated a 271

disturbed reaction mechanism of the cyclization of α-terpineol towards 1,8-cineole. Therefore, 272

we hypothesize a major role of T278 in the formation of cineole by fixing the intermediate 273

α-terpineol and supporting auto-protonation of its double bond. One sequence difference 274



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between N. forgetiana CIN and N. langsdorffii TER in position 279 and 277, respectively, is 275

closest to the obviously important T278, which is conserved in all CINs and TERs (Fig. 2). 276

The mutation of this residue (T279A) did not change the product composition but led to an 277

overall increase of activity in the N. forgetiana CIN (Table S9). 278

Beside these theoretical considerations regarding reaction mechanism and pathways, we 279

analysed the enantiomers of α-terpineol produced by recombinant wild type enzymes of five 280

Nicotiana species (Table 2). All five enzymes synthesized both enantiomers, however the S-281

enantiomer was always dominant. Interestingly, the ratios appear conserved in the different 282

species, for the CIN enzymes of N. bonariensis, N. forgetiana and N. suaveolens the S/R 283

ratios were 6-8, for the TER of N. alata it was 3, and in N. langsdorffii TER it was 11. These 284

results indicate that the route with the S-enantiomer is the preferred one. 285

286

Table 2: Amounts and ratios of α-terpineol antipods 287

Species (S)-(-)-α-Terpineol (ng/µl) (R)-(+)-α –Terpineol (ng/µl) S/R ratio N. langsdorffii TER 0.330 0.029 11.2 : 1

0.588 0.054 10.8 : 1 N. alata TER 0.081 0.025 3.2 : 1

0.049 0.017 2.8 : 1 N. bonariensis CIN 0.488 0.067 7.3 : 1

1.121 0.176 6.4 : 1 N. forgetiana CIN 1.175 0.149 7.9 : 1

2.964 0.387 7.7 : 1 N. suaveolens CIN 1.266 0.191 6.6 : 1

2.244 0.346 6.5 : 1 Recombinant enzymes were overexpressed in E. coli. Crude extracts were incubated with the substrate GPP and 288 products were analyzed by GCMS using a stereoselective column (see method). Enantiomers were identified 289 using commercially available compounds. Concentrations were determined according to an internal standard. 290 performed. n=2. 291

292

Discussion 293

1. Phenylalanine 266 is relevant for the product outcome of the 1,8-cineole synthase 294

It was previously demonstrated that single amino acid alterations of terpene synthases resulted 295

in considerable product profile alterations. Kampranis et al. (2007) altered the 1,8-cineole 296

synthase of Salvia officinalis into an α-terpineol synthase and sesquiterpene synthase. Garms 297

et al. (2012) and Zhuang et al. (2012) demonstrated that a single amino acid substitution near 298

the active pocket altered the product outcome for the sesquiterpene synthases SbTPS1 and 299

SbTPS2 of Sorghum bicolor. Here we showed that a single amino acid mutation (F266S), 300

changing the large aromatic side chain of phenylalanine into the small, hydroxylated amino 301



11

acid serine, resulted in a significant reduction of the 2nd cyclization reaction in the 1,8-cineol 302

synthase (CIN) of N. suaveolens (Fig. 3). The reverse mutation from serine to phenylalanine 303

of the terpineol synthase (TER) of N. langsdorffii changed the profile towards 1,8-cineole as 304

the major compound. Furthermore, insertion of R147 and N148 and both insertions in 305

combination with a mutation A277T increased also 1,8-cineole levels compared to the wild 306

type TER. 307

308

It is unclear how the amino acid 266 and insertions at position 147/148 influence the catalytic 309

mechanism. A direct alteration of the active cavity seems unlikely, due to the large distance 310

between these amino acids and the active site of >10 Å and >20 Å, respectively. However, 311

second-tier amino acids can affect the active site geometry and catalysis of enzymes (Hyatt 312

and Croteau, 2005; Greenhagen et al., 2006), e.g., as reported for the oxidosqualene cyclase 313

where the catalytic distinction between cycloartenol and lanosterol synthase activity was 314

critical (Lodeiro et al., 2004). In the case of the position 266 in N. suaveolens the changed 315

product specificity might be a result of either a change in the relative spatial orientation of the 316

neighboring helices or a change in the hydrogen bond network. Position 266 is located in an 317

α-helix which also forms the active site and contains important amino acids such as T278 and 318

the DDXXD motif. That is why a change of the spatial orientation of this helix might affect 319

the active site as well. 320

Further mutational analyses at position 266 were performed to determine which of the 321

properties or structures of amino acids are responsible for the change in the product profile. 322

Interestingly, 1,8-cineole remained the major product in the F266Y and F266T mutants with 323

aromatic and/or hydroxylated amino acid substitutions (Table S5 – S7, Fig. 3) supporting the 324

hypothesis that the size differences of the amino acid side chain of F266S result in a loose 325

coordination of α-terpineol, which could be subsequently released before being converted into 326

1,8-cineole. However, not in line with this interpretation is the observation that the small 327

amino acid valine at this position did not favor α-terpineol release. At this point we can only 328

speculate that additional, yet unknown features beside size and H-bond network influence the 329

reactions in the active pocket. Crystal structure analysis might shed light on this issue. 330

Contradictory results were also obtained when N. suaveolens F266C and other monoterpene 331

synthases with cysteine at this position were compared. The α-terpineol synthases from 332

Magnolia grandiflora (Lee and Chapell, 2008) or Vitis vinifera (Martin and Bohlmann, 2004) 333

favor α-terpineol synthesis, while the mutant enzyme F266C of N. suaveolens, like the wild 334

type enzyme, produced more 1,8-cineole (data not shown). Also the Citrus unshiu 1,8-cineole 335



12

synthase carries a cysteine at the corresponding position (Shimada et al., 2005). At the present 336

state of knowledge, we conclude that cysteine at this position in the helix has similar 337

structural and functional properties as phenylalanine. 338

With the help of domain swapping experiments it has been possible to show, that the N-339

terminal domain does not determine the product spectra of monoterpene synthases (Peters and 340

Croteau, 2003). Therefore it is hard to evaluate potential structural changes caused by the 341

insertion of R147 and N148 in N. langsdorffii. 342

343

344

2. Proposed catalytic mechanism 345

Homology modelling and semiempiric calculations enabled us to suggest an enzymatic 346

reaction mechanism for the formation of α-terpineol and 1,8-cineole in N. forgetiana CIN. 347

After ionization and intramolecular addition of the linalyl diphosphate, the resulting α-terpinyl 348

cation is stabilized by cation-π interactions with W253. A water molecule, which forms 349

hydrogen bonds between the side chain of T278 and H502 was detected in the active site 350

based on the homology modelling also includes explicit water molecules. This water molecule 351

is activated by proton abstraction supported by the catalytic dyad H502 and E249. During the 352

nucleophilic attack of a water molecule on the α-terpinyl cation one proton is transferred to a 353

putative catalytic dyad comprising H502 and E249, resulting in α-terpineol. Finally, a 354

hydroxyl group of an amino acid side chain (Y496 or T278) within the active site provides a 355

proton relay to facilitate the auto-protonation of the double bond of α-terpineol. The following 356

ring closure yields 1,8-cineole (Fig. 1). 357

A tryptophan, which is able to stabilize the intermediate cation, can be found in all 358

monoterpene synthases of known structure at a position corresponding to W253 in N. 359

forgetiana. Mutation of this tryptophan decreased the amount of limonene and increased the 360

amount of acyclic compounds in the product composition of the (4S)-limonene synthase 361

(Srividya et al., 2015). The authors also discussed a potential role of the tryptophan as 362

interaction partner of the intermediate cation. In the same enzyme a histidine residue, 363

corresponding to H502 of N. forgetiana, was proposed to either provide stabilization of the 364

cation or to act as base, abstracting a proton at the final step of limonene biosynthesis 365

