On the mechanism of ophiobolin F synthase and the absolute configuration of its product by isotopic labelling experiments

Article abstract of DOI:10.1039/d0ob01470b

An ophiobolin F synthase homolog was discovered from Aspergillus calidoustus CBS121601. The cyclisation mechanism of this terpene synthase was investigated by extensive isotopic labelling experiments and the absolute configuration of its product ophioboli

Full text of DOI:10.1039/d0ob01470b

View Article Online
View Journal
Organic &
Biomolecular
Chemistry
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: Z. Quan and J.
Dickschat, Org. Biomol. Chem., 2020, DOI: 10.1039/D0OB01470B.
Volume 15
Number 47
21 December 2017
Pages 9945-10124
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
Organic &
Biomolecular
Chemistry
Accepted Manuscripts are published online shortly after acceptance,
rsc.li/obc
before technical editing, formatting and proof reading. Using this free
service, authors can make their results available to the community, in
citable form, before we publish the edited article. We will replace this
Accepted Manuscript with the edited and formatted Advance Article as
soon as it is available.
You can find more information about Accepted Manuscripts in the
Information for Authors.
Please note that technical editing may introduce minor changes to the
text and/or graphics, which may alter content. The journal’s standard
Terms & Conditions and the Ethical guidelines still apply. In no event
ISSN 1477-0520
PAPER
I . J. Dmochowski et al.
Oligonucleotide modifi cations enhance probe stability for
shall the Royal Society of Chemistry be held responsible for any errors
single cell transcriptome in vivo analysis (TIVA)
or omissions in this Accepted Manuscript or any consequences arising
from the use of any information it contains.
rsc.li/obc

Please do not adjust margins
Organic & Biomolecular Chemistry
Page 1 of 7
View Article Online
DOI: 10.1039/D0OB01470B
COMMUNICATION
On the mechanism of ophiobolin F synthase and the absolute
configuration of its product by isotopic labelling experiments
Zhiyang Quana and Jeroen S. Dickschat*a
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
An ophiobolin F synthase homolog was discovered from Aspergillus II TCs start from the protonation of an olefinic double bond or
calidoustus CBS121601. The cyclisation mechanism of this terpene an epoxide. After substrate ionisation a multistep cationic
synthase was investigated by extensive isotopic labelling cascade leads to the terpene cyclase product. This process
experiments and the absolute configuration of its product involves typical cation chemistry such as cyclisations by
ophiobolin F was elucidated by enantioselective deuteration.
intramolecular attack of an olefinic double bond to a cationic
centre, Wagner-Meerwein rearrangements, hydride or proton
shifts, and eventually nucleophilic attack by water. The complex
cyclisations of terpenes can be investigated theoretically by
quantum chemical calculations5,6 or experimentally by enzyme
crystallization and site-directed mutagenesis,7 and isotopic
labelling experiments.8
Terpenes originate from two simple isoprene units,
dimethylallyl pyrophosphate (DMAPP) and isopentenyl
pyrophosphate (IPP), and constitute the largest class of natural
products with an exceptional structural diversity. They are
widely distributed in nature from bacteria and fungi to higher
plants, and also exhibit various bioactivities as fragrances,
pheromones, antibiotics, anticancer or antimalaria drugs, as
demonstrated by the prominent examples of taxol1 and
artemisinin.2 Terpenes can be classified according to their
numbers of isoprene units as hemiterpenes (C5), monoterpenes
