Structural Basis of Catalysis in the Bacterial Monoterpene Synthases Linalool Synthase and 1,8-Cineole Synthase

Article abstract of DOI:10.1021/acscatal.7b01924

Terpenoids form the largest and stereochemically most diverse class of natural products, and there is considerable interest in producing these by biocatalysis with whole cells or purified enzymes, and by metabolic engineering. The monoterpenes are an impo

Full text of DOI:10.1021/acscatal.7b01924

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Article
Structural basis of catalysis in the bacterial monoterpene
synthases linalool synthase and 1,8-cineole synthase
Vijaykumar Karuppiah, Kara Ranaghan, Nicole G.H. Leferink, Linus O. Johannissen, Muralidharan
Shanmugam, Aisling Ni Cheallaigh, Nathan Bennett, Lewis Kearsey, Eriko Takano, John
Gardiner, Marc Van der Kamp, Sam Hay, Adrian J. Mulholland, David Leys, and Nigel S. Scrutton
ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.7b01924 • Publication Date (Web): 09 Aug 2017
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Structural basis of catalysis in the bacterial
monoterpene synthases linalool synthase and
1,8-cineole synthase
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Vijaykumar Karuppiah,† Kara E. Ranaghan,‡ Nicole G. H. Leferink,† Linus O.
Johannissen,† Muralidharan Shanmugam,† Aisling Ní Cheallaigh,† Nathan J. Bennett,†
Lewis J. Kearsey,† Eriko Takano,† John M. Gardiner,† Marc W. van der Kamp,‡ Sam
Hay,† Adrian J. Mulholland,‡ David Leys† and Nigel S. Scrutton*,
†
†BBSRC/EPSRC Manchester Synthetic Biology Research Centre for Fine and
Specialty Chemicals (SYNBIOCHEM), Manchester Institute of Biotechnology,
School of Chemistry, Faculty of Science and Engineering, University of Manchester,
131 Princess Street, Manchester M1 7DN, U.K.
‡Centre for Computational Chemistry, School of Chemistry, University of Bristol,
Cantock’s Close, Bristol BS8 1TS, U.K.
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ABSTRACT
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Terpenoids form the largest and stereochemically most diverse class of natural
products, and there is considerable interest in producing these by biocatalysis with
whole cells or purified enzymes, and by metabolic engineering. The monoterpenes are
an important class of terpenes and are industrially important as flavours and
fragrances. We report here structures for the recently discovered Streptomyces
clavuligerus monoterpene synthases linalool synthase (bLinS) and 1,8-cineole
synthase (bCinS) and show that these are active biocatalysts for monoterpene
production using biocatalysis and metabolic engineering platforms. In metabolically
engineered monoterpene-producing E. coli strains use of bLinS leads to 300-fold
higher linalool production compared with the corresponding plant monoterpene
synthase. With bCinS, 1,8-cineole is produced with 96% purity compared to 67%
from plant species. Structures of bLinS and bCinS, and their complexes with
fluorinated substrate analogues, show that these bacterial monoterpene synthases are
similar to previously characterised sesquiterpene synthases. Molecular dynamics
simulations suggest that these monoterpene synthases do not undergo large-
scale conformational changes during the reaction cycle making them attractive targets
for structured-based protein engineering to expand the catalytic scope of these
enzymes towards alternative monoterpene scaffolds. Comparison of the bLinS and
bCinS structures indicates how their active sites steer reactive carbocation
intermediates to the desired acyclic linalool (bLinS) or bicyclic 1,8-cineole (bCinS)
products. The work reported here provides the analysis of structures for this important
class of monoterpene synthase. This should now guide exploitation of the bacterial
enzymes as gateway biocatalysts for the production of other monoterpenes and
monoterpenoids.
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KEYWORDS: monoterpene synthase, monoterpenoids, sesquiterpene synthase,
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terpenes, protein crystallography, molecular dynamics simulations
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GRAPHICAL ABSTRACT
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INTRODUCTION
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Terpenoids are the most abundant and largest class (>75000) of natural products.
Most are commonly found in plants and their biological roles range from interspecies
communication to intracellular signaling and defense against predatory species1. Their
use is wide ranging as pharmaceuticals, herbicides, flavorings, fragrances and
biofuels2. Despite the commercial interest in terpenoids, efforts to produce these in
high yields have been hampered by lack of availability of sufficiently robust and high
activity terpene synthase enzymes, although efforts to synthesise terpenoids by
synthetic biology routes have gathered pace in the recent years3-8.
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Terpenoids are synthesised from the isoprene building blocks dimethylallyl
pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP). Combination of
DMAPP and IPP generates pyrophosphate substrates of varying carbon lengths,
which are then utilised by terpene cyclases to produce either monoterpenes (C10),
sesquiterpenes (C15), diterpenes (C20) and others. Geranyl pyrophosphate (GPP), the
substrate used by monoterpene synthases is formed by coupling one molecule of
DMAPP with IPP, while farnesyl pyrophosphate (FPP), the substrate for
sesquiterpenes, is synthesised by coupling three individual isoprene precursors9.
The class I terpene synthases share a common α-helical fold and use a cluster of three
Mg2+ ionsto assist with substrate ionisation and release of the pyrophosphate moiety
(PPi). This generates a reactive allylic carbocation and triggers a cyclisation cascade
that likely involves multiple carbocation intermediates10. In many cases, substrate and
Mg2+ binding lead to a closed active site conformation, which guides substrate
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orientation and protects the carbocation intermediates from premature quenching11.
The exact architecture and mobility of the active site is thought to control the
cyclisation cascade to the final carbocation intermediate with high fidelity. The latter
is usually subject to deprotonation or addition of a water molecule, leading to
formation of a single product. However, some natural terpene synthases and
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engineered variant forms have been shown to form multiple reaction products12-13
.
To date, available crystallographic structures for the monoterpene cyclases/synthases
(mTC/S) that accept GPP as the substrate has been derived only for plant enzymes.