(Srividya et al., 2015). Earlier experiments using histidine inhibitors already pointed towards 366

a relevance of this amino acid in terpene synthases (Rajaoarivony et al., 1992). However, 367

despite the need of a proton acceptor in most monoterpene synthases, this amino acid is not 368

conserved throughout the monoterpene synthase family. 369



13

Our proposed mechanism is in accordance with experimental results of Wise et al. (2002), 370

which revealed a syn addition to the double bond in the 1,8-cineole synthase of Salvia 371

officinalis. In our theory the auto-protonation of the double bond does not happen directly, as 372

proposed by Wise el al. (2002), but via a proton relay. They also suggested, that the 373

deprotonation of the hydroxyl group takes place after the final ring closure to 1,8-cineole. 374

Although this idea is very intriguing because of the higher acidity of the α-terpinyl hydronium 375

ion, in all our simulations the proton is abstracted during the nucleophilic attack of the water. 376

377

3. (R)-(+)- or (S)-(-)-(α)terpinyl cation intermediate 378

There are characterized monoterpene synthases specifically synthesizing the R- or S-379

intermediate, e.g. the CIN of Salvia officinalis also produces (R)-α-terpineol (Wise et al., 380

1998) whereas the Arabidopsis thaliana CIN and the Vitis vinifera TER produce (S)-α-381

terpineol, thus suggesting the (R)- and (S)-α-terpinyl cation being the intermediate, 382

respectively (Martin and Bohlmann, 2004,; Chen et al., 2004). However, the stereochemistry 383

of the intermediate α-terpinyl cation of other characterized cineole synthases, e.g. Citrus 384

unshiu and Lavandula sp., is still unknown (Shimada et al., 2005; Demissie et al., 2012). Even 385

though the open-chain cationic intermediates are achiral, Croteau et al. (1994) pointed out, 386

that the stereochemical fate of the intermediate is already fixed upon binding and 387

isomerization of the substrate GPP. Recently, in vitro experiments in self-assembled cavities 388

clearly support that a direct isomerization of a geranyl cation to the cisoid isomer is possible 389

(Zhang and Tiefenbacher, 2015). 390

As we could determine the (S)-enantiomer of α-terpineol as the dominant compound released 391

from the cineole and terpineol synthases of five Nicotiana species (Table 2), this convincingly 392

supported and clarified the actual stereochemistry of this reaction mechanism. Furthermore, 393

the hypothesis of a (S)-α-terpinyl cation being attacked by a water molecule, resulting in (S)-394

α-terpineol, which protonates itself by a proton relay via threonine 278 and cyclizes to 1,8-395

cineole (Fig. 6) was also deduced from the mutant T278A, which specifically diminished the 396

production of 1,8-cineole and subsequently pointed out the special role of this amino acid in 397

the second cyclization reaction (Fig. 3, 5) 398

399

Material and methods 400

Site directed mutagenesis 401



14

For mutation experiments, N. forgetiana CIN and N. suaveolens CIN (Fähnrich et al., 2012; 402

Roeder et al., 2007) were cloned into the pET SUMO expression vector (Invitrogen, 403

Karlsruhe, Germany). Nucleotide changes were generated by using the Quick Change® Site 404

Directed Mutagenesis Kit (Agilent, Santa Clara, CA., U.S,A,) according to manufacturer’s 405

recommendation. PCR parameters for N. forgetiana CIN were as follows: a two minutes 406

initial denaturation at 95 °C was followed by 16 cycles of denaturation at 95 °C for 30 407

seconds, annealing at 55°C for 60 seconds and elongation at 68 °C for 12 minutes. Reactions 408

were finished by final elongation of 68 °C for 10 minutes. PCR parameters for N. suaveolens 409

CIN were slightly different: a two minutes initial denaturation at 97° was followed by 18 410

cycles of denaturation at 97 °C for 50 seconds, annealing at 55°C for 50 seconds, an 411

elongation at 68 °C for 12 minutes, and a final elongation of 10 min at 68 °C. Used primers 412

are shown in Table S13. PCR reactions were followed by adding 1 µl Dpn I (10 U/µl) 413

restriction enzyme to the reaction tubes for digestion of methylated and unmutated parental 414

DNA templates. The digestion was carried out for 90 minutes at 37 °C. 1µl of mutated 415

plasmids was used for the transformation into E. coli TOP10 cells (Invitrogen, Karlsruhe, 416

Germany). Plasmids were re-isolated from single E. coli TOP10 clones using the 417

NucleoSpin® Plasmid Easy Pure Kit (Machery-Nagel GmbH, Berlin, Germany) and mutated 418

sequences were confirmed by Sanger sequencing (GATC Biotech AG, Konstanz, Germany). 419

420

Heterologous protein expression 421

The proteins were expressed using the ChampionTM pET SUMO Protein Expression System 422

(Invitrogen, Karlsruhe, Germany). Expression and purification were carried out as described 423

in Hippauf et al. (2010). E. coli HMS 174 (DE3) strain (Novagene) was used for 424

overexpression of His6-tagged proteins. Overexpressed protein was obtained after a pre-425

incubation of 150 ml culture at 37 °C until OD600 of 0.6 and 1 was reached for N. forgetiana 426

and N. suaveolens, respectively. E. coli HMS 174 (DE3) cells were induced with 0.5 mM 427

isopropylthio-ß-galactoside and the incubation continued for 20 h at 20 °C. Crude extracts 428

were obtained by incubating the cell pellet with lysozyme (final concentration: 1 mg/ml), 429

sonication, and centrifugation in order to separate cell debris from the enzyme containing 430

soluble fraction. The overexpressed protein was purified by Ni–NTA affinity chromatography 431

(Qiagen, Hilden, Germany) according to the manufacturer instructions. Protein concentrations 432

were measured using the standard Bradford assay. Protein purification was checked using 433



15

SDS polyacrylamide gel electrophoresis and Western blotting (anti-His tag antibody, anti-434

rabbit IgG; Sigma, Munich, Germany) 435

436

Enzyme assay 437

For enzyme tests using N. forgetiana CIN wild type and mutated enzymes, 60 µg of purified 438

enzyme, 60 µl assay buffer (250 mM Hepes/KOH buffer (pH 8), 100 mM MgCl2, 2.5 mM 439

MnCl2, 50 % glycerol), 5 µl of DTT (1 M) and 40 µl of GPP (1 µg/µl; Echelon Biosciences, 440

Salt Lake City. U.S.A) were supplemented with water to a final volume of 250 µl and 441

incubated at 39 °C for 3 h. The assay samples were overlaid with 250 µl hexane containing 5 442

ng/µl cis-nerolidol (Carl Roth, Karlsruhe, Germany) as internal standard. The products were 443

extracted by vortexing for 30 seconds followed by centrifugation (5 min at 4000 x g). 1 µl of 444

the hexane phase was used for GC/MS analysis. 445

For enzyme tests using N. suaveolens CIN wild type and mutated enzymes, 10 µg of E. coli 446

crude extract and purified enzyme, respectively, 40 µl assay buffer (250 mM Hepes/KOH 447

buffer (pH 8), 100 mM MgCl2, 2.5 mM MnCl2, 50 % glycerol), 5 µl of DTT (1M) and 4 µl of 448

GPP (1 µg/µl; Echelon Biosciences, Salt Lake City, U.S.A.) were mixed with water to a final 449

volume of 200 µl and incubated at 41.5 °C for 3 h. The assay samples were overlaid with 200 450

µl hexane supplemented with 5 ng/µl cis-nerolidol (Carl Roth, Karlsruhe, Germany) as 451

internal.. The products were extracted by vortexing for 30 seconds and centrifugation (1 min, 452