(C10), sesquiterpenes (C15), diterpenes (C20), sesterterpenes (C25),
triterpenes (C30) and tetraterpenes (C40). The biosynthesis of
terpenes is divided into two stages, i. e. the elongation stage in
which DMAPP and IPP are fused to give geranyl pyrophosphate
(GPP), farnesyl pyrophosphate (FPP), geranylgeranyl
pyrophosphate (GGPP), and geranylfarnesyl pyrophosphate
(GFPP); these reactions are catalysed by prenyltransferases
(PT).3 In the cyclisation stage GPP, FPP, GGPP, and GFPP are
converted into often highly complex polycyclic terpenes by a
terpene cyclase (TC).4 Two main classes of enzymes are known
for terpene cyclisations, including class I TCs for which the
cyclisation cascades are initiated by the departure of the
pyrophosphate anion, while the cyclisations catalyzed by class
In 2007, the synthase for fusicoccadiene (1, PaFS, Figure 1), the
first chimeric enzyme containing both a PT and a TC domain,
was characterized from the plant-pathogenic fungus Phomopsis
amygdali.9 Subsequently, several other bifunctional di- and
sesterterpene cyclases were discovered, such as the diterpene
synthases for phomopsene (2, PaPS),10 variediene (3, EvVS),11
the cyclopiane type diterpene (4, PcCS),12 dolastadiene (5,
CgDS)13 and myrothecenol (6, MgMS),6 and the sesterterpene
synthases for ophiobolin F (7, AcOS),14 sesterfisherol (8, NfSS),15
stellatatriene (9, EvSS),16 astellifadiene (10, EvAS),17
quiannulatene (11, EvQS),18 preasperterpenoid A (12, PvPS),19
sesterbrasiliatriene (13, PbSS),19 Bm1 (14, BmTS1),20 Bm2 (15,
BmTS2),20 Bm3 (16, BmTS3),20 Pb1 (17, PbTS1)20 and
mangicdiene (18, FgMS).21 Among the products of these
enzymes, the absolute configurations of several molecules were
established by various methods such as total synthesis (for 1),22
Mosher’s method (for 3),11 X-ray crystal diffraction analysis (for
4, 9 and 10),12,17,23 circular dichroism (CD) in conjunction with
density functional theory (DFT) calculations (for 6, 8, 11 and
14),6,15,18,20 and by chemical correlation through an
enantioselective deuteration strategy (for 2, 3, 5, 6, 12 and
13).6,13,19,24,25 For 7 that was first isolated from Cochliobolus
heterostrophus,26 the relative configuration was secured by X-
a. Kekulé-Institut für Organische Chemie und Biochemie, Rheinische Friedrich-
Wilhelms-Universität Bonn, Gerhard-Domagk-Straße 1, 53121 Bonn, Germany.
E-mail: dickschat@uni-bonn.de; Tel: +49 228 735797.
Electronic Supplementary Information (ESI) available: Cloning of gene, construction
of plasmids, details of biological experiments, conditions of fermentations, isolation ray crystallography,14 while the absolute configuration has not
of the natural product and NMR spectra, isolation of mRNA, reverse-transcription
been formally established, but it can be deduced through
biosynthetic considerations from the known absolute
configuration of its highly bioactive derivative ophiobolin A. For
of mRNA, synthesis of isotopically labelled GGPPs, details of isotopic labelling
experiments, and NMR and GC/MS spectra from labelling experiments. See
DOI: 10.1039/x0xx00000x
This journal is © The Royal Society of Chemistry 2020
Org. Biomol. Chem., 2020, 00, 1-3 | 1
Please do not adjust margins

Please do not adjust margins
Organic & Biomolecular Chemistry
Page 2 of 7
COMMUNICATION
Organic & Biomolecular Chemistry
this compound the absolute configuration was assigned by X-
View Article Online
27
DOI: 10.1039/D0OB01470B
ray crystallography of its methoxybromo derivative and was
confirmed by total synthesis.28 Here we report on a new
H
H
H
ophiobolin
F (7) synthase homolog AcldOS (Aspergillus
H
H
calidoustus ophiobolin F synthase), the absolute configuration
of its product and the cyclisation mechanism towards 7
investigated by isotope labelling experiments.