Structures have been reported for bornyl diphosphate synthase (Salvia officinalis)14,
limonene synthase (Mentha spicata15 and Citrus sinensis)16-17, 1,8-cineole synthase
(Salvia fruticosa)18 and γ-terpinene synthase (Thymus vulgaris)19. Without exception,
plant mTC/S contain two domains: a C-terminal α-helical catalytic domain that
belongs to the class I terpenoid fold, and a N-terminal α-barrel domain with unclear
function and appears to be relictual. Though the overall sequence conservation is low,
the structure of the α-helical fold is highly conserved. The active site has two
conserved regions, the aspartate rich (DDXX(X)(D,E)) motif and the NSE
(NDXXSXX(R,K)(E,D)) triad, required for binding three catalytically essential Mg2+
ions. Structures of bornyl diphosphate synthase and limonene synthases have been
solved in complex with substrate analogues. In each case, GPP-analogues bind with
their pyrophosphate moieties coordinated by the Mg2+ ions and a network of residues
that are proposed to assist with catalysis.
Recent reports have shown that terpene synthases are also widely distributed in
bacteria, but the majority of these accept FPP as substrate and produce
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sesquiterpenes20-21. Ohnishi and coworkers characterised two bacterial mTC/S from
Streptomyces calvuligerus, namely 1,8-cineole synthase22 and linalool/nerolidol
synthase, which can accept either GPP and FPP as substrate, leading to linalool and
nerolidol products, respectively23. Heterologous expression of these enzymes in
Streptomyces avermitilis resulted in 1,8-cineole synthase (bCinS) producing 1,8-
cineole, and linalool/nerolidol synthase (bLinS) producing only linalool indicating
that bLinS is likely to function only as a mTC/S in this host20. The sequences of both
bCinS and bLinS reveal they comprise ~330 amino acids in a single catalytic domain
and lack the additional N-terminal α-barrel domain characteristic of plant enzymes.
Surprisingly, no closely related homologs of both enzymes have been found in other
bacteria24. The bacterial mTC/S 2-methylisoborneol synthase is present in many
bacteria. It accepts 2-methyl-GPP as substrate to produce 2-methylisoborneol. Unlike
the bacterial mTC/S reported here, 2-methylisoborneol synthase has a considerably
longer amino acid sequence (~400-500) and crystal structures have revealed a N-
terminal proline-rich domain that is disordered along with a class I terpenoid fold C-
terminal domain25.
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Linalool is mainly used as a fragrance material in 60-80% of perfumed hygiene
products. It is widely used in cosmetic products like perfumes, lotions, soaps and
shampoos and also in non-cosmetic products like detergents and cleaning agents.
Furthermore, during the manufacturing process of Vitamin E, linalool is a vital
intermediate. As an important ingredient in a wide range of commercial products, the
worldwide use of linalool exceeds 1000 metric tonnes per annum26. Both R and S
isomers of linalool are found in nature with R-(–)-linalool being the most widely
distributed in plant and flower extracts. To our knowledge, for industrial use as a
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fragrance, the isomeric mixture is used. 1,8-cineole (also called eucalyptol) is used as
a flavoring in food products, in cosmetics and also has medicinal properties27.
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This study integrates synthetic biology with biocatalysis and analysis of enzyme
structures and mechanisms. Here we describe high-resolution crystal structures of
bLinS and bCinS from Streptomyces calvuligerus, and complexes with fluorinated
substrate analogues. These structures define the active site architectures required to
steer reactive carbocation intermediates to the desired product outcomes. Expression
of bLinS and bCinS in E. coli monoterpene producing strains leads to improved
production of linalool and 1,8-cineole compared with plant monoterpene synthases
and the structures help to both rationalise product outcomes and guide future
exploitation of these enzymes in monoterpene/monoterpenoid production.
EXPERIMENTAL SECTION
Expression and Purification of bCinS and bLinS
The full-length genes coding for 1,8-Cineole synthase (bCinS; WP_003952918) and
Linalool synthase (bLinS; WP_0003957954) from Streptomyces clavuligerus ATCC
27064 were codon optimised and synthesised from GeneArt (Life Technologies). The
genes were amplified using PCR and sub-cloned into pETM11 vector digested with
NcoI and XhoI using Infusion cloning (Clontech). The final construct coded for either
1,8-Cineole synthase (bCinS) or Linalool synthase (bLinS) with a 6X-His tag
followed by a TEV protease cleavage site at the N-terminus. The expression and
purification method explained below was identical for both the proteins. The plasmid
was transformed into E. coli ArcticExpress (DE3) cells (Agilent) and a few colonies
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were inoculated into 100 ml 2X-YT media containing 40 μg/ml of kanamycin and 20
μg/ml of gentamycin and grown for 3-4 h at 37 °C. The culture was diluted into 3
litres of fresh 2X-YT media containing 40 μg/ml of kanamycin and allowed to grow
at 37 °C until the OD at 600 nm reached 0.6-0.8. At this stage, the temperature was
reduced to 16°C and 0.1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) was
added and incubated for 14-18 h. The cells were harvested by centrifugation at 6000 g
for 10 min and the pellet was resuspended in buffer A (25 mM Tris pH 8.0, 150 mM
NaCl, 1 mM DTT, 4 mM MgCl2 and 5% (v/v) glycerol). The cells were lysed by
sonication and the debris was removed by centrifugation at 30,000 g for 30 min. The
supernatant was filtered through a 0.2 μm filter and loaded onto a 5 ml HisTrap
column (GE Healthcare) pre-equilibrated with buffer A. The column was washed with
buffer A containing 10 mM imidazole (pH 8.0) and increasing up to 40 mM imidazole
by step gradients with 3 column volume for each concentration. Increasing the
concentration of imidazole to 200-500 mM eluted the protein. The purified protein
was desalted using a Centripure P100 column (emp Biotech GmbH) equilibrated with
buffer A. To remove the His tag, TEV protease was added (1:1000 (w/w)) to the
protein and incubated at 4 °C with gentle mixing for 24 h. The TEV protease was
removed by passing the protein mixture through a 5 ml HisTrap column and the flow
through was collected. The His-tag removed protein was concentrated and loaded
onto a Hiload Superdex (26/60) S75 column (GE Healthcare) pre-equilibrated with
buffer A. Pure fractions from the gel filtration column were concentrated to 13-15
mg/ml and stored at -80 °C as aliquots. Samples for EPR experiments were prepared
as explained above except buffer A was lacking MgCl2.
