Alternate Cyclization Cascade Initiated by Substrate Isomer in Multiproduct Terpene Synthase from Medicago truncatula

Article abstract of DOI:10.1021/acs.joc.6b02696

Promiscuity of terpene synthases results in the enormous diversity of terpenes found in nature. Multiproduct sesquiterpene synthase MtTPS5 isolated from Medicago truncatula generates 27 optically pure products from its natural substrate (2E,6E)-farnesyl diphosphate (FDP). In order to study the promiscuity of MtTPS5, (2Z,6E)-FDP, an analogue of presumptive reaction intermediates from natural reaction cascade, was utilized as a substrate. This stereoisomer induced a novel cyclization pathway leading to sesquiterpenes based on humulane, amorphene, and himachalane skeletons. Interestingly, none of these products matched those observed on incubation of MtTPS5 with natural (2E,6E)-FDP. Further determination of the absolute configuration of each product helped rebuild the stereochemical route of the reaction cascade. Interestingly, the presence of only one enantiomer of each product was observed, indicating the highly stereospecific nature of the enzymatic reaction. Substrate promiscuity of terpene synthases provides organism access to novel chemical bouquets of high optical purity by utilizing existing enzymes. The presence of this mechanism was indicated by the presence of these alternate products in natural herbivore-induced volatiles of M. truncatula.

Full text of DOI:10.1021/acs.joc.6b02696

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Article
Alternate Cyclization Cascade Initiated by Substrate Isomer
in Multiproduct Terpene Synthase from Medicago truncatula
Abith Vattekkatte, Stefan Garms, and Wilhelm Boland
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.6b02696 • Publication Date (Web): 14 Feb 2017
Downloaded from http://pubs.acs.org on February 16, 2017
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Alternate Cyclization Cascade Initiated by Substrate
Isomer in Multiproduct Terpene Synthase from
Medicago truncatula
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Abith Vattekkatte,# Stefan Garms,# and Wilhelm Boland *
Department of Bioorganic Chemistry, Max Planck Institute for Chemical Ecology, Beutenberg Campus,
HansꢀKnöllꢀStrasse 8, Dꢀ07745 Jena, Germany
boland@ice.mpg.de
# Equal Contribution
*To whom correspondence should be addressed. Fax: +49(0) 3641 571 202
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Abstract:
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Promiscuity of terpene synthases results in the enormous diversity of terpenes found in nature.
Multiproduct sesquiterpene synthase MtTPS5 isolated from Medicago truncatula generates 27 optically
pure products from its natural substrate (2E,6E)ꢀfarnesyl diphosphate (FDP). In order to study the
promiscuity of MtTPS5, (2Z,6E)-FDP an analogue of presumptive reaction intermediates from natural
reaction cascade was utilized as a substrate. This stereoisomer induced a novel cyclization pathway
leading to sesquiterpenes based on humulane, amorphene and himachalane skeletons. Interestingly,
none of these products matched with those observed on incubation of MtTPS5 with natural (2E,6E)ꢀ
FDP. Further determination of absolute configuration of each product helped rebuild the stereochemical
route of the reaction cascade. Interestingly, the presence of only one enantiomer of each product was
observed indicating the highly stereospecific nature of the enzymatic reaction. Substrate promiscuity of
terpene synthases provides organism access to novel chemical bouquets of high optical purity by
utilizing existing enzymes. Presence of this mechanism was indicated by the presence of these alternate
products in natural herbivoreꢀinduced volatiles of Medicago truncatula.
