A Case of Convergent Evolution: The Bacterial Sesquiterpene Synthase for 1‐epi‐Cubenol from Nonomuraea coxensis

A terpene synthase from Nonomuraea coxensis was identified as (+)‐1‐epi‐cubenol synthase. The enzyme is phylogenetically unrelated to the known enzyme of the same function that is widespread in streptomycetes. Isotopic labelling experiments were performed to unambiguously assign the NMR data and to investigate hydrogen migrations during terpene cyclisations. Epoxidations of (+)‐1‐epi‐cubenol and of the plant derived compounds (−)‐cubenol and (−)‐1‐epi‐cubenol confirmed the structure of a natural product isolated from the brown alga Dictyopteris divaricata and allowed to conclude on its absolute configuration. The crystal structures of the epoxides from (+)‐ and (−)‐1‐epi‐cubenol and the acid catalysed conversion into an isomeric ketone are reported.


Introduction
(À )-Cubenol ( 1) and (À )-1-epi-cubenol (2, Figure 1) were both first isolated from cubeb oil (Piper cubeba). [1]Both compounds were later reisolated from various plants including Cedrela toona, [2] Cinnamomum camphora, [3] Pilgerodendron uvifera, [4] Juniperus oxycedrus, [5] and Cryptomeria japonica, [6] and brown algae of the genus Dictyopteris. [7,8]Cubenol was also found in the dinoflagellate Gymnodinium nagasakiense. [9]The same study reported a cell destroying activity of (À )-1 isolated from Dictyopteris divaricata against the producer and several other planktonic species, but it is not clearly mentioned which enantiomer is produced by G. nagasakiense. [9]hortly after the first description from plants the enantiomer of (+)-2 was discovered from Streptomyces (this report erroneously shows the structure of (+)-1), [10] giving another example for the finding that sesquiterpenes from plants and bacteria often show opposite absolute configurations. [11]The terpene synthase for (+)-2 has been isolated from Streptomyces cell lysates, [12] followed by cloning of the gene from Streptomyces griseus and expression of the recombinant enzyme. [13]Epicubenol synthase is a widespread enzyme in the genus Streptomyces, [14] and accordingly, compound 2 is frequently observed in this genus. [15]The liverwort Scapania undulata is another source of (+)-2, [16] and also the plant Solidago canadensis is a producer of (+)-2. [17]Only one report mentions the occurrence of (+)-1 in the scale insect Ceroplastes ceriferus. [18]he structures for 1 and 2 were originally resolved by chemical correlations that indicated their epimeric nature with respect to the configuration at C1.The epimers were then distinguished based on dehydrations with thionyl chloride in pyridine, which yielded three elimination products 3-5 from 1, but only one (3) from 2 (Scheme 1A). [1]Because this reaction proceeds through a trans elimination, [19] the structures of 1 for cubenol and 2 for epi-cubenol were assigned. [1]he structure of 1 was later confirmed from the conversion of the mixture of epoxides 6 (major compound) and 7 (minor compound) with Li in EtNH 2 , yielding the major products Tcadinol (8) and T-muurolol (9) besides minor amounts of a material identical to cubenol (Scheme 1B). [4]This led to the conclusion that the minor product obtained from 7 should represent the structure of 1, confirming the assigned structure of cubenol. [20]e Groot and coworkers have reported on the conversion of bicyclogermacrane-1,8-dione (10) into two keto alcohols 11 and 12 by flash vacuum pyrolysis. [21]These workers noted that it was not possible to assign the configurations at C1 through NMR spectroscopy (NOE and analysis of coupling constants).Indirect conclusions were drawn from the smooth epimerisation at C4 of 12 upon treatment with NaOMe in MeOH, while 11 did not react.Based on these grounds it was concluded that all alkyl substituents in 11 are in equatorial positions in a transfused ring system.Furthermore, the ring junction of 12 was suggested to be cis, with an axially oriented Me group at C4 that could easily be epimerised.Compound 11 was then further converted into 1, which supported the original structural assignment. [21]ere we report on a bacterial (+)-1-epi-cubenol synthase from Nonomuraea coxensis that is phylogenetically unrelated to the reported enzyme from S. griseus, [13] and investigations on the absolute configuration and cyclisation mechanism of the product (+)-2 through isotopic labelling experiments.Investigations on the chemistry of cubenol and epi-cubenol from different sources resulted in the isolation of oxidation products, including an epoxide that is known from the brown alga Dictyopteris divaricata.