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Biotransformations
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Biotransformation reactions (0.25 ml) were prepared using buffer A and setup in glass
vials containing 2 mM GPP and 20 μM of bCinS or bLinS. The vials were incubated
at 25°C with gentle shaking for 16 h. The vials were cooled to 4°C and 0.25 ml of
ethyl acetate containing 0.01% (v/v) sec-butyl benzene as internal standard was
added. The samples were vortexed for 2 min and then spun at 18,000 g for 5 min.
Supernatant fractions containing the ethyl acetate layer were removed and dried over
anhydrous magnesium sulfate. Samples were analysed by GC-MS.
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Monoterpenoid Production in E.coli
Both bLinS and bCinS genes, including RBS, were amplified from their respective
pETM-11 expression vectors using primers pET_IF_Fw (5’-CAT CCC CAC TAC
TGA GAA TC-3’) and pET_IF_Rv (5’- GGT GGT GGT GCT CGA GTT A-3’) and
cloned using InFusion (Takara) into plasmid pGPPSmTC/S15 (Table S1), which was
PCR linearised using the primer pair Vector_IF_Fw (5’-TAA CTC GAG CAC CAC
CAC CAC C-3’) and Vector_IF_Rv (5’-TCA GTA GTG GGG ATG TCG TAA
TCG-3’) resulting in plasmids pGPPSmTC/S38 and pGPPSmTC/S39, respectively
(Table S1). Correct insertion was confirmed by automated sequencing (Eurofins).
For monoterpenoid production, the pGPPSmTC/S plasmids were co-transformed with
pMVA into E. coli DH5α and grown as described before3. Briefly, expression strains
were inoculated in terrific broth (TB) supplemented with 0.4% glucose in glass screw
capped vials, and induced for 72 h at 30°C with 50 μM IPTG and 25 nM anhydro-
tetracycline. A 20% n-nonane layer was added to capture the volatile terpenoids
products. After induction, the nonane overlay was collected, dried over anhydrous
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MgSO4 and mixed at a 1:1 ratio with ethyl acetate containing 0.1% (v/v) sec-butyl
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benzene as internal standard.
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GC-MS Analysis
Samples were injected onto an Agilent Technologies 7890B GC equipped with an
Agilent Technologies 5977A MSD. The products were separated on a DB-WAX
column (30 m x 0.32 mm i.d., 0.25 μm film thickness, Agilent Technologies). The
injector temperature was set at 240°C with a split ratio of 20:1 (1 μl injection). The
carrier gas was helium with a flow rate of 1 ml/min and a pressure of 5.1 psi. The
following oven program was used: 50 °C (1 min hold), ramp to 68 °C at 5 °C/min (2
min hold), and ramp to 230 °C at 25 °C/min (2 min hold). The ion source temperature
of the mass spectrometer (MS) was set to 230 °C and spectra were recorded from m/z
50 to m/z 250. Compound identification was carried out using authentic standards and
comparison to reference spectra in the NIST library of MS spectra and fragmentation
patterns as described previously3.
GC Analysis
To determine the chirality of linalool and nerolidol produced by bLinS, samples were
analysed by gas chromatography on an Agilent Technologies 7890A GC system
equipped with an FID detector, a 7693 autosampler and a CP-Chirasil-DEX-CB
column (25 m x 0.25 mm i.d., 0.25 μm film thickness). The biotransformation
samples and isomers of linalool and nerolidol standards were analysed using GC. In
this method, the injector temperature was at 180°C and 1 μl of sample was injected
split-less. The carrier gas was helium with a flow rate of 1 ml/min and a pressure of
11.3 psi. For nerolidol containing samples, the program began at a temperature of
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70°C and then increased to 150 °C at 8°C/min (2 min hold). This was followed by an
increase in temperature to 190 °C at 10 °C/min (3 min hold). For linalool containing
samples, the program began at a temperature of 70 °C which was then increased to 90
°C at 8°C/min. This was followed by an increase in temperature to 150°C at a rate of
2 °C/min and then to 190 °C at 40 °C/minute (1 min hold). The FID detector was
maintained at a temperature of 200°C with a flow of hydrogen at 30 ml/min.
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Chemical Synthesis of Fluorinated Substrate Analogues
Unless otherwise stated, all reactions were carried out in oven-dried glassware.
Reactions were monitored by Thin Layer Chromatography (TLC) on silica gel 60 F254
plates, visualised with phosphomolybdic acid stain (10 g Phosphomolybdic acid in
100 ml ethanol). Column chromatography was performed on Merck silica Gel 60
1
(particle size 40-63µm). H NMR, 13C NMR, 31P and 19F spectra were obtained
using a combination of 400 MHz and 500 MHz spectrometers and are reported as
chemical shift on the parts per million scale. Multiplicity abbreviated (br = broad, s =
singlet, d = doublet, dd = double doublet, t = triplet, m = multiplet, etc.) and coupling
constants were obtained in Hertz. Assignments were aided by COSY and HSQC. All
mass spectrometry results are reported as the mass to charge ratio and are reported
with % abundance against the base peak (100 %).