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Introduction:
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Terpenes with more than 55,000 known members are found in almost all forms of life and
constitute the most diverse class of natural products.1-3 They serve many biological functions,
such as hormones (abscisic acid, gibberellins, steroids);4 structural components of membranes
(phytosterols); pollinator attractants;5 toxins;6 deterrents for feeding; or oviposition to a variety
of insects.7 Terpenes are not only significant in chemical ecology but are also commercially
significant as medicines, materials, fuels, chemicals, and especially as flavors and fragrances.8-10
Volatile terpenoids which constitutes a substantial class of induced volatiles are generated by
enzymes known as terpene synthases. This class of enzymes has been extensively investigated in
recent times, including cloning and characterization of a number of cDNAs encoding terpenoid
synthases involved in primary and secondary metabolism in plants.11,12
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Enzymes with promiscuous functions have long been believed to evolve and acquire increased
specificity and activity, and this plasticity has been achieved with minor substitutions in amino
acids.13 This promiscuity gives us the opportunity to study the evolution of enzymes and design
terpene synthases as better catalysts. Catalytic promiscuity has long been known to be one of the
key features of terpene synthases, especially sesquiterpene synthases.9,10 All known
sesquiterpenes are based on 300 basic hydrocarbon skeletons formed by sesquiterpene synthases
from the universal precursor (2E,6E
) farnesyl diphosphate (FDP).14 This structural diversity is
the result of an enzymatic environment that holds the pliable isoprenoid substrate in a desirable
conformation to enforce specific CꢀC bond formation trajectory. Besides terpene synthases
generating single products, multiproduct terpene synthases are also known for producing a
collection of cyclic and acyclic products from a single substrate.11 Currently,
δꢀselinene synthase
and ꢀhumulene synthase from Abies grandis hold the record for generating 52 and 34 different
γ
sesquiterpenes from FDP.15 Such biosynthetic promiscuity is assumed to be possible as a result
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of active site permitting alternative conformations of the both substrate and reaction
intermediates.
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Figure 1: (2E,6E)-Farnesyl diphosphate (FDP) and (2Z,6E)- farnesyl diphosphate (FDP). The
initial steps of farnesyl carbocation formation and corresponding isomerization double bond.
During terpenoid biosynthesis, the unsaturated diphosphates (FDP, etc.) dissociate into highly
reactive cations and remainder diphosphate anions. These electrophilic carbocations react with
nearby electronꢀrich double bounds, followed by intramolecular cyclizations; rearrangements,
including methyl or hydride shifts prior to stabilization by either reaction with a nucleophile; or
deprotonation.16 Due to the (
progresses through the remote double bonds. In contrast, the direct intramolecular cyclization of
(2 ,6 )ꢀFDP with the neighboring C(5)ꢀC(6) double bond to cyclohexenyl cations is not
E)ꢀconfiguration of the substrate, this intramolecular cyclization
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E
possible and requires a preceding isomerization. This isomerization is achieved by a suprafacial
migration of the diphosphate moiety in the substrate that leads to a tertiary allylic intermediate
bound to the enzyme active site (Figure 1).17ꢀ19 It was believed that there is a high freeꢀenergy
barrier for the direct rotation of the allylic cation but recently geranyl cation isomerization has
shown to be feasible in a selfꢀassembled cavity.20,21 Presence of this intermediate prompted the
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use of (2
to study the substrate promiscuity of terpene synthases (Figure 1). The efficient synthesis of
(2 ,6
)ꢀFDP, has been previously described,22 and used to study the kinetics of various terpene
Z,6E)ꢀFDP as a substrate mimicking the secondary cisoid nerolidyl cation intermediate
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synthases, but few studies have compared the cyclization with highly promiscuous multiproduct
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enzymes.23ꢀ26 For example, Cop4 and Cop6 from Coprinus cinereus resulted in the opposite
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enantiomer (6S)ꢀβꢀbisabolene from (2
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E
)ꢀFDP rather than (6R)ꢀβꢀbisabolene and (2
E,6E)ꢀ
germacradienyl carbocation, respectively, from (2E,6E
)ꢀFDP.27 In our previous work with
multiproduct terpene synthases from Zea mays, we observed that this rotation had huge turnover
benefits but generated an identical product profile.28 Hence, we wanted to test the substrate
promiscuity of MtTPS5 enzyme with (2
among its 27 optically pure products.