1
2
3
OH
AcldOS, the gene encoding a candidate bifunctional terpene
synthase with 66 % identity to AcOS, was heterologously
expressed in Aspergillus oryzae NSAR1. After cultivation of the
A. oryzae transformant harboring acldOS, the metabolites
accumulated in its mycelium were extracted and analysed by
GC/MS (Figure S1). Compared with the negative control strain
that was transformed with an empty vector, the GC/MS results
indicated the presence of a sesterterpene. The compound was
isolated from a large scale fermentation with a yield of 17 mg/L
and identified as 7 by one- and two-dimensional NMR
spectroscopy (Table S2, Figures S2 – S9). Our extensive structure
elucidation showed that some of the earlier assignments need
correction (for 13C and 1H of C1, C5, and C13, and 1H of C9 and
C17).11
H
HO
H
H
H
H
4
5
6
H
H
H
OH
H
H
H
H
HO
7
8
H
In order to reveal the absolute configuration and to investigate
the cyclisation mechanism of 7, recombinant AcldOS for in vitro
incubations was desired. For this purpose, mRNA was isolated
from the A. oryzae transformant harboring acldOS and used as
H
H
H
a
template with reverse transcriptase to obtain the
9
10
corresponding cDNA. The cDNA of AcldOS was cloned into the
pYE-Express vector to generate the Escherichia coli expression
plasmid pYE-AcldOS (detailed sequences of genomic DNA and
cDNA of acldOS are given in Figure S10). The N-terminally His6-
tagged sesterterpene synthase AcldOS was expressed in E. coli
BL21(DE3) and purified by Ni2+-NTA affinity chromatography
(Figure S11). Incubation of the purified enzyme with
DMAPP+IPP, GPP+IPP, FPP+IPP, GGPP+IPP, and GFPP revealed
formation of the same product 7 in all cases (Figure S12).
H
H
H
H
H
H
H
11
12
H
H
H
H
The absolute configuration of 7 was determined by incubation
experiments with stereoselectively deuterated and
simultaneously 13C-labelled isotopomers of IPP, followed by
HSQC analysis of the obtained products. Carbons carrying
deuterium in the substrates introduce additional stereocenters
into the products during conversion by the terpene synthase
with a strictly defined stereochemical course, i. e. inversion of
configuration at C1 of allyl diphosphates and bond formation to
C4 of IPP with attack to its Si face.29,30 Through these reactions
the successive elongations of DMAPP with IPP units to GFPP,
shown for the sake of brevity in one step catalysed by the PT
domain in Scheme 1, stereoselectively deuterated GFPP
isotopomers can be obtained from corresponding IPP
isotopomers with a stereoselective deuteration at C1 (HR or HS
H
13
14
H
H
OH
H
15
16
H
H
H
H
=
2H) or C4 (HE or HZ 2H). Determination of the relative
=
orientation of the additional stereocenters introduced with
these substrates to the original stereocenters based on NOESY
data can resolve the absolute configuration of the target
molecule. The additional 13C-label in the substrates at the
deuterated carbons strongly enhances the sensitivity for the
hydrogen signals in the HSQC spectra for small scale reactions
without the need of compound purification.
17
18
Figure 1. Products of characterised bifunctional fungal terpene
synthases.
2 | Org. Biomol. Chem., 2020, 00, 1-3
This journal is © The Royal Society of Chemistry 2020
Please do not adjust margins

Page 3 of 7
Organic & Biomolecular Chemistry
Please do not adjust margins
Organic & Biomolecular Chemistry
COMMUNICATION
A)
Isotope labelling experiments were also conducted to
investigate the biosynthetic mechanism to 7. It is hypothesized
View Article Online
DOI: 10.1039/D0OB01470B
that the first stage of biosynthesis for 7 begins with the fusion
of one DMAPP and four IPP units to GFPP by the PT domain of
AcldOS. At the second stage catalysed by the TC domain, the
cyclisation of GFPP starts with the departure of the OPP- group,
followed by a 1, 11-10,14-cyclisation to cation A. Then, a 1,5-
hydride shift results in cation B, followed by a 2,6-cyclisation to
cation C. Final attack by water results in the product 7 (Scheme
2). This hypothesis was first presented by Oikawa and
coworkers,14 but experimental or theoretical investigations are
currently lacking.
PPO
OPP
HE
HE
PT
HZ
HZ
OPP
PPO
HZ
HE
HZ
HE
OPP
To experimentally address this biosynthetic hypothesis, 25
singly 13C-labelled substrates in combination with non-labelled
precursors for the enzymatic preparation of all 25 isotopomers
of (13C)GFPP were incubated with AcldOS in order to introduce
a 13C-label into every position of 7 (Table S3, Figures S15 – S17).
The required labelled substrates were obtained by synthesis in
our previous work13,33-35 or as reported in the ESI (for (1-13C)-,
(3-13C)- and (4-13C)GGPP, Schemes S1 and S2). The results from
these experiments confirmed the overall model regarding the
GFPP fold leading to 7 and also further supported the
reassignments of 13C-NMR data as mentioned above.