Synthesis of 2-fluorogeraniol and 2-fluoronerol: Sodium hydride (538 mg, 60%
dispersion, 13.5 mmol) was washed with petroleum ether and suspended in THF (40
ml). The suspension was cooled to 0 °C and a solution of the ethyl
(diethoxyphosphoryl) fluoroacetate (2.48 ml, 12.2 mmol) in THF (13.4 ml) was added
dropwise over 10 min. The reaction was stirred for 30 min before adding 6-methyl-5-
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hepten-2-one (1.5 ml, 10.2 mmol) dropwise over 30 min. The reaction was stirred
overnight at room temperature. The reaction was cooled back to 0 °C and quenched
by pouring on to ice water. The product was extracted with diethyl ether (3 x 30 ml),
dried over MgSO4 and then reduced to dryness. The crude product was then dissolved
in THF (64 ml), cooled to 0 °C and LiAlH4 (541 mg, 14.3 mmol) was added. The
reaction was stirred at room temperature for 3 hours then quenched with the addition
of saturated aqueous NH4Cl. The solution was extracted with diethyl ether (3 x 30 ml)
and the subsequent combined organic phases were washed with brine (30 ml). The
product was purified by column chromatography (hexane/diethyl ether, 95/5, v/v) to
give 2-fluorogeraniol (783 mg, 41%) and 2-fluoronerol (856 mg, 45%) with a total
yield of 86%25, 28 (Scheme S1). 2-fluorogeraniol: 1H NMR (400 MHz, CDCl3) δ 5.17
– 5.05 (m, 1H, H7), 4.24 (dd, J = 22.3, 3.2 Hz, 2H, H1), 2.10 (m, 4H, H5, H6), 1.68
(s, 3H, H4), 1.67 (d, J = 2.9 Hz, 3H, H9/10), 1.61 (s, 3H, H9/10). 13C NMR (101
MHz, CDCl3) δ 132.2, 123.6, 116.1 (d, J = 16.2 Hz), 58.2 (d, J = 29.3 Hz), 29.8 (d, J
= 7.07 Hz), 25.9 (d, J = 4.0 Hz), 25.7, 17.6, 15.4 (d, J = 6.01 Hz). 19F NMR (471
MHz, CDCl3) δ -121.3 (t, J = 22.4 Hz) (Figure S1a-e). 2-fluoronerol: 1H NMR (400
MHz, CDCl3) δ 5.16 – 5.02 (m, 1H, H7), 4.19 (dd, J = 22.9, 5.3 Hz, 2H, H1), 2.16 –
2.00 (m, 4H, H5, H6), 1.70 (s, 3H, H4), 1.60 (s, 3H, H9/10), 1.57 (s, 3H, H9/10). 13C
NMR (101 MHz, CDCl3) δ 133.3, 123.5, 115.9 (d, J = 14.1 Hz) , 58.0 (d, J = 29.2
Hz), 31.9 (d, J = 5.1 Hz), 26.6 (d, J = 5.1 Hz), 25.8, 17.9, 13.7 (d, J = 9.1 Hz). 19F
NMR (471 MHz, CDCl3) δ –119.4 (t, J = 22.6 Hz) (Figure S2a-e).
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Synthesis of 2-fluorogeranyl pyrophosphate (FGPP) and 2-fluoroneryl
pyrophosphate (FNPP): Acetonitrile (60 ml) was added to 2-fluorogeraniol or 2-
fluoronerol (400 mg, 2.3 mmol). To this, trichloroacetonitrile (2 ml) was added
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followed by H3PO4(Et3N)2 salt (2.1 g). The reaction was stirred overnight. It was then
poured on to diethylether (50 ml) and washed with concentrated aqueous ammonia (3
x 100 ml). The ammonia washes were combined and washed once with diethyl ether
(50 ml). The aqueous phase was reduced to dryness. The crude product was loaded on
to a silica gel column and the starting material was recovered using petroleum
ether/diethyl ether (9/1, v/v). The eluent system was then switched to
propanol/concentrated aqueous ammonia/water (7/2/1, v/v/v) to isolate mono and
pyrophosphate derivatives (Scheme S1). When 2-fluorogeraniol was used, 2-
fluorogeranyl monophosphate (93 mg, 0.37 mmol, 16%) and 2-fluorogeranyl
pyrophosphate (74 mg, 0.2 mmol, 10%) were obtained. When 2-fluoronerol was used,
2-fluoroneryl monophosphate (117 mg, 0.47 mmol, 20%) and 2-fluoro neryl
pyrophosphate (200 mg, 0.54 mmol, 27%) were obtained. 2-fluorogeraniol
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pyrophosphate: H NMR (500 MHz, D2O) δ 5.29 – 5.15 (m, 1H, H7), 4.59 (dd, J =
23.6, 6.2 Hz, 2H, H1), 2.22 – 2.09 (m, 4H, H5, H6), 1.72 (d, J = 2.8 Hz, 3H, H4),
1.69 (s, 3H, H9/10), 1.62 (s, 3H, H9/10). 13C NMR (126 MHz, D2O) δ 133.9 (s),
123.7 (s), 119.5 (d, J = 15.0 Hz), 60.9 (dd, J = 31.6, 5.0 Hz), 29.1 (d, J = 6.4 Hz), 25.1
(s), 24.8 (s), 16.9 (s), 14.6 (d, J = 4.9 Hz). 31P NMR (162 MHz, D2O) δ –8.37 (d, J =
19
21.5 Hz), –10.95 (d, J = 21.6 Hz). F NMR (471 MHz, D2O) δ -120.44 (t, J = 23.6
Hz). HRMS ESI C10H18FO7P2 [M–H]– calculated: 331.0512, found: 331.0517 (Figure
S3a-g). Data was found to be in accordance with the literature16, 29. 2-fluoronerol
1
pyrophosphate: H NMR (400 MHz, D2O) δ 5.14 – 5.06 (m, 1H, H7), 4.50 (dd, J =
23.6, 6.1 Hz, 2H, H1), 2.07 (br s, 4H, H5, H6), 1.62 (s, 6H, H4, H9/10), 1.54 (s, 3H,
H9/10). 13C NMR (126 MHz, D2O) δ 134.3 (s), 123.3 (s), 119.1 (d, J = 13.2 Hz), 60.4
(dd, J = 31.0, 4.3 Hz), 30.8 (d, J = 4.6 Hz), 25.7 (d, J = 2.7 Hz), 24.8 (s), 16.9 (s),
12.8 (d, J = 8.6 Hz) 31P NMR (162 MHz, D2O) δ –7.64 (d, J = 21.4 Hz), –10.93 (d, J
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= 20.9 Hz) 19F NMR (471 MHz, D2O) δ –119.13 (t, J = 23.1 Hz). HRMS ESI
C10H18FO7P2 [M–H]– calculated: 331.0512, found: 331.0517 (Figure S4a-h). Data
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was found to be in accordance with the literature16, 29
.