Results and Discussion
Despite the fact that many bacterial terpene synthases have been characterised during the past years, a phylogenetic tree constructed from the amino acid sequences of 4018 bacterial terpene synthase homologs obtained through BLAST searches using the amino acid sequences of all characterised bacterial terpene synthases as query sequences reveals still many mono-phyletic branches of enzymes with unknown functions (Figure S1).For this study, a terpene synthase homolog from Nonomuraea coxensis residing in an uncharacterised branch of the tree was selected for investigation (accession number WP_ 157383393, gene locus tag Nocox_30630).The amino acid sequence of this enzyme showed all highly conserved residues that are required for enzyme functionality, including the aspartate-rich motif ( 91 DDAFCE), the NSE triad ( 232 NDLLSYAKE), the pyrophosphate sensor (Arg186) and the 318 RY pair (Figure S2).The closest characterised enzyme is the 4-epi-cubebol synthase from S. roseum with an amino acid sequence identity of 48 %. [22] Gene cloning, heterologous expression, and incubation of the purified terpene synthase homolog from N. coxensis (Figure S3) with oligoprenyl diphosphates resulted in the efficient conversion of FPP into a single product, while GPP, GGPP, and GFPP were not accepted by the enzyme (Figure S4).A preparative scale incubation of FPP (50 mg, 115 μmol) gave a high yield of a sesquiterpene alcohol (20 mg, 81 μmol, 70 %) whose structure elucidation by NMR spectroscopy (Table S1, Figures S5-S12) revealed the planar structure of cubenol.The relative configuration was assigned from the observed NOESY correlations that were well explained from an energy minimised structure using the MM2 function of Chem3D (Figure 2).In one case the literature reports different 13 C-NMR data for 2, [5] suggesting that these data may have been recorded for another cubenol stereoisomer, while all other reports are consistent, [13,16,23] but may require some reassignments (Table S2).The absolute configuration of (1R,6S,7R,10S)-2 was evident from its positive optical rotation ([α] D 25 = + 87.0, c 0.17, EtOAc) that is opposite to the optical rotation of 2 from cubeb oil ([α] D 26 = À 95.7, c 0.86, CHCl 3 ). [1]Taken together, the characterised enzyme was identified as Nonomuraea coxensis (+)-1-Epi-Cubenol Synthase (NcECS).Despite the fact that NcECS and the (+)-1-epi-cubenol synthase from S. griseus (GecA) [13] are both highly selective in the formation of (+)-2 from FPP, the two enzymes show an amino acid sequence identity of only 29 %, and the highly conserved motifs are different (for GecA the aspartate-rich motif is 81 DDQLDD and the NSE triad reads 226 NDVYSFEKE).
None of the previous papers reporting the NMR data of 2 has made a full assignment of all hydrogen signals.Such an assignment is particularly hindered for C3, because the hydro-  gens connected to this carbon showed no conclusive NOESY correlations (Figure 2).Stereoselective deuteration experiments can resolve this situation and can further secure the assignments for other pairs of diastereotopic hydrogens.Specifically, the labelled terpene monomers (R)-and (S)-(1-13 C,1-2 H)IPP [24] can be converted with DMAPP, Escherichia coli isopentenyl diphosphate isomerase (IDI) [25] and FPP synthase (FPPS) from Streptomyces coelicolor [26] into stereoselectively deuterated FPP isotopomers with a well known stereochemical course. [27]onversion into 2 with NcECS and product analysis by HSQC spectroscopy allowed for an assignment of the hydrogens at C8 (from C9 of FPP), while the hydrogens at C2 (from C5 of FPP) are isochronic (Scheme 2A, Figure S13).Corresponding experiments with (E)-and (Z)-(1-13 C,1-2 H)IPP [28] revealed the hydrogen signals for C3 and C9 (from C4 and C8 of FPP) (Scheme 2B, Figure S14).
The findings regarding the specific 1,3-hydride shift of the 1-pro-S hydrogen to C11 are in line with earlier observations made for a series of sesquiterpene synthases whose cyclisation cascades proceed through the intermediates A and B. [31] The formation of (S)-A requires (R)-NPP as a precursor, because the 1,10-ring closure is an anti-S N 2' reaction, while the formation of (R)-NPP is only possible by syn-allylic transposition of diphosphate, with the shown stereochemical fate for the 1-pro-R and 1-pro-S hydrogens.After cyclisation to A the 1-pro-S hydrogen will be closer to the cation at C11 than the 1-pro-R hydrogen, and consequently the 1-pro-S hydrogen migrates towards C11.For the opposite enantiomer of (À )-2 as it occurs in plants the migration of the 1-pro-R hydrogen can be expected.This mechanistic view was further supported by incubation of (R)and (S)-NPP (synthesised as in reference [32]) with NcECS that resulted in the more efficient formation of (+)-2 from (R)-NPP in comparison to (S)-NPP (Figure S18).The partial conversion of (S)-NPP into (+)-2 can be explained by its enzyme catalysed isomerisation to FPP.After a conformational change another isomerisation can then lead to the on-pathway intermediate (R)-NPP. [33]fter cyclisation by a terpene synthase, terpenes are often further modified by oxidative chemistry.For instance, an epoxide derived from cubenol has been reported from the marine brown alga Dictyopteris divaricata, [34] but its absolute configuration remained unassigned.To investigate the structure of this compound, (À )-1 and (À )-2 were isolated from cubeb oil and both converted with m-CPBA into the epoxides (À )-13 and (À )-14 (Scheme 3).The NMR data of 13, but not of 14, matched those of the algal compound (Tables S4 and S5, Figures S19-S34).Also the optical rotation of [α] D 25 = À 4.5 (c 0.13, EtOAc) showed the same sign as determined for the algal compound ([α] D 21 = À 8.2 (c 0.13, CHCl 3 ), establishing its structure as that of (À )-13.Treatment of 14 with p-TsOH resulted in the epoxide rearrangement to the ketone 16 (Table S6, Figures S35-S42).Under the same conditions the rearrangement of 13 to 15, known as a synthetic compound, [21] proved to be impossible.Notably, the epoxide (À )-14 readily crystallised and single crystals were analysed by CuKα X-ray, and also the crystal structure of its enantiomer could be obtained through epoxidation of the NcECS product (+)-2 (Figure 3, Tables S7 and S8).