Z,6E)ꢀFDP through variations in catalytic mechanisms
Results and Discussion:
We had previously elucidated the complexities involved in the mechanistic pathway of MtTPS5
reaction cascade with (2 ,6 )ꢀFDP using a combination of techniques, including labelling
E
E
experiments.29 In brief, the disassociation of diphosphate moiety initiates the reaction cascade
leading to the formation of a highly reactive farnesyl carbocation. The ring closure involving C1
to C11 carbons of the farnesyl carbocation generates the humulyl cation, which leads to
compounds such as αꢀhumulene and βꢀcaryophyllene. Most of the other products follow the
initial C1 to C10 closure to generate the germacrenꢀ11ꢀyl cation; further cyclization of this
cation leads to products like germacrene D and germacrenylꢀbased alcohols. (2Z,6E)ꢀgermacrenꢀ
1ꢀyl cation and cadinanꢀ7ꢀyl cation are the other key cationic intermediates leading to about 80%
products which are generated by the isomerization of FDP to the nerolidyl diphosphate (NDP).
Substitution of even single amino acid in MtTPS5 has a dramatic effect on the product profile;
the substitution of tyrosine with phenylalanine stops the formation of a key intermediate via the
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protonation of germacrene D as a neutral intermediate. The 27 optically pure products are the
result of a series of cyclization steps and hydride shifts under the tight control of the enzyme.
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effects of the initial isomerization step on the reaction cascade. These alterations in product
profile via different reaction channels depend on the nature of the initial carbocationic
intermediates; ease of deprotonation and their stability.
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Figure 2: GC-FID Chromatogram of major sesquiterpenes after the incubation of recombinant
MtTPS5 with (2E,6E)-FDP and (2Z,6E)-FDP. The compounds were identified by their Kovats indices
and mass spectra, and compared to authentic references. Only major sesquiterpenes are labelled.
(2E,6E)-FDP: (1) αꢀcopaene, (2) βꢀcubebene, (3) (E)ꢀ βꢀcaryophyllene, (4) cadinaꢀ3,5ꢀdiene, (5) alloꢀ
aromadendrene, (6) γꢀmuurolene, (7) germacrene D, (8) αꢀmuurolene, (9) cubebol, (10) δꢀcadinene,
(11) copanꢀ3ꢀol,(12) 4αꢀhydroxygermacraꢀ1(10),5ꢀdiene, (13) copaborneol, (14) torreyol.
(2Z,6E)-FDP: (15) αꢀylangen, (16) αꢀhimachalene, (17) isobicyclogermacrene, (18) αꢀamorphene, (19)
γꢀhumulene, (20) γꢀhimachalene, (21) βꢀhimachalene, (22) δꢀamorphene, (25) humulaꢀ4,9ꢀdienꢀ8ꢀol and
(27) 2ꢀhimachalenꢀ7ꢀol. Sesquiterpene alcohols (23), (24) and (26) (C15H26O IIꢀIV) could not identified.
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Interestingly, unlike the incubation of (2
which showed only quantitative differences within the natural product profile, MtTPS5 gave a
completely distinct 23 product profile with no similarity to the (2 ,6 )ꢀFDP product profile
Z,6E)ꢀFDP with maize sesquiterpene synthases,
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(Figure. 2). Out of these 23 compounds in GCꢀFID chromatogram, 13 sesquiterpenes were
isolated and identity of eight compounds were confirmed based on a comparison of their mass
spectra and retention indices with authentic references and two terpenoids were identified by
other means. The most interesting aspect of these results was the absence of the dominant
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terpenoid skeletons that were observed with (2E,6E)ꢀFDP. The product profile of (2Z,6E)ꢀFDP
consisted predominantly of monoꢀ and bicyclic sesquiterpenes comprising humulane,
amorphene and himachalane skeletons, none of which were observed in the reaction with the
natural substrate. These results suggested that product formation with (2Z,6E)ꢀFDP by MtTPS5
followed a completely novel pathway as compared to natural mechanistic pathway.