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Crystallisation of bCinS and bLinS
Crystallisation trials containing 200 nl of protein and 200 nl of precipitant solution
were setup in 3-well swissci plates using a mosquito robot (TTP Labtech). Five
commercial screens namely Morpheus I and II, JCSG+, PACT premier and SG1
(Molecular Dimensions Ltd) were used in initial trails. For both enzymes, three
distinct samples were screened: the apo-enzyme, the enzyme in presence of 2 mM
FGPP, and the enzyme in presence of 2 mM FNPP. The bCinS-FNPP crystallised in
Morpheus II A4 condition (90mM of LiNaK (0.3M lithium sulphate, 0.3M sodium
sulphate, 0.3 M potassium sulphate), 0.1M of buffer system 4 (1M MOPSO, 1M Bis-
Tris) pH 6.5 and 50% precipitant mix 8 (10% PEG 20000, 50% trimethyl propane,
2% NDSB 195)). The bLinS-FGPP crystallised in Morpheus D7 condition (0.12 M
Alcohols (0.2M 1,6-hexanediol, 0.2M 1-nutanol, 0.2M 1,2-propanediol, 0.2M 2-
propanol, 0.2M 1,4-butanediol, 0.2M 1,3-propanediol), 0.1 M Buffer System 2 (1.0M
sodium HEPES, MOPS (acid)) pH 7.5 and 50% v/v precipitant Mix 3 (40% v/v
glycerol, 20% w/v PEG 4000)). The apo-LinS crystallised in SG1 E2 condition (25%
w/v PEG3350). Although apo-bCinS crystallised, optimisation of growth conditions
failed to produce single crystals of sufficient size for further study. In an attempt to
obtain the bCinS-FGPP structure, bCinS-FNPP crystals were soaked overnight in
presence of 2mM FGPP prior to cryo-cooling. The apo-bLinS crystals were cryo-
protected by soaking in mother liquor supplemented with 20% glycerol. For all FGPP
and FNPP complexes, the ligands were included in the cryo-solution.
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Structure Solution
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All data were collected at Diamond Light Source (DLS). Diffraction images were
integrated and scaled by xia230 automated data processing pipeline, using XDS31 and
XSCALE. Crystals of bCinS contained two molecules in the asymmetrical unit and
belonged to P1 space group. Crystals of bLinS belonged to the tetragonal system
(spacegroup I4) and also contained two molecules in asymmetrical unit. The bLinS
structures (apo-bLinS and bLinS-FGPP) were solved by molecular replacement using
the Pentalenene synthase structure (PDB 1PS132) as the search model in Phaser33. The
bCinS-FNPP structure was solved by model replacement using the apo-bLinS
structure as the search model. The apo-bLinS, bLinS-FGPP, bCinS-FNPP and bCinS-
FNPP/FGPP models were built using Autobuild in Phenix34. The structures were
completed using iterative rounds of manual model building in coot35 and refinement in
phenix.refine36. The structures were analysed using PDB_REDO37 and validated using
molprobity tools38. The refinement statistics are provided in Table 1. The atomic
coordinates and structure factors have been deposited in the Protein Data Bank with
accession codes 5NX4, 5NX5, 5NX6 and 5NX7.
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Table 1: X-ray data collection and refinement statistics
bCinS-FNPP
bLinS (apo)
bLinS-FGPP
bCinS-FNPP/FGPP
Data collection
Space group
I4
I4
P1
P1
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Unit cell dimensions
a=b=140.15 Å, c=87.18
a=b=139.37 Å, c=86.06
a=60.81 Å, b=60.83 Å,
c=64.10 Å; α=90.04°,
β=92.89°, γ=101.98°
DLS I04
a=60.75 Å, b=60.83 Å,
c=64.26 Å; α=92.67°,
β=89.98°, γ=101.77°
DLS I04
Å; α=β=γ=90°
Å; α=β=γ=90°
X-ray source
Wavelength (Å)
Resolution range (Å)
Multiplicity
I/σ I
DLS I04-1
DLS I04
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0.92819
0.99
0.99
0.9795
50.90-2.38 (2.42-2.38)
4.5 (3.9)
36.62-1.82 (1.85-1.82)
6.8 (6.9)
32.27-1.63 (1.66-1.63)
1.8 (1.8)
64.19-1.51 (1.53-1.51)
2 (2)
14.6 (1.7)
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10 (2.1)
Completeness (%)
Rmerge
99.7 (99.7)
100 (100)
96 (94.6)
95.9 (93.4)
0.082 (0.766)
0.093 (0.889)
0.043 (0.44)
0.998 (0.576)
152818 (6641)
33849 (1697)
0.057 (1.371)
0.062 (1.483)
0.024 (0.563)
0.999 (0.51)
502331 (25222)
73739 (3654)
0.09 (0.639)
0.127 (0.904)
0.09 (0.639)
0.987 (0.479)
193529 (9539)
107492 (5258)
0.035 (0.315)
0.047 (0.428)
0.032 (0.287)
0.999 (0.824)
270436 (13510)
136704 (6737)
Rmeas
Rpim
CC1/2
Total observations
Total unique
Refinement
R-work
0.1850
0.2240
0.002
0.45
0.1667
0.1950
0.009
0.94
0.1662
0.20
0.1492
0.1830
0.008
0.96
R-free
RMS (bonds)
RMS (angles)
Average B-factor (Å2)
Ramachandran plot
Favored
0.013
1.15
52.5
42.8
24.8
27.8
98.94
1.06
0
99.29
0.71
0
98.98
1.02
0
98.45
1.35
0.2
Allowed
Outliers
EPR Spectroscopy
Electron Paramagnetic Resonance (EPR) measurements were carried out using a
Bruker ELEXSYS-500 X-band EPR spectrometer operating in both cw and pulsed
modes, equipped with an Oxford variable-temperature unit and ESR900 cryostat with
Super High-Q resonator. All EPR samples were prepared in the quartz capillary tubes
(outer diameter; 4.0 mm, inner diameter 3.0 mm) and frozen in liquid N2. The X-band
EPR tubes were then transferred into the EPR probe head, which was pre-cooled to 20
K. The low-temperature EPR spectra were measured at 20 K as a frozen solution. A
microwave power of 36 dB (50 mW) and modulation of 5 G appear to be optimal for
recording the EPR spectrum of the bLinS and bCinS protein samples prepared using
various ratio of protein to Mn2+ concentration in the presence of 10-fold excess of
FGPP. The concentrations of the proteins (bLinS and bCinS) and FGPP in all the
samples were 0.400 mM and 1.5 mM respectively, whereas the ratio to the Mn2+
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concentration was systematically varied from 1 to 6. The low temperature EPR
spectra were acquired using the following conditions: sweep time of 84 s, microwave
power of 50 mW, time constant of 41 ms and modulation amplitude of 5 G. All the
spectra have been normalised to account for the different numbers of scans
accumulated for each sample. The data analysis was performed using EasySpin
toolbox for the Matlab program package.