HE
HZ
HE
16
16
HE
HE
HZ
TC
HZ
HZ
8
8
H
H
H
12
12
HZ
HZ
4
4
HE
HE
H
HE
HE
OPP
HZ
HZ
HO
B)
IDI
OPP
HR
HS OPP
HR
OPP
HR
HS
HS
20
PT
19
21
18
17
PPO
HR
OPP
DMAPP
OPP
HS HR
16
HR
PT
15
TC
HS
22
HS
+
8
9
-OPP-
24
5
7
14
OPP
10
13
6
x 4
11
OPP
1
2
12
IPP
23
4
OPP
3
HS
HR
HS
HR
25
17
17
GFPP
HR
HR
TC
HS
HS
9
9
13
HS
HR
HS
HR
H
1
13
H
HS
HR
H
HS
HR
H
H
HR
HS
OPP
1,5-H-~
H
2,6-cyc
HR
5
H
5
HS
H
H 15
1
15H
HS
8
HR
8
HO
6
Scheme 1. Determination of the absolute configuration of 7 by
using an enantioselective deuteration strategy. Incubation of A)
DMAPP + (E)- or (Z)-(4-13C,4-2H)IPP + AcldOS, and B) (S)- or (R)-
(1-13C,1-2H)IPP + IDI + AcldOS.
2
A
B
20
19
21
Three new stereocenters, at C4, C12, and C16, were introduced
into 7 when DMAPP and (E)- or (Z)-(4-13C,4-2H)IPP24 were used
as precursors, and five new stereocenters, at C1, C5, C9, C13,
and C17, were introduced when (R)- or (S)-(1-13C,1-2H)IPP31 was
converted by AcldOS and isopentenyl diphosphate isomerase
(IDI) from Serratia plymuthica32 (for a summary of all labelling
experiments cf. Table S3). HSQC spectroscopy revealed the
chemical shifts of the diastereotopic pro-S and pro-R hydrogens
for all eight methylene groups (Scheme 1, Figures S13 and S14).
Together with their NOESY correlations with hydrogens of the
original stereocenters the absolute configuration of
(2S,3R,6S,10S,11R,14R,15S)-7 was assigned.
18
17
15
16
22
H
8
9
H
H
24
H
7
-H+
H
14
10
13
H
H
11
6
5
2
12
1
3
23
H
4
H
3
25
HO
C
7
H2O
Scheme 2. Proposed biosynthetic mechanism for 7.
This journal is © The Royal Society of Chemistry 2020
Org. Biomol. Chem., 2020, 00, 1-3 | 3
Please do not adjust margins

Please do not adjust margins
Organic & Biomolecular Chemistry
Page 4 of 7
COMMUNICATION
Organic & Biomolecular Chemistry
To investigate the 1,5-hydride shift from A to B, (7-13C)FPP33 was
incubated with (E)- or (Z)-(4-2H)IPP32 in order to obtain 13C-2H
double labelled 7. The labelled products were analyzed by 13C
NMR without purification. While the combinations of (7-13C)FPP
plus IPP and (7-13C)FPP plus (E)-(4-2H)IPP gave a strong 13C
singlet signal for the labelled C15 of 7, in agreement with a non-
deuterated carbon, the combination of (7-13C)FPP plus (Z)-(4-
2H)IPP resulted in a slightly upfield shifted triplet for C15,
indicating a direct 13C-2H bond that was formed by the
deuterium migration of HR-4 to C15 (Figure S18). The GC/MS
spectrum of the molecules obtained with AcldOS from (7-
13C)FPP plus (E)-(4-2H)IPP and (7-13C)FPP plus (Z)-(4-2H)IPP also
supported the same conclusion through localisation of the
deuterium label in the core structure of 7 from (E)-(4-2H)IPP,
and in the side chain of 7 from (Z)-(4-2H)IPP, based on a
fragment ion arising by loss of the side chain (Figure S19).
Notes and references
View Article Online
1
M. C. Wani, H. L. Taylor, M. E. Wall, P. DCOogI:g1o0.n10a3n9d/DA0.OTB.01470B
McPhail, J. Am. Chem. Soc., 1971, 93, 2325.