4000 x g). 1 µl of the hexane phase was used for GC/MS analysis. 453

The biochemical parameters vmax, Km and Kcat were determined by using different substrate 454

concentrations (10 nM, 20 nM, 30 nM, 40 nM, 50 nM and 60 nM). The product formation 455

was monitored between 2 min and 10 min. The initial rate (=linear range) of each substrate 456

concentration was plotted in a Lineweaver-Burk diagram. The assay was performed in a total 457

volume of 50 μl with 10 μl 5x Hepes buffer, 1 μl substrate 3H-GPP (1 mCi/ml, Hartmann 458

Analytics, Braunschweig, Germany), 1 μg enzyme, 2.5 mM DTT, overlaid with 180 ml 459

hexane, and incubated at 41.5 oC. The reaction was stopped by transferring it on ice for 15 s, 460

mixing (1 min), and centrifugation (2 min,13000 rpm). 50 μl of the organic phase was added 461

to 2 ml scintillation solution (Ultima GoldTM, Perkin Elmer, Rodgau, Germany) and product 462

formation was counted in Tri-carb 2810TR (Perkin Elmer, Rodgau, Germany). 463

464



16

GC/MS analysis 465

The volatile compounds were analyzed with a Shimadzu QP5000 gas chromatograph 466

connected to a mass spectrometer (GC/MS) for identification (Shimadzu, Tokyo, Japan). 467

Separation was performed on a DB-5MS column (60 m x 0.25 mm x 0.25 µm; Agilent, Santa 468

Clara, CA., U.S.A.)) with helium as carrier gas (flow rate of 1.1 ml min-1) using an injection 469

temperature of 200 °C and a temperature gradient from 35 °C (2 min hold) to 280 °C (15 min 470

hold) with a ramp of 10 °C min-1. Mass spectra were obtained by using the scan mode (total 471

ion count, 40–280 mz-1). Compound identity was confirmed by comparison of obtained 472

spectra with spectra in the library of the National Institute of Standards and Technology 473

(NIST147) and with spectra obtained from authentic standards. Enantiomers of α-terpineol 474

were separated employing the GC 7890A coupled to a 5975C mass selective detector 475

(GC/MSD; Agilent, Santa Clara, CA., U.S.A.) which was equipped with an enantioselective 476

column coated with heptakis(6-O-tert-butyldimethylsilyl-2,3-di-O-methyl)-β-cyclodextrin 477

(50% in OV17, w/w; 25 m × 0.25 mm i.d.; König et al., 1994). The sample was injected in 478

split mode with a split flow of 20 ml min-1, an injection temperature of 250 °C, and a column 479

flow of 1.2 ml min-1. The speration started with an initial temperature of 50 °C for 1 min 480

followed by a ramp of 2 °C min-1 to a final temperature of 170 °C. The mass range was set 481

from 45 to 300 AMU with a scan speed of 5.4 scans sec-1. Identity was confirmed by 482

comparing the mass spectra with the NIST library and with those of authentic standards. 483

484

Homology modelling 485

Sequences of N. forgetiana CIN, N. suaveolens CIN and N. langsdorffii TER were published 486

recently (Fähnrich et al., 2011; 2012; Roeder et al., 2007) and correspond to UNIPROT Codes 487

I7CTV3, A5Y5L5, and H2ELN1, respectively. The sequences have been truncated to the 488

mature protein, starting with RR(x)8W motif. For homology model creation, the automated 489

modelling by YASARA (Krieger et al., 2009) has been used with default settings. Due to errors 490

in the interpretation of ligand double bonds, the templates were not taken from pdb redo 491

(default) but downloaded from rcsb pdb and provided manually. For further work, the hybrid 492

models, containing features derived from all given templates, were used. All homology 493

models were energy optimized using the force field YASARA2 (Krieger at al., 2002) as 494

implemented in YASARA. The model of N. forgetiana CIN was refined by a molecular 495

dynamics simulation (MD) for 7.5 ns using the force field AMBER03 (Duan et al., 2003). Due 496

to problems regarding the backbone dihedrals in loop N160-A168, this loop has been taken 497



17

over from the model of N. langsdorffii TER into the mean structure of the production phase of 498

the MD. To minimize deviations from planarity of the peptide bonds, in a second MD (175 ns, 499

AMBER03) the force constant of the peptide dihedral was increased by factor 1.5. After each 500

modelling step the models have been evaluated by the model quality assessment programs 501

PROSA (Sippl, 1990), PROCHECK (Laskowski et al., 1993) and QMEAN (Benkert et al., 2008). 502

Due to missing parameters for the carbocation in established docking programs the reactive 503

intermediates (R)- and (S)-α-terpinyl cation have been positioned in the putative active site 504

manually. Since the automated parameterization of YASARA failed for the intermediates, these 505

intermediates were parameterized with AM1-BCC (Jakalian et al., 2002) and GAFF (Wang et 506

al., 2004) using ANTECHAMBER and SQM (Case et al., 2013). The models have been put into 507

water boxes and, while fixing all heavy atoms of the receptor and ligand, the system was 508

relaxed with an MD (2 ns, Amber03). 509

For the semi-empirical calculations the model was reduced to active site residues and ligands. 510

The elucidation of the reaction mechanism was performed with the help of grid calculations 511

using PM7 (Stewart, 2013) as implemented in MOPAC (Stewart, 1990). For both stereoisomers 512

of the intermediate, different combinations of reaction coordinates have been examined. 513

514

List of supplemental material 515

Supplemental Figure S1A-G: Analysis of volatile profiles (% distribution of monoterpenes), 516

enzyme activities (pkat/mg) and terpineol/cineole ratios in the mutants and wildtype cineole 517

synthase (E. coli crude extract) of Nicotiana suaveolens 518

Supplemental Figure S1A-G: Analysis of volatile profiles (% distribution of monoterpenes), 519

enzyme activities (pkat/mg) and terpineol/cineole ratios in the mutants and wildtype cineole 520

synthase (E. coli crude extract) of Nicotiana suaveolens 521

Supplemental Figure S2: SDS-PAGE and Western blot of the purified wild type and mutant 522

(F266S, F266T, F266V) enzymes of Nicotiana suaveolens 523

Supplemental Figure S3: Analysis of volatile profiles (pie chart: % distribution of 524

monoterpenes), enzyme activities (pkat/mg) and terpineol/cineole ratio of mutants of terpineol 525

synthase (E.coli crude extract) of Nicotiana langsdorffii 526

Supplemental Figure S4: Purification of the recombinant wild type and mutant cineole 527

synthase of Nicotiana forgetiana 528



18

Supplemental Figure S5A-D: Supporting calculations for 3D model of cineole synthase of 529

Nicotiana forgetiana 530

A) Ramachandran plot of the final model of the cineole synthase of Nicotiana forgetiana 531

generated with PROCHECK (Laskowski et al., 1993). 532

B) PROSA-plot of the homology model of the cineole synthase Nicotiana forgetiana after 533

initial (red) modelling and refinement (blue). For comparison the main template of 534

limonene synthase of Mentha spicata (2ONH) is shown (cyan). 535

C) Scores of model evaluation programs. 536

D) Comparison of QMEAN results of homology models of the cineole synthase of N. 537

forgetiana and the crystal structure of the limonene synthase of Mentha spicata and a 538

reference dataset provided by QMEAN (Benkert et al., 2008). 539

Supplemental Figure S6: MOPAC grid calculations to examine the energy barriers of the 540

reaction path from α-terpineol to 1,8-cineole of Nicotiana forgetiana 541

Reaction coordinates are marked in yellow and green, respectively, and differ between 542

(R)- and (S)-α-terpineol (A: (S)-α-terpineol, B) (R)-α-terpineol). Interestingly, the starting 543

geometries differ considerably. Energies in tables are presented relative to the energy of the 544

starting geometry of the putative reaction path (in kcal/mol). 545

Supplemental Figure S7: Reaction path from α-terpinyl cation to 1,8 cineole in Nicotiana 546

forgetiana 547

Proposed reaction path considering the R stereoisomer (A) and the S stereoisomer (B) 548