The absolute configuration of αꢀ and δꢀamorphene (18, 22), α ꢀ and γꢀ himachalane (16, 20), as well as
of αꢀylangen (15), was determined by GCꢀMS with a chiral phase column (Figure 3, supporting
information). The stereochemical analysis of the enzymatic products with (2Z,6E)ꢀFDP revealed the
retention of configuration among products with the same hydrocarbon skeleton also between related
structures. One common factor observed with both geometric isomers is that the enantiomeric
composition of all substances had a high degree of optical purity. The absolute configuration and
identity of individual compounds was confirmed by comparison with authentic standards obtained from
various sources (supporting information). Accordingly, the formation of only one enantiomer of the
products shows that MtTPS5 exerts strong control on the initial conformation of the farnesyl cation. In
contrast, the trichodiene synthase from Fusarium sporotrichioides and the multiproduct terpene
synthase TPS4 and TPS5 from Zea mays generate a mixture of diastereomeric and racemic products.30,31
For αꢀamorphene (18), the determination of the enantiomeric excess was not possible because the (+)ꢀ
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enantiomer coꢀeluted with that of (+)ꢀδꢀamorphene 22 (supporting information). All compounds
generated from MtTPS5 with (2E,6E)ꢀFDP as a substrate shared (S)ꢀconfigured stereochemistry at C10.
In contrast, the bridgehead hydrogen atoms (C1ꢀH, C6ꢀH) of αꢀylangen (15), as well as αꢀ and δꢀ
amorphene (18, 22) of (2Z,6E)ꢀFDP had an opposite orientation to those from volatiles generated by
(2E,6E)ꢀFDP (1, 8 and 10) (Figure. 3).
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Figure 3: Determination of the absolute configurations of some products obtained after the
incubation of MtTPS5 with (2Z,6E) -FDP. The separation of the enantiomers was carried out by GCꢀ
MS on a chiral phase (shown for 15 (A), 20 (B) and 22 (C)). D. Juxtaposition of isomeric compounds
starting from (2Z,6E)ꢀFDP (top row: isobicyclogermacrene (17)) and (2E,6E)ꢀFDP (bottom
row: bicyclogermacrene (28)).
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Figure 4: Structure elucidation of humula-4,9-dien-8-ol (25). GCꢀMS chromatograms, which were
obtained by reactions of a column chromatography on silica gel, purified a fraction of 25 and its
secondary products. (A): an 25ꢀenriched fraction, (B) products by catalytic hydrogenation of 25, (C)
products after acid catalyzed dehydration of 29, (D) products by catalytic hydrogenation of 30a-e, (E)
reference, obtained from 19 by hydrogenation 32a, b.
The available mass spectra libraries and Kovats indices were unsuccessful in identifying the major
product 25. The structure of compound 25 was elucidated by classical stepꢀbyꢀstep derivatization
(Figure. 4). The mass spectrum of 25 (supporting information) showed a weak molecular peak at
m/z=222 and fragment ions at m/z =207 [MꢀMe]+ and m/z =204 ([MꢀH2O]+), indicating the structure of a
sesquiterpene. The catalytic hydrogenation of a highly enriched fraction 25 yielded compound 29
(Figure. 4B) with an increased mass (4 amu) of the fragment ion [MꢀH2O]+ m/z = 208 (Figure 4). This
increase in mass indicated that compound 25 is monocyclic in nature wherein two double bonds were
reduced. After acidꢀcatalyzed dehydration of 29 with trifluoroacetic anhydride, five compounds (30a-e)
were separated by gas chromatography (Figure. 4C), which had the expected molecular peak at m/z =
208. The detection of five stereoisomeric products helped us conclude that the hydroxy group could
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only be located on a tertiary carbon atom, as a secondary alcohol, would have provided only four
products. Further proof that 25 is as an 11ꢀ membered ring system was observed after the hydrogenation
of γꢀhumulene (19) (Figure. 4E). The products diastereomeric humulane (32a,b) matched exactly with
the hydrogenation products of the mixture of substances (30a-e) (Figure. 4D,E). Connecting all the
observed results, the product can be identified as humulaꢀ4,9ꢀdienꢀ8ꢀol (25), which was isolated by
Pentegova et al., from the resin of silver fir (Abies alba).32
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Figure 5: Proposed mechanism of formation of the products after incubation of (2Z,6E) -FDP with
MtTPS5.