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Simulations of Apo-bCinS and bLinS
Molecular dynamics (MD) simulations of apo-bCinS and bLinS were carried out in
AMBER14 using the CHARMM27 force field39-40. The protonation states of titratable
residues were estimated using the PDB2PQR server with proPKA, and the enzymes
were solvated using a box of minimum 12 Å around the protein with counter-ions
added. Two sets of isothermal-isobaric ensemble (NPT) MD simulations were
performed at 298 K for each enzyme, using different starting velocities, following the
system setup. Langevin dynamics was used for temperature control (collision
frequency of 5 ps-1 for equilibration and 2 ps-1 for production), and pressure was
controlled by coupling to an external bath (AMBER14 default settings) for NPT
conditions. The system setup consisted of: (i) energy minimisation of the solvent; (ii)
50 ps of (NPT) solvent equilibration; (iii) energy minimisation of the entire system
with positional restraints of 5 kcal mol-1 Å-2 applied to all Cα atoms; (iv) canonical
ensemble (NVT) thermalisation to 298 K over 20 ps with positional restraints of 5
kcal mol-1 Å-2 on Cα atoms; (v) 40 ps of NPT equilibration with decreasing restraints
on the Cα atoms; (vi) 1 ns unconstrained NPT equilibration; (vii) 100 ns production
simulation. Average linkage hierarchical clustering (after alignment of structures
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based on Cα positions) was used to identify representative structures to illustrate
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protein conformational sampling during the simulations.
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Simulations of the Ternary Complexes of bCinS with 3 Mg2+ Ions and GPP or
NPP
The protonation states of titratable residues were estimated using PropKA3.141-42 and
the enzyme was solvated using a box of TIP3P43 water molecules (with a minimum
buffer or 13 Å around the protein) using the solvate plugin of the VMD package44.
Counter-ions were added to neutralize the system using autoionize plugin of VMD44.
The CHARMM27 forcefield39 was used to describe the protein with parameters for
GPP and NPP were adapted from those used for FPP in the work of van der Kamp et
al.45. The position of the GPP or NPP substrate was based on the position of the
fluorinated analogue resolved in the crystal structure. Due to the minimal differences
in the structure of the inhibitor and substrate (F vs H), the position in the crystal
structure was considered a suitable starting point for the simulations. It has been
suggested that many terpene cyclase/synthase structures contain substrates bound in
unreactive conformations46-47; however structures containing the larger and more
flexible FPP, the building block for sesquiterpenes are more prevalent than
monoterpenes. The parameter set developed by Allner et al.48 was used to describe the
three Mg2+ ions. The setup of the model consisted of: (i) minimisation of the positions
of the hydrogen atoms (all heavy atoms fixed); (ii) minimisation of the solvent (with
all protein heavy atoms fixed); (iii) energy minimisation of the entire system with
positional restraints of 5 kcal mol-1 Å-2 applied to all Cα atoms; (iv) canonical
ensemble (NVT) thermalisation to 300 K over 20 ps with positional restraints of 5
kcal mol-1 Å-2 on Cα atoms; (v) thermal equilibration at 300K for 100 ps with
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positional restraints of 5 kcal mol-1 Å-2 on Cα atoms; (vi) 140 ps of NPT equilibration
with decreasing restraints on the Cα atoms; (vi) 100 ns production simulation. Two
sets of isothermal-isobaric ensemble (NPT) MD simulations were performed at 300 K
for each enzyme, repeating steps (iv)-(vi) to obtain 2 models with different initial
conditions. MD simulations were carried out on GPUs using the PMEMD code49 of
AMBER1650. Langevin dynamics was used for temperature control (collision
frequency of 5 ps-1 for equilibration and 2 ps-1 for production), and pressure was
controlled by coupling to an external bath (AMBER16 default settings) for NPT
conditions. Average linkage hierarchical clustering (after alignment of structures
based on positions of active site residues) was carried out using the CPPTRAJ utility
of AMBERTOOLS 1650 to identify representative structures of the ternary complex
over the course of the simulations.
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Simulations of the Ternary Complexes of bLinS with 3 Mg2+ Ions and GPP or
FPP
The models of bLinS were built from the coordinates of chain B of the protein, with
positions of the Mg2+ ions determined based on alignment with the structures of
sesquiterpene synthases aristocholene synthase (ATAS, PDB 4KUX51) and Epi-
isozizaene synthase (PDB 3KB952). GPP was built into the model based on the
position of the phosphate ion observed in the bLinS chain B structure and using the
geometry of FGPP observed in the bCinS-FGPP structure. The FPP model was
generated based on the position of farnesyl thiolodiphosphate FSPP in ATAS51. Some
positional restraints were then applied to the Mg2+ ions and coordinating protein
residues in the NSD and DDXXD motifs in order to form the correct binding pattern.
The Mg2+ to oxygen atom distance (for Asn218, Ser222, Asp226 and Asp79) was
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restrained to a value of 2.3 Å with a force constant k = 20 kcal mol-1 Å-2. The same
procedure as used for the bCinS models was then followed to perform the MD
simulations of bLinS with GPP and FPP.
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RESULTS AND DISCUSSION
Linalool and 1,8-cineole Production in E. coli
Biotransformation reactions showed that purified bLinS and bCinS produced linalool
and 1,8-cineole, respectively, when supplied with GPP. No by-products were
observed when analysed by GC-MS (Figure 1). To investigate the suitability of both
enzymes for monoterpenoid production in engineered E. coli strains, bLinS and bCinS
were inserted in an E. coli ‘plug-and-play’ monoterpenoid production platform, which
consists of two gene modules3. The first module (pMVA) contains a hybrid
mevalonate (MVA) pathway under regulation of IPTG-inducible promoters53 and the
second (plasmid series pGPPSmTC/S, Table S1) comprises a refactored, N-
terminally truncated geranyl diphosphate synthase (GPPS) gene from Abies grandis
(AgtrGPPS2) followed by an mTC/S gene (in this case bLinS or bCinS, respectively)
under control of a tetracycline-inducible promoter. Strains containing both the pMVA
and pGPPS-bLinS or pGPPS-bCinS plasmids, respectively, were grown in a two-
phase shake flask system using glucose as the feedstock and n-nonane as an organic
phase to facilitate product capture. Products accumulated in the organic phase were
identified and quantified by GC-MS analysis.