Table S1: Terpineol and cineole synthase amino acid sequence identities (%) and 549

number of different amino acids of various Nicotiana species 550

Table S2: Amino acid alterations in terpineol and cineole synthases of Nicotiana species 551

Table S3: Mutants constructed of cineole synthase of Nicotiana suaveolens 552 Table S4: Distribution of monoterpene products (%), specific enzyme activities, and 553

α-terpineol/1,8-cineole ratios of the wildtype and mutated enzymes of Nicotiana suaveolens 554

Table S5: Amounts of products synthesized by the wildtype and mutated enzymes of 555 Nicotiana suaveolens 556

Table S6: Specific enzyme activities of the wildtype enzyme and mutated enzymes of 557 Nicotiana suaveolens 558

Table S7: Relative enzyme activities of Nicotiana suaveolens wildtype and mutant enzymes 559



19

Table S8: Amounts of product synthesized by the wildtype and mutated enzymes of Nicotiana 560 forgetiana 561

Table S9: Specific enzyme activities of the wildtype and mutated enzymes of Nicotiana forgetiana 562

Table S10: Relative enzyme activities of Nicotiana forgetiana wildtype and mutant enzymes 563

Table S11: Templates used for homology modelling 564

Table S12: Biochemical parameters of cineole synthases of different plant species and a 565 limonene synthase of Mentha spicata 566

Table S13A: Primer sequences to generate Nicotiana forgetiana mutants 567

Table S13B: Primer sequences to generate Nicotiana suaveolens mutants 568

569

Acknowledgements 570

The authors thank the University of Rostock, the Institute for Plant Biochemistry (Halle) and 571

the Deutsche Forschungsgemeinschaft (Pi 153/22) for financial support. We are thankful to 572

Claudia Dinse (Rostock) for technical assistance; Francel Verstappen (Wageningen) for 573

enantiomer separation; Marco Kai, Sybille Lorenz for analysing wild type enzymes with 574

terpineol as substrate and Aleš Svatoš for allowing to use his GC/MS for the latter 575

measurements. We are especially grateful to the master students Madeleine Neumann and 576

Madlen Klodt who performed the experiments with Nicotiana langsdorffii. 577

578

Figure legends 579

Figure 1: Synthesis of “cineole cassette” monoterpenes 580

The substrate geranyl pyrophosphate (GPP) is ionized by diphosphate elimination resulting in 581

the geranyl cation. Subsequently, this cation is converted into the linalyl cation and α-terpinyl 582

cation. The synthesis of the acyclic β-myrcene might proceed via the geranyl cation or via the 583

linalyl cation by deprotonation. The intermediate α-terpinyl cation is the precursor for all 584

cyclic monoterpenes. The 2,7 ring closure results in the pinyl cation which is deprotonated to 585

synthesize β-pinene and α-pinene. Sabinene, with a cyclopropane ring, is released after two 586

carbocation formations and 2,6 ring closure. α-terpineol is formed after water capture of the 587

α-terpinyl cation. Broken lines indicate possible reactions leading to 1,8-cineole. A 588

cyclization reaction resulting in 1,8-cineole uses α-terpineol as precursor (Degenhardt et al., 589

2009, modified) 590



20

Figure 2. Amino acid sequence alignment of the cineole and terpineol synthases of seven 591

Nicotiana species 592

Amino acid sequence alignment of the cineole synthases of N. suaveolens, N. forgetiana, N. 593

bonariensis, N. longiflora, N. mutabilis and the terpineol synthases of N. alata and N. 594

langsdorffii. Sequences of putative mature enzymes were aligned using CLUSTALW 2.0.10 595

(Thompson et al., 1994). Conserved sequence motifs of monoterpene synthases are 596

highlighted in yellow. Black circle above sequence: amino acids of the active pocket of 597

monoterpene synthase according to Fähnrich et al. (2012). Asterisks (*): identical amino 598

acids, spaces: amino acids different, double dots: amino acids differ from each other but have 599

similar properties, one dot below sequence: amino acids differ from each other but have little 600

similar properties (EMBL-EBI 2014). Red letters: amino acid differences compared to the 601

sequence of N. forgetiana. Cyan highlights: amino acid mutations reported in this publication. 602

Mutated residues and sequence motifs correspond to Fig. 4B and C. 603

604

Figure 3. Volatile profiles and specific enzyme activities of wild type and mutant 605

enzymes of Nicotiana forgetiana, N. suaveolens and N. langsdorffii 606

Enzymes of N. forgetiana (A), N. suaveolens (B) and N. langsdorffii (C) were overexpressed 607

in E. coli and purified via affinity chromatography (Fig. S3, S4) (crude extracts were used for 608

N. langsdorffii wt, 147R/148N and S264F). The pie chart presents the distribution (%) of each 609

monoterpene released by wild type and mutant enzymes. Specific enzyme activities (pkat/mg) 610

were calculated for each monoterpene and summed up to total specific activities (middle 611

panel). The α-terpineol/1,8-cineole ratio is presented in the lower panel. 612

613

Figure 4: Homology models of 1,8-cineol synthases of Nicotiana forgetiana and Nicotiana 614

suaveolens 615

A) The helices of the C- and N-terminal domains of the N. forgetiana cineol synthase are 616

shown in green and light green, respectively. The α-terpinyl cation (cyan) and the 617

diphosphate (orange) are presented as sticks. Violet dots represent the three magnesium 618

ions (magnesium cluster). The mesh shows the surface of the cavity, which contains the 619

putative active site. 620



21

B) C-terminal domain of N. forgetiana 3D model. Stick representation of mutated amino 621

acids. The trinuclear magnesium cluster (violet) marks the active site. Colored cartoon 622

regions represent conserved motifs, RR(X)8W motif: pink; RWW motif: blue; RDR motif: 623

green; NALV motif: yellow; DDxxD motif: orange; NSE/DTE motif: cyan, CYMNE 624

motif: purple. Mutated amino acids and motifs shown correspond to Fig. 2. Additionally 625

shown: N-terminus covering the active site (pink line). 626

C) C-terminal domain of N. suaveolens 3D model with mutated amino acids and motifs. 627

Coloring as in Fig. 4B. 628

629

Figure 5. Active site of the cineole synthase of Nicotiana forgetiana 630

Amino acid side chains are shown in grey sticks. The back of the reactive intermediate 631

α-terpinyl cation (cyan) is located on a hydrophobic patch (grey mesh), while the tertiary 632

carbocation in C7 (green-cyan) exhibits a cation-pi stack upon the indole ring of W253. The 633

C3 atom, where the prenyl moiety has been cleaved off in the last ionization step is still in 634

spatial proximity of the diphosphate (red-orange). A water molecule (red-white) is fixed by 635

H502 and T278 and ready to perform a nucleophilic attack on the cationic C7 of the α-terpinyl 636

cation. 637

638

Figure 6: Proposed mechanism for the formation of 1,8-cineole 639

Depending on the stereochemistry of the intermediate the protonation of the double bond of 640

the α-terpinyl cation is provided by a proton relay via the hydroxyl groups of either Tyr496 or 641