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Previous labelling and mechanistic studies of MtTPS5 with (2E,6E)ꢀFDP showed that the enzyme is
capable of generating cadalane sesquiterpenes via two different pathways.29 A large proportion of the
released compounds (~33%) from (2Z,6E)ꢀFDP had a cadalane backbone; however, these cadalenes
possessed the opposite absolute configuration (1S, 6R) at the bridgehead carbon atoms in contrast to the
natural products. The determination of the absolute configurations of some of the products helped us
reconstruct the stereochemical course of the reaction cascade from the starting conformation of the (2Z)ꢀ
isomeric substrate (Figure. 5). The highly reactive (2Z,6E)ꢀfarnesyl cation, which is formed after the
cleavage of the diphosphate group of (2Z,6E)ꢀFDP, predominantly undergoes (~57%) a C1ꢀC11 ring
closure to generate the (2Z,6E)ꢀhumulꢀ10ꢀyl cation. The transfer of a positive charge from C10 to C1 is
possible only by a direct 1,3ꢀhydride shift. The presence of a quaternary carbon atom between these two
positions eliminates the possibility of two consecutive 1,2ꢀhydride shifts. The deprotonation of the
(2Z,6E)ꢀhumulꢀ1ꢀyl cation (36) generates γꢀhumulene (19); γꢀhumulene (19) on further reaction with
water results in the main product (25). Furthermore, the himachalanes 16, 21, 20 and 27 are released
from 36 through an electrophilic attack on the si face of the C6ꢀC7 double bond, followed by the
elimination of a proton or by reaction with a water molecule.
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Another reaction channel, which starts from the (2Z,6E)ꢀfarnesyl cation, passes through the tenꢀ
membered macrocyclic (2Z,6E)ꢀgermacreneꢀ11ꢀyl cation (33) after the initial C1ꢀC10 cyclization. In
this step, the (R) configuration at C10 is initially established, which is conserved in all the cadalane
sesquiterpenes. Analogous to the proposed reaction mechanism with (2E,6E)ꢀFDP (a 1,3ꢀdeprotonation
of C1ꢀHRe leading to bicyclogermacrene), a similar mechanism is observed with (2Z,6E)ꢀFDP, wherein
the isomer isobicyclogermacrene (17) is formed. In addition, the absolute configuration at C1 of
isobicyclogermacrene (17) suggests that a shift of 1ꢀ HRe from C1 to C11 generates the (2Z,6E)ꢀ
germacreneꢀ11ꢀyl cation (33). 1,3 hydride shift in carbocation (33) yields 35 and a further C1ꢀC6 ringꢀ
closure leads to cadinaneꢀ7ꢀyl cation (37c) and the further loss of a proton in the two double bond
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resulting in amorphenes 22 and 18. The tricyclic rings are formed after a C2ꢀC7 additional cyclization
of 37c followed by deprotonation to (ꢀ)ꢀαꢀylangen (15).
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A close correlation exists between the two cyclization cascades, starting from both (2E,6E)ꢀ and
(2Z,6E)ꢀFDP. The essential elements, such as the 1,3ꢀhydride shift, initial 1,10ꢀ or 1,11ꢀ cyclization and
the formation of bicyclic compounds by a secondꢀring closure, are found in both reaction mechanisms.