Conclusions
The enzyme NcECS for (+)-1-epi-cubenol represents the first characterised terpene synthase from the genus Nonomuraea, an actinomycete isolated from sandy soil from Cox's Bazar, Bangladesh. [35]The genus has only scarcely been investigated for its secondary metabolites, but is known for the production of glycopeptide antibiotics, [36,37] while no terpenes have been reported from Nonomuraea to date.Notably, NcECS is not related to the known (+)-1-epi-cubenol that is widespread in streptomycetes, [13,14] giving another example of convergent evolution in bacterial terpene biosynthesis besides the known case of the distantly related (+)-T-muurolol synthases from Streptomyces clavuligerus and Roseiflexus castenholzii. [38,39]Interestingly, both (+)-1-epi-cubenol synthases and (+)-T-muurolol synthases are also not related to each other (Figure S1), although the biosyntheses of 1-epi-cubenol and T-muurolol proceed through the same cationic intermediates, i. e. Tmuurolol is formed by attack of water to cation C (Scheme 2).These findings, together with the occurrence of the enantiomers of (À )-cubenol and (À )-1-epi-cubenol in plants, demonstrate that the cadinane skeleton of these sesquiterpene alcohols is a privileged structural motif in nature.Future research may give deeper insights into the question of how phylogenetically distinct enzymes with very low sequence identity can realise the same terpene skeletons.