Thr278. 642

643

References 644

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Rajaonarivony JI, Gershenzon J, Miyazaki J, Croteau R (1992) Evidence for an essential 750 histidine residue in 4S-limonene synthase and other terpene cyclases. Arch Biochem Biophys 751 299: 77-82 752 753 Roeder S, Hartmann AM, Effmert U, Piechulla B (2007) Regulation of simultaneous 754 synthesis of floral scent terpenoids by the 1,8-cineole synthase of Nicotiana suaveolens. Plant 755 Mol Biol 65: 107–124 756 757 Shimada T, Endo T, Fujii H, Hara M, Omura M (2005) Isolation and characterization of 758 (E)-beta-Ocimene and 1,8-cineole synthases in Citrus unshiu Marc. Plant Sci 168: 987-995 759 760 Sippl MJ (1990) Calculation of conformational ensembles from potentials of mean force. An 761 approach to the knowledge-based prediction of local structures in globular proteins. J Mol 762 Biol 213: 859-883 763 764 Srividya N, Davis EM, Croteau RB, Lange BM (2015) Functional analysis of (4S)-765 limonene synthase mutants reveals determinants of catalytic outcome in a model monoterpene 766 synthase. Proc Natl Acad Sci USA 112: 3332-3337 767 768 Starks CM, Back K, Chappell J, Noel JP (1997) Structural basis for cyclic terpene 769 biosynthesis by tobacco 5-epi-aristolochene synthase. Science 277: 1815-1820 770 771 Stewart JJP (1990) MOPAC: a semi-empirical molecular orbital program. J Comput Aided 772 Mol Des 4: 1-105 773 774 Stewart JJP (2013) Optimization of parameters for semi-empirical methods VI: more 775 modifications to the NDDO approximations and re-optimization of parameters. J Mol Model 776 19: 1-32 777 778 Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of 779 progressive multiple sequence alignment through sequence weighting, position-specific gap 780 penalties and weight matrix choise. Nucl Acids Res 22: 4673-4680 781 782 Turner G, Gershenzon J, Nielson EE, Froehlich FE, Croteau R (1999) Limonene 783 synthase, the enzyme responsible for monoterpene biosynthesis in peppermint, is localized in 784 leucoplasts of oil gland secretory cells. Plant Physiol 120: 879-886 785 786 Wang J, Wolf RM, Caldwell JW, Kollman PA, Case DA (2004) Development and testing 787 of a general amber force field. J Comput Chem 25: 1157-1174 788 789 Williams D, McGarvey DJ, Kathira EJ, Croteau R (1998) Truncation of limonene 790 synthase preprotein provides a fully active ‘pseudomature’ form of this monoterpene cyclase 791 and reveals the function of the amino-terminal. Biochem 37: 12213-12220 792 793 Wise ML, Savage TJ, Katahira E, Croteau R (1998) Monoterpene synthases from common 794 sage (Salvia officinalis). cDNA isolation, characterization, and functional expression of (+)-795 sabinene synthase, 1,8-cineole synthase, and (+)-bornyl diphosphate synthase. J Biol Chem 796 273: 14891-14899 797 798



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Wise ML, Urbansky M, Helms GL, Coates RM, Croteau R (2002) Syn stereochemistry of 799 cyclic ether formation in 1,8-cineole biosynthesis catalyzed by recombinant synthase from 800 Salvia officinalis. J Am Chem Soc 124: 8546-8547 801 802 Yahyaa M, Matsuba Y, Brandt W, Doron-Faigenboim A, Bar E, McClain A, 803 Davidovich-Rikanati R, Lewinsohn E, Pichersky E, Ibdah M (2015) Identification, 804 functional characterization, and evolution of terpene synthases from a basal dicot. Plant 805 Physiol 169:1683-97 806 807 Zhang Q, Tiefenbacher K (2015) Terpene cyclization catalysed inside a self-assembled 808 cavity. Nature Chemistry 7: 197-201 809 810 Zhuang X, Köllner TG, Zhao N, Li G, Jiang Y, Zhu L, Ma J, Degenhardt J, Chen F 811 (2012) Dynamic evolution of herbivore-induced sesquiterpene biosynthesis in Sorghum and 812 related grass crops. Plant J 69: 70-80 813



1

1

Figure 1: Synthesis of “cineole cassette” monoterpenes 2

The substrate geranyl pyrophosphate (GPP) is ionized by diphosphate elimination resulting in 3

the geranyl cation. Subsequently, this cation is converted into the linalyl cation and α-terpinyl 4

cation. The synthesis of the acyclic β-myrcene might proceed via the geranyl cation or via the 5

linalyl cation by deprotonation. The intermediate α-terpinyl cation is the precursor for all 6

cyclic monoterpenes. The 2,7 ring closure results in the pinyl cation which is deprotonated to 7

synthesize β-pinene and α-pinene. Sabinene, with a cyclopropane ring, is released after two 8

carbocation formations and 2,6 ring closure. α-terpineol is formed after water capture of the 9

α-terpinyl cation. Broken lines indicate possible reactions leading to 1,8-cineole. A 10

cyclization reaction resulting in 1,8-cineole uses α-terpineol as precursor (Degenhardt et al. 11