The huge structural difference in the products is mainly due to the different starting conformation of the
two substrates, as each of the reactive moieties occupies a different spatial arrangement, which needs
further examination with help of docking studies. The large influence of the initial folding of farnesyl
cation in the active site, depending on the configuration of the C2ꢀC3 double bond, has already been
shown for the 5ꢀepiꢀaristolochene synthase (TEAS) of tobacco.33 The availability of crystal structures of
enzymeꢀsubstrate complexes of TEAS with fluorine analogs of (2E,6E)ꢀ and (2Z,6E)ꢀFDP helped us
understand the structural basis of this conformational difference. However, in case of multiproduct
terpene synthases lack of available crystal structures hinders the structural explanation for different
stereoꢀand regiochemical reaction cascades resulting from the two starting conformations.33,34 Work is
in progress to coꢀcrystalize the 3ꢀbromo analogs of FDP with MtTPS5 to decipher the structural basis of
this diversity and substrate promiscuity.35
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The isolation of a (2Z,6E)ꢀFDP synthase from Mycobacterium tuberculosis, which is involved in the
bacterial cell wall biosynthesis, indicates the relevance of (2Z,6E)ꢀFDP in other biological systems.36,37
In a recent work, an novel terpene synthase gene family from evolutionary perspective has been
reported in the striped flea beetle, these enzymes have evolved from transꢀisoprenyl diphosphate
synthases and are involved in the biosynthesis of a sesquiterpene which acts as a maleꢀspecific
aggregation pheromone.38 Interestingly, a novel sesquiterpene biosynthesis pathway from other isomer
(2Z,6Z)ꢀFDP in the wild tomato Solanum habrochaites has also been reported.39 Natural presence of
(2Z,6E)ꢀFDP in other systems invoked our interest in looking for presence of these alternate volatiles
from MtTPS5 in the herbivore induced terpenoid volatiles of Medicago truncatula. Interestingly, we
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found five hydrocarbons α, β, γ himachalanes (16, 21, 20), γꢀhumulene (19) and αꢀylangen (15) in the
volatile blends upon Spodoptera littoralis or Tetranychus urticae infestation which were emitted by
Medicago truncatula.40 These compounds are absent in the product profile of the known terpene
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synthases from Medicago truncatula on incubation with (2E,6E
)ꢀFDP.41 As a natural substrate
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(2Z,6E)ꢀFDP may lead to volatile production in Medicago truncatula. Presence of these compounds in
natural volatile blends also indicates the possibility of a "sleeping" cyclization pathway starting from
(2Z,6E)ꢀFDP, but none has been discovered yet.
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Conclusion
In this work, we probe the reaction mechanism of multiproduct terpene synthase from Medicago
truncatula with an analogue that mimics the isomerization via an NDP intermediate of the C2=C3 π
bond of (2E,6E)ꢀFDP. The results show that MtTPS5 is a promiscuous enzyme, generating 23 alternate
cyclization products starting from (2Z,6E)ꢀFDP. The (2Z,6E)ꢀFDP product profile consisted
predominantly of monoꢀ and bicyclic sesquiterpenes based on humulane, amorphene and himachalane
ring skeletons. We showed that MtTPS5 retains the exceptional stereochemical control over the reaction
cascade through determination of the absolute configuration of the final products which indicated the
presence of only one enantiomer. Additionally, the detection of some alternate products in herbivoreꢀ
induced volatiles indicates the possible natural use of substrate promiscuity by terpene synthases. This
promiscuity provides organisms with access to novel chemical defenses that are adaptable with
available enzymes and possible evolutionary advantage. Along with help of siteꢀdirected mutagenesis,
these alternative geometric analogs can be used to generate novel cyclic products with known terpene
synthases
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Experimental Section
General methods.
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Reactions were performed under Argon gas. Solvents were dried based on standard procedures.
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Chemical shifts of H, 13C and 31P NMR was given in ppm (
δ
) based on solvent peaks. (1H NMR)
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CDCl3: 7.27 and 77.4 ppm (13C NMR). D2O/ND4OD: 4.79 (1H NMR); C NMR and P NMR were
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referenced to external standard 3ꢀ(trimethylsilyl)ꢀpropionic acidꢀd4 sodium salt (TSP; 3 % in D2O) and
phosphoric acid (H3PO4, 10 % in D2O), respectively. GCꢀMS: equipped with a DB5 (15 m
ꢀ
0.25 mm,
0.25 ꢀm); helium served as carrier gas. Molecular composition of prepared compounds was determined
by ESIꢀMS using a tandem quadrupole mass spectrometer (geometry quadrupoleꢀhexapoleꢀquadrupole)
equipped with an electrospray (ESI) source. Highꢀresolution ESIꢀMS (HRꢀEIꢀMS) were recorded at
resolution ca 2500. Highꢀresolution MS (EI) data were obtained in positive ion mode using 70 eV
ionization energy. A DBꢀ5 capillary column, 30 m × 0.25 mm, 0.25 µm film thickness using helium (1.5
mL sꢀ1) as carrier gas was used for separation.