Product profiles and titres obtained with bLinS and bCinS were compared with
previously obtained profiles using mTC/S enzymes obtained from plants (Figure 1),
i.e. LinS from Artemisia annua (RLinS_Aa) and CinS from Salvia fruticosa
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(CinS_Sf), Arabidopsis thaliana (CinS_At), and Citrus unshiu (CinS_Cu)3. Both
bacterial enzymes outperformed the plant enzymes: bLinS produced about 300-fold
more linalool than RLinS_Aa (363.3 ± 57.9 versus 1.3 mg Lorg-1). With bCinS, 1,8-
cineole was produced in considerably purer form compared to that produced using the
plant enzymes. Strains containing bCinS produced 116.8 ± 36.4 mg Lorg-1 (96% pure);
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-1
-1
this compares to 118.2 mg Lorg (67% pure) for CinS_Sf, 46.6 mg Lorg (42% pure)
for CinS_At, and 18.2 (63% pure) for CinS_Cu for the strains containing the
corresponding plant CinS enzymes.
As well as GPP formation catalysed by the heterologous GPPS, the engineered E. coli
strains also produce the sesquiterpene precursor farnesyl diphosphate (FPP) from
native host encoded enzymes54. Strains containing bLinS were able to convert FPP to
nerolidol (159.1 ± 7.3 mg Lorg-1), indicating that bLinS acts as both a monoterpene and
sesquiterpene synthase. We demonstrated that bLinS makes R-(–)-linalool and trans-
nerolidol with GPP and FPP, respectively (Figure S5a-f). In contrast, no
sesquiterpene products were detected with E. coli strains containing bCinS indicating
it is restricted to the production of monoterpene products. With each of the strains,
geraniol and farnesol (and their derivatives) were detected in organic overlays of
cultures alongside the expected terpenoids. An unidentified endogenous E. coli
pathway has previously been shown to convert both GPP and FPP into geraniol and
farnesol respectively3, which are subsequently converted into oxidative by-products
by endogenous dehydrogenation and isomerisation reactions55. In particular, E. coli
PhoA phosphatase was implicated in converting GPP to geraniol56 and two integral
membrane phosphatases (PgpB and YbjG) were shown to convert FPP to farnesol57.
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The reported product profiles and yields suggest that bacterial monoterpene synthases
are better suited compared to the corresponding plant enzymes for monoterpenoid
production using engineered E. coli strains. Armed with this information we set out
to determine the structures of bLinS and bCinS, in both ligand-free and complexed
with fluorinated substrate analogues, with the objective of informing on mechanism,
and guiding future engineering/exploitation in biocatalysis and metabolic engineering
programmes.
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Figure 1. GC-MS analysis of bCinS and bLinS. A) bCinS product profile when
inserted in an engineered E. coli strain capable of overproducing GPP. B) bCinS
conversion of GPP (2 mM) in vitro. C) bCinS conversion of NPP (2 mM) in vitro. D)
1,8-cineole standard (0.1 mg/ml). E) bLinS product profile when inserted in an
engineered E. coli strain capable of overproducing GPP. F) bLinS conversion of GPP
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(2 mM) in vitro. G) R-(–)-linalool standard (0.1 mg/ml). H) cis- and trans-nerolidol
standards (0.1 mg/ml). IS = internal standard (sec-butyl benzene).
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Structure of the bCinS FNPP Complex
Crystals of bCinS were obtained when co-crystallised with 2-fluoro neryl
pyrophosphate (FNPP), a fluorinated GPP isomer. Unfortunately, bCinS crystallised
poorly when not bound to a substrate analogue. This suggests a conformational
change occurs between an open (apo)-form and a closed (substrate-inhibitor bound)
complex similar to that seen with other terpene cyclases45, 58. Previous studies have
indicated that some terpene cyclases/synthases can also accept neryl pyrophosphate
(NPP) as substrate16. In the case of bCinS, incubation with NPP also leads to 1,8
cineole (Figure 1). As observed with other terpene synthases, fluorination of the
substrate blocks the key ionisation step, blocking diphosphate release and formation
of the geranyl/neryl cation28. The bCinS-FNPP structure was determined to 1.63 Å
and reveals the enzyme is a dimer of a typical class I terpenoid α-helical domain, with
the active sites oriented in an anti-parallel fashion (Figure 2a). Analysis of the bCinS
dimer revealed a total buried surface area of 4114Å2, indicating the oligomeric state is
biologically relevant (using PISA59). Both monomers are similar in structure (r.m.s.d.
of 0.25 Å over 315 Cα atoms), with residues that constitute one of the loops close to
the active site disordered. The bound FNPP is clearly defined in the electron density
of both active sites, with no significant differences in conformation between both
monomers (Figure 2b). The pyrophosphate moiety of FNPP makes extensive
interactions with residues in the active site, in addition to coordination by two Mg2+
ions and interactions with several water molecules. While one Mg2+ is bound by the
conserved NSE motif (Mg2+ B), the other is bound by the aspartate rich motif (Mg2+
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A). No clear density could be observed that corresponds to the location of the third
metal ion (Mg2+ C).
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Figure 2. Structure of bCinS in complex with FNPP. A) Cartoon representation of
the bCinS dimer with the solvent accessible surface shown colour coded per
monomer. B) Stereoview of the FNPP-Mg2+ ion binding site. Key polar interactions
are shown by dotted lines. The electron density indicates multiple positions of the
diphosphate moiety as well as several Mg2+ binding residues. C) Stereoview of the
FNPP hydrophobic binding pocket. A single water molecule is present, located close
to the C6 atom.