D. L. Klayman, Science, 1985, 228, 1049.
B. A. Kellogg and C. D. Poulter, Curr. Opin. Chem. Biol., 1997,
1, 570.
2
3
4
5
D. W. Christianson, Chem. Rev., 2017, 117, 11570.
H. Sato, T. Mitsuhashi, M. Yamazaki, I. Abe and M. Uchiyama,
Angew. Chem. Int. Ed., 2018, 57, 14752.
6
7
F. Lin, L. Lauterbach, J. Zou, Y. Wang, J. Lv, G. Chen, D. Hu, H.
Gao, X. Yao and J. S. Dickschat, ACS Catal., 2020, 10, 4306.
M. Chen, W. K. W. Chou, T. Toyomasu, D. E. Cane and D. W.
Christianson, ACS Chem. Biol., 2016, 11, 889.
J. S. Dickschat, Eur. J. Org. Chem., 2017, 4872.
T. Toyomasu, M. Tsukahara, A. Kaneko, R. Niida, W.
Mitsuhashi, T. Dairi, N. Kato and T. Sassa, Proc. Natl. Acad.
Sci. U.S.A., 2007, 104, 3084.
8
9
10 T. Toyomasu, A. Kaneko, T. Tokiwano, Y. Kanno, Y. Kanno, R.
Niida, S. Miura, T. Nishioka, C. Ikeda, W. Mitsuhashi, T. Dairi,
T. Kawano, H. Oikawa, N. Kato and T. Sassa, J. Org. Chem.,
2009, 74, 1541.
11 B. Qin, Y. Matsuda, T. Mori, M. Okada, Z. Quan, T.
Mitsuhashi, T. Wakimoto and I. Abe, Angew. Chem. Int. Ed.,
2016, 55, 1658.
12 T. Mitsuhashi, T. Kikuchi, S. Hoshino, M. Ozeki, T. Awakawa,
S. Shi, M. Fujita and I. Abe, Org. Lett., 2018, 20, 5606.
13 G. Bian, J. Rinkel, Z. Wang, L. Lauterbach, A. Hou, Y. Yuan, Z.
Deng, T. Liu and J. S. Dickschat, Angew. Chem. Int. Ed., 2018,
57, 15887.
14 R. Chiba, A. Minami, K. Gomi and H. Oikawa, Org. Lett., 2013,
15, 594.
15 Y. Ye, A. Minami, A. Mandi, C. Liu, T. Taniguchi, T. Kuzuyama,
K. Monde, K. Gomi and H. Oikawa, J. Am. Chem. Soc., 2015,
137, 11846.
16 Y. Matsuda, T. Mitsuhashi, Z. Quan and I. Abe, Org. Lett.,
2015, 17, 4644.
17 Y. Matsuda, T. Mitsuhashi, S. Lee, M. Hoshino, T. Mori, M.
Okada, H. Zhang, F. Hayashi, M. Fujita and I. Abe, Angew.
Chem. Int. Ed., 2016, 55, 5785.
18 M. Okada, Y. Matsuda, T. Mitsuhashi, S. Hoshino, T. Mori, K.
Nakagawa, Z. Quan, B. Qin, H. Zhang, F. Hayashi, H. Kawaide
and I. Abe, J. Am. Chem. Soc., 2016, 138, 10011.
19 T. Mitsuhashi, J. Rinkel, M. Okada, I. Abe and J. S. Dickschat,
Chem. Eur. J., 2017, 23, 10053.
20 K. Narita, H. Sato, A. Minami, K. Kudo, L. Gao, C. Liu, T. Ozaki,
M. Kodama, X. Lei, T. Taniguchi, K. Monde, M. Yamazaki, M.
Uchiyama and H. Oikawa, Org. Lett., 2017, 19, 6696.
21 G. Bian, Y. Han, A. Hou, Y. Yuan, X. Liu, Z. Deng and T. Liu,
Metab. Eng., 2017, 42, 1.
22 N. Kato, C. Zhang, T. Matsui, H. Iwabuchi, A. Mori, A. Ballio
and T. Sassa, J. Chem. Soc., Perkin Trans. 1, 1998, 2473.