Heterologous expression of terpene synthases.
Strains of E. coli (BL21ꢀCodonPlus(DE3)) containing the recombinant vectors of MtTPS541 with an Nꢀ
terminal His8ꢀtag were cultivated to optical density(at 600 nm) A600 = 0.5 at 37 °C in LuriaꢀBertani (LB)
broth ꢀmedium with kanamycin (50 ꢁg mL−1) stirring at 200 rpm. The expression medium was induced
with isopropyl βꢀdꢀ1ꢀthiogalactopyranoside (IPTG, final concentration 1 mM), and the cultures were
shaken overnight at 16 °C at 200 rpm. Cells were centrifuged at 4000 rpm for 20 min, and the cell pellet
was resuspended in lysis buffer (300 mM NaCl,10 mM imidazole, 50 mM NaH2PO4, pH 8.0) and
incubated for 1 h at 4 °C with lysozyme (1 mg mL−1). The cell disruption was performed by sonication
for 2 × 2 min. The cell debris was separated by centrifugation at 10000g for 30 min. A column of Ni2+ꢀ
NTAꢀAgarose, was equilibrated with eight bed volumes of lysis buffer, and the supernatant after
centrifugation was passed over it. It was further washed twice with four bed volumes of washing buffer
(300 mM NaCl,50 mM NaH2PO4, 20 mM imidazole, pH 8.0), the protein was eluted with two bed
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volumes of elution buffer ( 300 mM NaCl, 50 mM NaH2PO4 250 mM imidazole, pH 8.0). The purified
protein was directly desalted) by passing it through a NAP 25 column into a TRISꢀbuffer (50 mM TRIS,
pH 7.5, 10 mM NaCl, 10% glycerol, diluted to reach a concentration of 1 mg mL−1 and stored at −20
°C. The protein concentration was quantified by using the method explained by Bradford et al.42
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Identification of enzyme products
A ZBꢀ5 capillary column equipped instrument (0.25 mm i.d. × 15 m with 0.25 ꢁm film) was used to
perform GC−MS analysis. 1µl of the sample was injected in splitless mode at 220 °C injection port
temperature. The oven temperature was initially maintained at 50 °C for 2 min then followed by a ramp
up of 10 °C min−1 until 240 °C followed by an additional ramp of 30 °C min−1 upto 280 °C and finally
maintained for 2 min. Ionization potential was set at 70 eV, and with a scanning range from 40 to 250
amu. Carrier gas was helium at a flow rate of 1.5 mL min−1 .
Compound identification was performed by comparison of individual mass spectra and Kováts indices
(retention indices) with those of published reference spectra in Adams' terpene library43, MassFinders’
(software version 3.5) and the NIST database. Moreover, retention indices (RI) of sesquiterpene peaks
calibrated with GC runs of C8−C20 alkane standard mixture were compared with authentic reference
compounds RI values (Table Aꢀ1, supporting information). Essential oils containing relevant
sesquiterpenoids were generously provided by Prof. Dr. Stephan von Reuss or bought from commercial
supplier.
Quantification of products
For quantification of enzymatic products, the products were first separated by gas chromatography
(injection volume 2 ꢁL, H2 carrier gas 1.5 mL min−1) under the above described conditions and further
analyzed on a flame ionization detector (FID) at 250°C. Calibration curves obtained from samples with
different concentrations of (E)ꢀβꢀcaryophyllene and torreyol was used for correction of the different
response factors of sesquiterpene hydrocarbons and alcohols. The average and standard deviations of
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The Journal of Organic Chemistry
relative ratios were determined by at least four independent samples setting the sum of identified
compounds to 100%.