Spectroscopy and optical rotation:
1 H NMR, and 13 C NMR spectra were recorded on a Bruker Avance I 500 MHz spectrometer and a Bruker Avance III HD 700 MHz Cryo spectrometer.Chemical shifts were referenced to the residual proton signal of C 6 D 6 (δ = 7.16 ppm) for 1 H NMR and the 13 C signal of C 6 D 6 (δ = 128.06)for 13 C NMR. [40] IR spectra were recorded on a Bruker α infrared spectrometer with a diamond ATR probe head.Peak intensities are given as s (strong), m (medium), w (weak), and br (broad).Optical rotations were recorded on a Modular Compact Polarimeter MCP 100 (Anton Paar, Graz, Austria).The optical rotation parameters are 1) temperature: 25 °C, 2) wavelength: 589 nm (the sodium D line), and 3) the path length: 10 cm.The compound concentrations c are given in g 100 mL À 1 .
Protein expression and purification: E. coli BL21 (DE3) was transformed with plasmid pYE-NcECS for protein expression.A preculture of the transformant was cultivated in LB medium with kanamycin (50 μg mL À 1 ).The preculture (20 mL) was incubated at 37 °C overnight with shaking at 160 rpm and then used to inoculate an expression culture (1 L) in LB medium with kanamycin.The culture was grown at 37 °C for ca. 4 h until an OD 600 of 0.4-0.6 was reached and then cooled to 18 °C.IPTG solution (400 mm, 1 mL) was added to induce protein expression.The expression was carried out at 18 °C with shaking at 160 rpm for 20 h.The culture was centrifuged (3,600×g, 40 min) to separate the cells from the medium.The supernatant was discarded and the cell pellet was resuspended in binding buffer (20 mL; 20 mm Na 2 HPO 4 , 500 mm NaCl, 20 mm imidazole, 1 mm MgCl 2 , pH = 7.4, 4 °C).Cell lysis was carried out through ultrasonication (6×1 min).The resulting suspension was centrifuged (14,610×g, 2×10 min) to remove the cell debris.The supernatant was filtered and transferred to a Ni 2 + -NTA affinity column (5 mL resin volume; Super Ni-NTA, Generon, Slough, UK).Undesired proteins were eluted with binding buffer (2×10 mL).The desired protein was eluted with elution buffer (10 mL; 20 mm Na 2 HPO 4 , 500 mm NaCl, 500 mm imidazole, 1 mm MgCl 2 , pH = 7.4, 4 °C).The protein purity was checked by SDS-PAGE and the concentration was determined by Bradford assay (yield: 10 mL protein preparation, 2.5 mg mL À 1 ). [44]all scale reactions with NcECS (substrate scope): Enzymatic test reactions were performed using freshly prepared NcECS and different substrates dissolved in 25 mm NH 4 HCO 3 (10 mg mL À 1 ).The enzyme solution obtained from a Ni 2 + -NTA affinity chromatography was diluted with incubation buffer (50 mm TRIS, 10 mm MgCl 2 , 20 % glycerol, pH 8.2, final enzyme concentration 0.4 mg mL À 1 ).To 1 mL of this enzyme solution 0.5 mg of each substrate (GFPP, GGPP, FPP, and GPP) was added.The reaction mixture was incubated at 30 °C overnight, followed by extraction with hexane for GC/MS analysis.The results are shown in Figure S3.
Incubation experiments with labelled substrates: Incubation experiments were performed with substrates (each ca.0.5 mg) and enzyme preparations as listed in Table S3.For enzyme preparations and incubation conditions the same protocol as mentioned above for small-scale reactions was used.If necessary, isopentenyl diphosphate isomerase (IDI) [24] and farnesyl pyrophosphate synthase (FPPS) [26] were added with a final concentration of 0.2 mg mL À 1 .After overnight incubation, the reaction mixtures were extracted three times with benzene and analysed by GC/MS.The extracts were then concentrated to dryness with a stream of Argon.C 6 D 6 (0.5 mL) was added to the dried extract and the samples were subjected to NMR analyses.
X-ray crystallography of (+)-14 and (À )-14: Compound (+)-14 or (À )-14 (each 3 mg) was dissolved in Et 2 O (500 μL) and the solution was stored at À 20 °C.After 3 weeks, crystals were obtained for X-ray analysis.The data collection was performed on a Bruker D8 Venture diffractometer ((À )-14) or on a STOE StadiVari diffractometer ((+)-14) using in both cases Cu-K α (λ = 1.54178Å) radiation.Both diffractometers were equipped with a low-low-temperature device (Cryostream 800 series, Oxford Cryosystems, 100(2) K).Intensities were measured by fineslicing φ-and ω-scans and corrected for background, polarisation, and Lorentz effects.Multi-scan absorption corrections following Blessing's method were applied for both data sets. [45]he structures were solved by intrinsing-phasing methods and refined anisotropically by the least-squares procedure implemented in the SHELX program system. [46]The hydrogen atoms were included isotropically using the riding model on the bound carbon atoms.The absolute configuration was determined by inspection of the Flack-parameter and by using Bayesian statistics on Bijvoet differences. [47]Deposition Numbers 2289117 (for (À )-14) and 2289118 (for (+)-14) contain the supplementary crystallographic data for this paper.These data are provided free of charge by the joint Cambridge Crystallographic Data Centre and Fachinformationszentrum Karlsruhe Access Structures service.

Scheme 1 .
Scheme 1. Previously reported chemical transformations for structure elucidation of 1 and 2. A) Dehydrations with SOCl 2 , B) reductive epoxide openings performed with the mixture of major 6 and minor 7, C) chemical correlation of 10 through 11 with 1.

Figure 2 .
Figure 2. Observed NOESY correlations for 2 and their structural interpretation using an energy minimised structure of 2 (MM2 function of Chem3D).

Scheme 2 .
Scheme 2. Cyclisation mechanism for (+)-2 and isotopic labelling experiments.A) The experiment with (R)-and (S)-(1-13 C,1-2 H)IPP allows to assign the NMR data for the hydrogens at C2 and C8 and demonstrates the 1,3hydride shift from A to B. B) The experiment with (E)-and (Z)-(4-13 C,4-2 H)IPP allows to assign the NMR data for the hydrogens at C3 and C9.C) The experiments with (7-13 C)GPP and (R)-or (S)-(1-13 C,1-2 H)IPP give additional support for the 1,3-hydride shift from A to B. D) The experiment with (3-13 C,2-2 H)GPP gives evidence for the 1,2-hydride shift from C to D. Coloured hydrogens are substituted by deuterium, 13 C-labelled carbons are indicated by black and purple dots.