2009, modified 12



1

N. suaveolens CIN RRSGNYGPTMWDFEYIQSIHNDYTEKKYMNRLNKLKEEMKKMIMAEGSQELEKLELIDNL 60 1 N. forgetiana CIN RRSGNYQPTMWDFEYIQSIHNDYAGDKYMKRFNELKEEMKKMIMAEGSQELEKLELIDNL 60 2 N. bonariensis CIN RRSGNYQPTMWDFEYIQSIHNDYAGDKYMKRFNELKEEMKKMIMAEGSQELEKLELIDNL 60 3 N. longiflora CIN RRSGNYQPTMWDFEYIQSIHNDYAGDKYMKRFNELKEEMKKMIMAEGSQELEKLELIDNL 60 4 N. mutabilis CIN RRSGNYQPTMWDFEYIQSIHNDYAGDKYMKRFNELKEEMKKMIMAEGSQELEKLELIDNL 60 5 N. alata TER RRSGNYQPTMWDFEYIQSIHNDYAGDKYMKRFNELKEEMKKMIMAEGSQELEKLELIDNL 60 6 N. langsdorffii TER RRSGNYQPTMWDFEYIQSIHNDYAGDKYMKRFNELKEEMKKMIMAEGSQELEKLELIDNL 60 7 ****** ****************: .***:*:*:************************** 8 9 N. suaveolens CIN QRLGVSYHFKHEIMQILSSIKQHSTPADSLYATALKFRLFREYGFHISQEIFGGLSETHT 120 10 N. forgetiana CIN QRLGVSYHFKHEIMQILSSIKQHSTPADSLYATALKFRLLREHGFHISQEIFDGLSETHT 120 11 N. bonariensis CIN QRLGVSYHFKHEVMQILSSIKQHSTRADSLYATALKFRLLREHGFHISQEIFDGLSETHT 120 12 N. longiflora CIN QRLGVSYHFKHEIMQILSSIKQHSTPADSLYATALKFRLLREHGFHISQEIFDGLSETHT 120 13 N. mutabilis CIN QRLGVSYHFKHEIMQILSSIKQHSTPADSLYATALKFRLLREQGFHISQEIFDGLSETHT 120 14 N. alata TER QRLGVSYHFKHEIMQILSSIKQHSTPADSLYATALKFRLLREHGFHISQEIFDGLSETHT 120 15 N. langsdorffii TER QRLGVSYHFKHEIMQILSSIKQHSTPADSLYATALKFRLLREHGFHISQEIFDGLSETHT 120 16 ************:************ *************:** ********* ******* 17 18 N. suaveolens CIN EDTKGMLYLYEASFLATEGESELEKARNWTEKHLREYLENKNDDQNVAELVHHALELPLH 180 19 N. forgetiana CIN KDTKGMLYLYEASFLATEGESELEQARNWTEKHLREYLKNKNIDQNEAKLVHRALELPLH 180 20 N. bonariensis CIN KDTKGMLYLYEASFLATEGESELEQARNWTEKHLREYLKNKNIDQNVAKLVHRALELPLH 180 21 N. longiflora CIN KDTKGMLYLYEASFLATEGESELEQARNWTEKHLREYLKNKNIDQNEAKLVHRALELPLH 180 22 N. mutabilis CIN KDTKGMLYLYEASFLATEGESELEQARNWTEKHLREYLKNKNIDQNEAKLVHRALELPLH 180 23 N. alata TER KDTKGMLYLYEASFLATEGESELEQA--WTEKHLREYLKNKNIDQNVAKLVHRALELPLH 178 24 N. langsdorffii TER KDTKGMLYLYEASFLATEGESELEQA--WTEKHLREYLKNKNIDQNVAKLVHRALELPLH 178 25 :***********************: **********:*** *** *:***:******* 26 27 N. suaveolens CIN WRMLRIEARWFINFYKKKQDMIPLLLELAILDFNIVQAAHIEDLKYVARWWKETCLAENL 240 28 N. forgetiana CIN WRMLRLEARWFISFYKKRQDMIPLLLELAILDFNIVQAAHIEDLKYVARWWKETGLAENL 240 29 N. bonariensis CIN WRMLRLEARWFIGFYKKRQDMIPLLLELAILDFNIVQAAHIQDLKYVARWWKETALAENL 240 30 N. longiflora CIN WRMLRLEARWFISFYKKRQDMIPLLLELAILDFNIVQAAHIEDLKYVARWWKETGLAENL 240 31 N. mutabilis CIN WRMLRLEARWFISFYKKRQDMFPLLLELAILDFNIVQAAHTEDLKYVARWWKETGLAENL 240 32 N. alata TER WRMLRLEARWFISFYKKRQDMIPLLLELAILDFNIVQAAHIQDLKYVARWWKETGLAENL 238 33 N. langsdorffii TER WRMLRLEARWFISFYKKRQDMIPLLLELAILDFNIVQAAHIQDLKYVARWWKETGLAENL 238 34 *****:****** ****:***:****************** :************ ***** 35 O O O OOOOO 36 N. suaveolens CIN PFARDRLVENFFWTIGVNFLPQYGYFRRIETKVNALVTTIDDVYDVFGTMDELQCFTHAF 300 37 N. forgetiana CIN PFARDRLVENFFWTIGVNFLPQYGYFRRIETKVNALVTTIDDVYDVFGTLDELQCFTDAI 300 38 N. bonariensis CIN PFARDRLVENFFWTIGVNFLPQYGYFRRIETKVNALVTTIDDVYDVFGTLDELQCFTDAI 300 39 N. longiflora CIN PFARDRLVENFFWTIGVNFLPQYGYFRRIETKVNALVTTIDDVYDVFGTLDELQCFTDAI 300 40 N. mutabilis CIN PFARDRLVENFFWTIGVNFLPQYGYFRRIETKVNALVTTIDDVYDVFGTLDELQCFTDAI 300 41 N. alata TER PFARDRLVENFFWTIGVNFLPQYGYFRRIETKVNALVTTIDDVYDVFGTLDELQCFTDAI 298 42 N. langsdorffii TER PFARDRLVENFFWTIGVNFLPQYGYSRRIETKVNALVTAIDDVYDVFGTLDELQCFTDAI 298 43 ************************* ************:**********:*******.*: 44 45 N. suaveolens CIN QRWNIDELDNLPDNMKMCYFALDNFINEVAGDAFEEHRVPILSYLRNAWTDLCKAYLREA 360 46 N. forgetiana CIN QRWNTDELDNLPDNMKMCYFALDDFINEVACDAL------IVPYLRNAWTDLCKSYLREA 354 47 N. bonariensis CIN QRWNTDELDNLPDNMKMCYFALDDFINEVACDAL------IVPYLRNAWRDLCKSYLREA 354 48 N. longiflora CIN QRWNTDELDNLPDNMKMCYFALDDFINEVACDAL------IVPYLRNAWTDLCKSYLREA 354 49 N. mutabilis CIN QRWNTDELDNLPDNMKMCYFALDDFINEVACDAL------IVPYLRNAWTDLCKSYLREA 354 50 N. alata TER QRWNTDELDNLPDNMKMCYFALDDFINEVACDAL------IVPYLRNAWTDLCKSYLIEA 352 51 N. langsdorffii TER QRWNTDELDNLPDNMKMCYFALDDFINEVACDAL------IVPYLRNAWTDLCKSYLIEA 352 52 **** ******************:****** **: *: ****** ****:** ** 53 O 54 N. suaveolens CIN KWYFSKYIPTMEEYMDNAWISISAPVILVHAYFLVANPVNKEVLHYLENYHDIIRWSALI 420 55 N. forgetiana CIN KWYFSKYIPTMEEYMDNAWISISAPVILVHAYFLIANPVNKEALHYLRNYHDIIRWSALI 414 56 N. bonariensis CIN KWYFSKYIPTMEEYTDNAWISISAPVILVHAYFLIANPVNKEALHYLRNYHDIIRWSALI 414 57 N. longiflora CIN KWYFSKYIPTMEEYMDNAWISISAPVILVHAYFLIANPVNKEALHYLRNYHDIIRWSALI 414 58 N. mutabilis CIN KWYFSKYIPTMEEYMDNAWISISAPVILVHAYFLIANPVNKEALHYLRNYHDIIRWSALI 414 59 N. alata TER KWYFSKYIPTMEEYMDNAWISISAPVILVHAYFLIANPVNKEALHYLRNYHDIIRWSALI 412 60 N. langsdorffii TER KWYFSKYIPTMEEYMDNAWISISAPVILVHAYFLIANPVNKEALHYLRNYHDIIRWSALI 412 61 ************** *******************:*******.****.************ 62 OOOOOOOOOOO 63 N. suaveolens CIN LRLANDLGTSSEELKRGDVPKSIQCYMNEKKVSEEEARQHIRLLISETWKKLNEAHNVAA 480 64 N. forgetiana CIN LRLANDLGTSSDELKRGDVPKSIQCYMNEKKVSEEEARQHIRLLISETWKKLNEAHNIAA 474 65 N. bonariensis CIN LRLANDLGTSSDELKRGDVPKSIQCYMNEKKVSEEEARQHIRLLISETWKKLNEAHNIAA 474 66 N. longiflora CIN LRLANDLGTSSDELKRGDVPKSIQCYMNEKKVSEEEARQHIRLLISETWKKLNEAHNIAA 474 67 N. mutabilis CIN LRLANDLGTSSDELKRGDVPKSIQCYMNEKKVSEEEARQHIRLLISETWKKLNEAHDIAA 474 68 N. alata TER LRLANDLGTSSDELKRGDVPKSIQCYMNEKKVSEEEARQHIRLLISETWKKLNEAHNVAA 472 69 N. langsdorffii TER LRLANDLGTSSDELKRGDVPKSIQCYMNEKKVSEEEARQHIRLLISETWKKLNEAHNVAA 472 70 ***********:********************************************::** 71 72 N. suaveolens CIN HPFPKMFVKCAMNLARMAQCMYQHGDGHGHGDLNSETTNHIMALLFESIPPA 532 73 N. forgetiana CIN HPFPKMFVKSAMNLARMAQCMYQHGDGHG--GQNSETQNSIMALVFESIPPA 524 74 N. bonariensis CIN HPFPKMFVKTAMNLARMAQCMYQHGDGHG--GQNSETQNRIMALLFESIPPA 524 75 N. longiflora CIN HPFPKMFVKSAMNLARMAQCMYQHGDGHG--GQNSETQNSIMALLFESIPPA 524 76 N. mutabilis CIN HPFPKMFVKSAMNLARMAQCMYQHGDGHG--GQNSETQNRIMALLFESIPPA 524 77 N. alata TER HPFPKMFVKSAMNLARMAQCMYQHGDGHG--GQNSETQNRIMALLFESIPPA 522 78 N. langsdorffii TER HPFPKMFVKSAMNLARMAQCMYQHGDGHG--GQNSETQNRIMALLFESIPPA 522 79 ********* ******************* **** * ****:******* 80 81

Figure 2. Amino acid sequence alignment of the cineole and terpineol synthases of seven 82

Nicotiana species 83



2

Amino acid sequence alignment of the cineole synthases of N. suaveolens, N. forgetiana, N. 84

bonariensis, N. longiflora, N. mutabilis and the terpineol synthases of N. alata and N. 85

langsdorffii. Sequences of putative mature enzymes were aligned using CLUSTALW 2.0.10 86