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Determination of the stereochemistry of the enzyme products
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The separation and identification of product enantiomers were performed by GC−MS using a heptakis
(2,3ꢀdiꢀOꢀmethylꢀOꢀtertꢀbutyldimethylsilyl)ꢀβꢀcyclodextrin column (50% in OV1701, w/w) (FSꢀ
Hydrodex βꢀ6TBDM) (0.25 mm i.d. × 25 m × 0.25 ꢁm film) and operated with helium at 1 mL min−1 as
carrier gas. A splitless injection of sample (1 ꢁL) at 220 °C and a temperature program was started from
60 °C and kept for 5 min, ramped up 2 °C min−1 to 160 °C followed by an additional ramping up of 30
°C min−1 to 220 °C with 2 min hold. Samples containing both enantiomers were prepared by mixing
pentane extracts of the liverwort Preissia quadrata44 or Scarpania undulata45 with the corresponding
reference or were gifts from Prof. Stephan von Reuss.
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Enzyme assays for product analysis
Standard assays were performed with 600 nM purified protein in assay buffer (25 mM HEPES, pH 7.5,
1 mM DTT, 10% glycerol, 10 mM MgCl2) with 50 ꢁM substrate (FDP) making final volume of 1
mL. The reaction mixture was overlayed with 100 ꢁL of pentane containing 1 ng ꢁL−1 of dodecane
(internal standard) on static platform to trap the volatile products. After incubation for 90 min at 30 °C,
the reaction was halted by vortexing (20 s) due to enzyme denaturation. The entire reaction mixture was
frozen in liquid nitrogen; the pentane layer extracted after thawing and analyzed by GC−MS.
Mass Spectra of Compound 25:
MS (ESI): m/z=222, 207 [MꢀMe]+, 204 ([MꢀH2O]+).
Preparative assays
For the identification of the unknown compound (25), two 10ꢀmL assays consisting 600 nM purified
enzyme in assay buffer and FDP (50 ꢁM) was overlayed with 10 mL of pentane. After overnight
incubation at 30 °C, the volatiles were further extracted with 5 mL of pentane three times. The
combined pentane extracts were passed through a Pasteur pipette containing Na2SO4, the volume was
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then reduced to a final volume of ∼100 ꢁL. Alcohols were separated using pentane/ether (6:1, v/v) for
elution on silica column (1 g, Pasteur pipette). Two fractions of highly enriched compound 25 were
obtained, and reduced to a volume of approximately 1 ml under a light argon stream.
Catalytic hydrogenation
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For the catalytic hydrogenation, the solution of the respective sesquiterpene alcohol was concentrated to
dryness under argon stream. The residue was taken up in 300 ꢁl of pentane, and a spatula tip catalyst
(5% rhodium on aluminum, ~10µg) was added. The gas space of the reaction vessel was flushed with
H2 for 10s, and the reaction mixture was stirred for a further 15 minutes under a H2 atmosphere (1 atm).
The suspension was then filtered over cotton, and the solid components were rinsed with 500 ꢁl of
pentane. The volume was reduced to ~ 200 ꢁl and the sample was analyzed by GCꢀMS.
Acid-catalyzed dehydration
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The sample (∼500ng GC) of the alcohol was reꢀdissolved in 200 ꢁL of dry CH2Cl2, and 5 ꢁL of
trifluoroacetic anhydride (derivatization grade) was added under argon atmosphere. The reaction
mixture was stirred for 30 min, and after the addition of pentane (1 ml), 10% NaHCO3 (1 ml) was added
to quench the reaction. The aqueous phase was extracted 2 x 1 mL of pentane. The combined organic
extracts were dried (Na2SO4) and reduced to ∼100 ꢁL under argon. Concentrated samples were then
analyzed by GC−MS.
Acknowledgments
Financial support by the Max Planck Society and help with authentic reference standards by Prof. Dr.
Stephan vonReuss is gratefully acknowledged.
Supporting information
Complete tables of relative ratios of products, retention indices, source of references. Spectra of enzyme
products in comparison with authentic reference standards. Mass spectrometric data from incubation
experiments with substrates and synthetic procedure of (2Z,6E)ꢀFDP.
References
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