EPR Reveals Binding of 3 Mn2+ Ions to bCinS
To ascertain whether bCinS binds to two or three Mg2+ ions, we employed EPR
spectroscopy by titrating bCinS purified in the absence of MgCl2 withMn2+. The Mn2+
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ion serves as a valuable probe of the Mg2+ ion binding sites60-62. This substitution
allowed application of cw-EPR spectroscopy to investigate the number of potential
metal binding sites in bCinS. Comparison of the EPR spectra of the aqueous MnCl2
and bCinS with and without the inhibitor FGPP indicates that the spectrum of the 1:1
bCinS-FGPP:Mn2+ sample contains a highly resolved multiplet structure (Figure 3a;
red spectrum). This multiplet structure is the 55Mn hyperfine coupling which is due to
the interaction of electron spin (S=5/2) of the Mn2+ ion with the nuclear spin (I = 5/2)
of 55Mn nucleus. It is a characteristic signature of binding of FGPP to Mn2+ ion, which
is centered at g ~ 2.0. This multiplet feature increases in intensity only until the ratio
of Mn2+ ion concentration relative to bCinS-FGPP reaches 3. However, where the
relative concentration of Mn2+ ion is greater than 3, the EPR spectra show overall
increase in intensity due to the contribution from free/unbound Mn2+ ion. The EPR
spectrum of the 1:6 bCinS-FGPP:Mn2+ sample (Figure 3a; magenta spectrum) can be
simulated (Fig 3a; cyan spectrum) by 1:1 addition of the EPR spectra of 1:3 bCinS-
FGPP:Mn2+ sample with (Figure 3a; blue spectrum) the 1:3 bCinS:Mn2+ (Figure 3a;
black spectrum). This indicates that there are 3 potential metal binding sites available
in bCinS. Detailed analysis and assignment of the various transitions in the EPR
spectra (Figures S6a-b and S7a-b) are provided in the supplementary information.
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Figure 3. EPR confirms binding of 3 Mn2+ in solution. cw-EPR spectra of ‘Mn2+’
substituted bCinS (A) and bLinS (B) protein samples with varying equivalents of
Mn2+ concentration with or without FGPP measured as a frozen solution along with
standard MnCl2. The plot shows the multiplet EPR signal arise from the Mn2+ ion
around the g = 2 region (from 250-400 mT).
Structure of the bCinS-FGPP Complex and bCinS Mechanism
Soaking of the bCinS-FNPP crystals with FGPP led to the partial exchange of the
inhibitor in both monomers (structure determined to 1.51 Å resolution). Besides the
obvious reorientation of the carbon skeleton, the presence of FGPP does not lead to
active site reconfiguration. However, the soaking protocol used has led to clear
electron density of a partially occupied third Mg2+ ion (Mg2+ C;Figure 4). This in turn
is accompanied by a modest change in conformation of the E155 region (Figure 4a),
bringing the E155 side chain into close contact with water molecules ligating Mg2+ C.
Given the partial occupancy of the inhibitors and of the E155/Mg2+ C, it is unclear
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whether there is a direct link between the nature of the ligand bound in the active site
and the binding of Mg2+ C. However, as both GPP and NPP act as substrates for
bCinS, presumably both requiring binding of 3 Mg2+ ions, it seems plausible the
soaking procedure used is responsible for the observed changes in the E155 region
and the associated Mg2+ C binding.
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Based on the bCinS-FNPP and bCinsS-FGPP/FNPP structures, a mechanism for the
bacterial 1,8-cineole synthesis can be proposed, by analogy to observations made with
plant monoterpene synthases18 (Figure 5). Unlike FGPP, the carbon chain
conformation of FNPP (and by extension the NPP substrate) is compatible with
cyclisation of the initial carbocation (in this case linalyl) derived from substrate
ionisation to form the (R)-terpinyl cation. Indeed, the FNPP C1 and C6 atoms are
placed at a distance of ~3.6 Å. In contrast, steric constraints require the FGPP carbon
skeleton to undergo an isomerisation step following substrate ionisation and geranyl
carbocation formation prior to cyclisation. For other monoterpene cyclase enzymes,
this has been proposed to occur via transient formation of linalyldiphosphate and
concomitant change from the transoid to cisoid configuration14-15. A second substrate
ionisation step then generates the linalyl carbocation species, which can proceed to
the cyclisation step. The fact that both GPP and NPP result in the same product
suggests the exact configuration of the respective linalyl carbocation species (GPP
versus NPP derived) and resulting terpinyl carbocation are similar, resembling the
carbon chain configuration of the FNPP inhibitor. However, recent solution studies
using labeled GPP have suggested bCinS proceeds via the (S)-terpinyl cation, in
contrast to the (R)-terpinyl configuration proposed on the basis of the bCinS-FNPP
crystal structure63. Following formation of the terpinyl carbocation, conversion via the
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final cyclisation step to the 1,8-cineol product is proposed to occur via a syn
addition63. With the exception of a single water molecule, coordinated by Trp58 and
Asn305, the hydrophobic binding pocket is devoid of solvent. This water molecule is
placed at a distance of ~3.6 Å to the C6 of FNPP (Figure 2c), and thus appears the
most likely candidate for nucleophilic attack on the terpinyl cation. MD simulations
show that this water molecule remains at an average distance of 3.84 (±0.45 run1,
(±0.53 run2) Å from C7 of GPP throughout the 100 ns simulation (Figure 6). The
water molecule interacts with Asn305, but no longer interacts with Trp58. Figure 6
(A-C) shows the different positions of the hydrocarbon tail of GPP and NPP in the
representative structure from the dominant cluster for the 100 ns simulations. The
hydrocarbon tail of NPP occupies the position adopted by the sidechain of Phe77 in
the simulations of bCinS with GPP. There are more water molecules near to C7 of
NPP and the shortest distance is not with a single water molecule throughout the
entire simulation, as was observed for GPP. However, simulations with NPP show
that the average position of the water molecule is more distant than in the bCinS/GPP
system with an average C7 – WAT O distance of 4.26 ±0.59 Å run1 and 4.42±0.59 Å
in run1 (Figure 6 (d-e)). Formation of the neutral α-terpineol through deprotonation
is avoided by the lack of any suitable acid-base group in close proximity of this water
molecule. Production of the bicyclic 1,8-cineole from the protonated α-terpineol
species is proposed to occur via intramolecular proton transfer to C2, followed by C2-
O bond formation leading to formation of the second cycle. Considering the relative
position of the water molecule and the C2 atom in the FNPP structure, this scenario
will require some conformational changes to occur. This is distinct from the proposed
mechanism for the plant 1,8 cineole synthase, for which a syn addition of water is
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