(Thompson et al 1994). Conserved sequence motifs of monoterpene synthases are highlighted 87

in yellow. Black circle above sequence: amino acids of the active pocket of monoterpene 88

synthase according to Fähnrich et al. (2012). Asterisks (*): identical amino acids, spaces: 89

amino acids different, double dots: amino acids differ from each other but have similar 90

properties, one dot below sequence: amino acids differ from each other but have little similar 91

properties (EMBL-EBI 2014). Red letters: amino acid differences compared to the sequence 92

of N. forgetiana. Cyan highlights: amino acid mutations reported in this publication. Mutated 93

residues and sequence motifs correspond to Fig. 3B and C. 94

95



1

1

Figure 3. Volatile profiles and specific enzyme activities of wild type and mutant 2

enzymes of Nicotiana forgetiana, N. suaveolens and N. langsdorffii 3

Enzymes of N. forgetiana (A), N. suaveolens (B) and N. langsdorffii (C) were overexpressed 4

in E. coli and purified via affinity chromatography (Fig. S3, S4) (crude extracts were used for 5

N. langsdorffii wt, 147R/148N and S264F). The pie chart presents the distribution (%) of each 6

monoterpene released by wild type and mutant enzymes. Specific enzyme activities (pkat/mg) 7

were calculated for each monoterpene and summed up to total specific activities (middle 8

panel). The α-terpineol/1,8-cineole ratio is presented in the lower panel. 9



1

A 1

2

B C 3

4

5

Figure 4: Homology models of 1,8-cineol synthases of Nicotiana forgetiana and Nicotiana 6

suaveolens 7

A) The helices of the C- and N-terminal domains of the N. forgetiana cineol synthase are 8

shown in green and light green, respectively. The α-terpinyl cation (cyan) and the 9

diphosphate (orange) are presented as sticks. Violet dots represent the three magnesium 10

ions (magnesium cluster). The mesh shows the surface of the cavity, which contains the 11

putative active site. 12



2

B) C-terminal domain of N. forgetiana 3D model. Stick representation of mutated amino 13

acids. The trinuclear magnesium cluster (violet) marks the active site. Colored cartoon 14

regions represent conserved motifs, RR(X)8W motif: pink; RWW motif: blue; RDR motif: 15

green; NALV motif: yellow; DDxxD motif: orange; NSE/DTE motif: cyan, CYMNE 16

motif: purple. Mutated amino acids and motifs shown correspond to Fig. 2. Additionally 17

shown: N-terminus covering the active site (pink line). 18

C) C-terminal domain of N. suaveolens 3D model with mutated amino acids and motifs. 19

Coloring as in Fig. 3B. 20

21

22



1

1

2

Figure 5. Active site of the cineole synthase of Nicotiana forgetiana 3

Amino acid side chains are shown in grey sticks. The back of the reactive intermediate 4

α-terpinyl cation (cyan) is located on a hydrophobic patch (grey mesh), while the tertiary 5

carbocation in C7 (green-cyan) exhibits a cation-pi stack upon the indole ring of W253. The 6

C3 atom, where the prenyl moiety has been cleaved off in the last ionization step is still in 7

spatial proximity of the diphosphate (red-orange). A water molecule (red-white) is fixed by 8

H502 and T278 and ready to perform a nucleophilic attack on the cationic C7 of the α-terpinyl 9

cation. 10

11



1

1

2

Figure 6: Proposed mechanism for the formation of 1,8-cineole 3

Depending on the stereochemistry of the intermediate the protonation of the double bond of 4

the α-terpinyl cation is provided by a proton relay via the hydroxyl groups of either Tyr496 or 5

Thr278. 6



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http://search.crossref.org/?page=1&rows=2&q=Benkert P, Tosatto SC, Schomburg D (2008) QMEAN: A comprehensive scoring function for model quality assessment. Proteins 71: 261-277

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http://scholar.google.com/scholar?as_q=QMEAN: A comprehensive scoring function for model quality assessment&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

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http://www.ncbi.nlm.nih.gov/pubmed?term=Bohlmann J%2C Keeling CI %282008%29 Terpenoid biomaterials%2E Plant J 54%3A656%2D669&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Bohlmann J, Keeling CI (2008) Terpenoid biomaterials. Plant J 54:656-669

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Bohlmann&hl=en&c2coff=1

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http://www.ncbi.nlm.nih.gov/pubmed?term=Case DA et al%2E %282013%29 AmberTools 13%2E University of California%2C San Francisco&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Case DA et al. (2013) AmberTools 13. University of California, San Francisco

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Peters RJ, Croteau RB (2003) Alternative termination chemistries utilized by monoterpene cyclases: chimeric analysis of bornyldiphosphate, 1,8-cineole and sabinene synthases. Arch Biochem Biophys 417: 203-211


Raguso RA, Schlumpberger BO, Kaczorowski RL, Holtsford TP (2006) Phylogenetic fragrance patterns in Nicotiana sections Alataeand Suaveolentes. Phytochem 67: 1931-1942


Rajaonarivony JI, Gershenzon J, Miyazaki J, Croteau R (1992) Evidence for an essential histidine residue in 4S-limonene synthaseand other terpene cyclases. Arch Biochem Biophys 299: 77-82


Roeder S, Hartmann AM, Effmert U, Piechulla B (2007) Regulation of simultaneous synthesis of floral scent terpenoids by the 1,8-cineole synthase of Nicotiana suaveolens. Plant Mol Biol 65: 107-124


Shimada T, Endo T, Fujii H, Hara M, Omura M (2005) Isolation and characterization of (E)-beta-Ocimene and 1,8-cineole synthasesin Citrus unshiu Marc. Plant Sci 168: 987-995


Sippl MJ (1990) Calculation of conformational ensembles from potentials of mean force. An approach to the knowledge-basedprediction of local structures in globular proteins. J Mol Biol 213: 859-883


Srividya N, Davis EM, Croteau RB, Lange BM (2015) Functional analysis of (4S)-limonene synthase mutants reveals determinantsof catalytic outcome in a model monoterpene synthase. Proc Natl Acad Sci USA 112: 3332-3337


Starks CM, Back K, Chappell J, Noel JP (1997) Structural basis for cyclic terpene biosynthesis by tobacco 5-epi-aristolochenesynthase. Science 277: 1815-1820


Stewart JJP (1990) MOPAC: a semi-empirical molecular orbital program. J Comput Aided Mol Des 4: 1-105Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Stewart JJP (2013) Optimization of parameters for semi-empirical methods VI: more modifications to the NDDO approximations andre-optimization of parameters. J Mol Model 19: 1-32


Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignmentthrough sequence weighting, position-specific gap penalties and weight matrix choise. Nucl Acids Res 22: 4673-4680


Turner G, Gershenzon J, Nielson EE, Froehlich FE, Croteau R (1999) Limonene synthase, the enzyme responsible formonoterpene biosynthesis in peppermint, is localized in leucoplasts of oil gland secretory cells. Plant Physiol 120: 879-886


Wang J, Wolf RM, Caldwell JW, Kollman PA, Case DA (2004) Development and testing of a general amber force field. J ComputChem 25: 1157-1174


Williams D, McGarvey DJ, Kathira EJ, Croteau R (1998) Truncation of limonene synthase preprotein provides a fully active www.plantphysiol.orgon July 13, 2020 - Published by Downloaded from Copyright © 2016 American Society of Plant Biologists. All rights reserved.

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http://search.crossref.org/?page=1&rows=2&q=Raguso RA, Schlumpberger BO, Kaczorowski RL, Holtsford TP (2006) Phylogenetic fragrance patterns in Nicotiana sections Alatae and Suaveolentes. Phytochem 67: 1931-1942

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http://search.crossref.org/?page=1&rows=2&q=Rajaonarivony JI, Gershenzon J, Miyazaki J, Croteau R (1992) Evidence for an essential histidine residue in 4S-limonene synthase and other terpene cyclases. Arch Biochem Biophys 299: 77-82

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http://search.crossref.org/?page=1&rows=2&q=Roeder S, Hartmann AM, Effmert U, Piechulla B (2007) Regulation of simultaneous synthesis of floral scent terpenoids by the 1,8-cineole synthase of Nicotiana suaveolens. Plant Mol Biol 65: 107-124

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http://www.ncbi.nlm.nih.gov/pubmed?term=Shimada T%2C Endo T%2C Fujii H%2C Hara M%2C Omura M %282005%29 Isolation and characterization of %28E%29%2Dbeta%2DOcimene and 1%2C8%2Dcineole synthases in Citrus unshiu Marc%2E Plant Sci 168%3A 987%2D995&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Shimada T, Endo T, Fujii H, Hara M, Omura M (2005) Isolation and characterization of (E)-beta-Ocimene and 1,8-cineole synthases in Citrus unshiu Marc. Plant Sci 168: 987-995

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http://search.crossref.org/?page=1&rows=2&q=Stewart JJP (1990) MOPAC: a semi-empirical molecular orbital program. J Comput Aided Mol Des 4: 1-105

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http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Turner&hl=en&c2coff=1

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http://www.ncbi.nlm.nih.gov/pubmed?term=Wang J%2C Wolf RM%2C Caldwell JW%2C Kollman PA%2C Case DA %282004%29 Development and testing of a general amber force field%2E J Comput Chem 25%3A 1157%2D1174&dopt=abstract

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'pseudomature' form of this monoterpene cyclase and reveals the function of the amino-terminal. Biochem 37: 12213-12220Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Wise ML, Savage TJ, Katahira E, Croteau R (1998) Monoterpene synthases from common sage (Salvia officinalis). cDNA isolation,characterization, and functional expression of (+)-sabinene synthase, 1,8-cineole synthase, and (+)-bornyl diphosphate synthase. JBiol Chem 273: 14891-14899


Wise ML, Urbansky M, Helms GL, Coates RM, Croteau R (2002) Syn stereochemistry of cyclic ether formation in 1,8-cineolebiosynthesis catalyzed by recombinant synthase from Salvia officinalis. J Am Chem Soc 124: 8546-8547


Yahyaa M, Matsuba Y, Brandt W, Doron-Faigenboim A, Bar E, McClain A, Davidovich-Rikanati R, Lewinsohn E, Pichersky E, Ibdah M(2015) Identification, functional characterization, and evolution of terpene synthases from a basal dicot. Plant Physiol 169:1683-97


Zhang Q, Tiefenbacher K (2015) Terpene cyclization catalysed inside a self-assembled cavity. Nature Chemistry 7: 197-201Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Zhuang X, Köllner TG, Zhao N, Li G, Jiang Y, Zhu L, Ma J, Degenhardt J, Chen F (2012) Dynamic evolution of herbivore-inducedsesquiterpene biosynthesis in Sorghum and related grass crops. Plant J 69: 70-80



http://www.ncbi.nlm.nih.gov/pubmed?term=Williams D%2C McGarvey DJ%2C Kathira EJ%2C Croteau R %281998%29 Truncation of limonene synthase preprotein provides a fully active %27pseudomature%27 form of this monoterpene cyclase and reveals the function of the amino%2Dterminal%2E Biochem 37%3A 12213%2D12220&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Williams D, McGarvey DJ, Kathira EJ, Croteau R (1998) Truncation of limonene synthase preprotein provides a fully active 'pseudomature' form of this monoterpene cyclase and reveals the function of the amino-terminal. Biochem 37: 12213-12220

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Williams&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Truncation of limonene synthase preprotein provides a fully active 'pseudomature' form of this monoterpene cyclase and reveals the function of the amino-terminal&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Truncation of limonene synthase preprotein provides a fully active 'pseudomature' form of this monoterpene cyclase and reveals the function of the amino-terminal&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Williams&as_ylo=1998&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Wise ML%2C Savage TJ%2C Katahira E%2C Croteau R %281998%29 Monoterpene synthases from common sage %28Salvia officinalis%29%2E cDNA isolation%2C characterization%2C and functional expression of %28%2B%29%2Dsabinene synthase%2C 1%2C8%2Dcineole synthase%2C and %28%2B%29%2Dbornyl diphosphate synthase%2E J Biol Chem 273%3A 14891%2D14899&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Wise ML, Savage TJ, Katahira E, Croteau R (1998) Monoterpene synthases from common sage (Salvia officinalis). cDNA isolation, characterization, and functional expression of (+)-sabinene synthase, 1,8-cineole synthase, and (+)-bornyl diphosphate synthase. J Biol Chem 273: 14891-14899

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Wise&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Monoterpene synthases from common sage (Salvia officinalis)&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Monoterpene synthases from common sage (Salvia officinalis)&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Wise&as_ylo=1998&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Wise ML%2C Urbansky M%2C Helms GL%2C Coates RM%2C Croteau R %282002%29 Syn stereochemistry of cyclic ether formation in 1%2C8%2Dcineole biosynthesis catalyzed by recombinant synthase from Salvia officinalis%2E J Am Chem Soc 124%3A 8546%2D8547&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Wise ML, Urbansky M, Helms GL, Coates RM, Croteau R (2002) Syn stereochemistry of cyclic ether formation in 1,8-cineole biosynthesis catalyzed by recombinant synthase from Salvia officinalis. J Am Chem Soc 124: 8546-8547

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Wise&hl=en&c2coff=1


http://scholar.google.com/scholar?as_q=&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Wise&as_ylo=2002&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Yahyaa M%2C Matsuba Y%2C Brandt W%2C Doron%2DFaigenboim A%2C Bar E%2C McClain A%2C Davidovich%2DRikanati R%2C Lewinsohn E%2C Pichersky E%2C Ibdah M %282015%29 Identification%2C functional characterization%2C and evolution of terpene synthases from a basal dicot%2E Plant Physiol 169%3A1683%2D97&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Yahyaa M, Matsuba Y, Brandt W, Doron-Faigenboim A, Bar E, McClain A, Davidovich-Rikanati R, Lewinsohn E, Pichersky E, Ibdah M (2015) Identification, functional characterization, and evolution of terpene synthases from a basal dicot. Plant Physiol 169:1683-97

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Yahyaa&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Identification, functional characterization, and evolution of terpene synthases from a basal dicot&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Identification, functional characterization, and evolution of terpene synthases from a basal dicot&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Yahyaa&as_ylo=2015&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Zhang Q%2C Tiefenbacher K %282015%29 Terpene cyclization catalysed inside a self%2Dassembled cavity%2E Nature Chemistry 7%3A 197%2D201&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Zhang Q, Tiefenbacher K (2015) Terpene cyclization catalysed inside a self-assembled cavity. Nature Chemistry 7: 197-201

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Zhang&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Terpene cyclization catalysed inside a self-assembled cavity&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Terpene cyclization catalysed inside a self-assembled cavity&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Zhang&as_ylo=2015&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Zhuang X%2C K�llner TG%2C Zhao N%2C Li G%2C Jiang Y%2C Zhu L%2C Ma J%2C Degenhardt J%2C Chen F %282012%29 Dynamic evolution of herbivore%2Dinduced sesquiterpene biosynthesis in Sorghum and related grass crops%2E Plant J 69%3A 70%2D80&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Zhuang X, K�llner TG, Zhao N, Li G, Jiang Y, Zhu L, Ma J, Degenhardt J, Chen F (2012) Dynamic evolution of herbivore-induced sesquiterpene biosynthesis in Sorghum and related grass crops. Plant J 69: 70-80

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Zhuang&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Dynamic evolution of herbivore-induced sesquiterpene biosynthesis in Sorghum and related grass crops&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Dynamic evolution of herbivore-induced sesquiterpene biosynthesis in Sorghum and related grass crops&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Zhuang&as_ylo=2012&as_allsubj=all&hl=en&c2coff=1


The α-terpineol to 1,8-cineole cyclization reaction of tobacco … › ... › 2016 › 10 › 11...

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