23 I. H. Qureshi, S. A. Husain, R. Noorani, N. Murtaza, Y. Iitaka,
S. Iwasaki and S. Okuda, Tetrahedron Lett., 1980, 21, 1961.
24 L. Lauterbach, J. Rinkel and J. S. Dickschat, Angew. Chem. Int.
Ed., 2018, 57, 8280.
Conclusions
In conclusion, we discovered a new ophiobolin F synthase
AcldOS from Aspergillus calidoustus, elucidated the absolute
structure of its product 7, and provided insights into the
biosynthetic cyclisation cascade from GFPP to 7 using an isotope
labelling approach. The biosynthetic mechanism is essentially as
proposed by Oikawa and coworkers. Our work addressed and
resolved specific details such as the question which of the two
diastereotopic at C8 of the cationic intermediate A migrates
into the side chain towards C15. Such aspects are difficult, if not
impossible, to predict, but could thoroughly be elucidated by
the experiments described here. While for compound isolation
of 7 a fairly high yielding fungal heterologous expression system
(in vivo) was applied, allowing to obtain 17 mg/L of pure
compound by fermentation, for the isotopic labelling
experiments an E. coli expression system (in vitro) was used. In
these experiments the purified enzyme is needed, because the
synthetic isotopically labelled isoprenoid diphosphates cannot
be administered in feeding experiments with the in vivo system.
Feeding experiments would be possible with earlier precursors
such as labelled glucose, acetate or mevalonate, but in these
cases the precision to accurately position the isotopic labels
according to the needs of the experiment to follow a specific
biosynthetic step cannot be reached. In our future work, we will
apply the powerful combination of methods used here to study
further terpene biosynthetic pathways from microorganisms.
Conflicts of interest
There are no conflicts to declare.
25 J. Rinkel, S. T. Steiner, G. Bian, R. Chen, T. Liu and J. S.
Dickschat, ChemBioChem, 2020, 21, 486.
26 S. Nozoe, M. Morisaki, K. Fukushima and S. Okuda,
Tetrahedron Lett., 1968, 9, 4457.
27 S. Nozoe, M. Morisaki, K. Tsude, Y. Iitaka, N. Takahashi, S.
Tamura, K. Ishibashi and M. Shirasaka, J. Am. Chem. Soc.,
1965, 87, 4968.
28 K. Tsuna, N. Noguchi and M. Nakada, Angew. Chem. Int. Ed.,
2011, 50, 9452.
Acknowledgements
This work was funded by the DFG (DI1536/7-2).
29 J. W. Cornforth, R. H. Cornforth, G. Popjak and L. Yengoyan,
J. Biol. Chem., 1966, 241, 3970.
4 | Org. Biomol. Chem., 2020, 00, 1-3
This journal is © The Royal Society of Chemistry 2020
Please do not adjust margins

Page 5 of 7
Organic & Biomolecular Chemistry
Please do not adjust margins
Organic & Biomolecular Chemistry
COMMUNICATION
30 J. W. Cornforth, R. H. Cornforth, C. Donninger and G. Popjak,
Proc. R. Soc. London Ser. B, 1966, 163, 492.
31 J. Rinkel and J. S. Dickschat, Org. Lett., 2019, 21, 2426.
32 J. Rinkel, L. Lauterbach, P. Rabe and J. S. Dickschat, Angew.
Chem. Int. Ed., 2018, 57, 3238.
View Article Online
DOI: 10.1039/D0OB01470B
33 P. Rabe, L. Barra, J. Rinkel, R. Riclea, C. A. Citron, T. A.
Klapschinski, A. Janusko and J. S. Dickschat, Angew. Chem.
Int. Ed., 2015, 54, 13448.
34 P. Rabe, J. Rinkel, E. Dolja, T. Schmitz, B. Nubbemeyer, T. H.
Luu and J. S. Dickschat, Angew. Chem. Int. Ed., 2017, 56,
2776.
35 J. Rinkel, L. Lauterbach and J. S. Dickschat, Angew. Chem. Int.
Ed., 2019, 58, 452.
This journal is © The Royal Society of Chemistry 2020
Org. Biomol. Chem., 2020, 00, 1-3 | 5
Please do not adjust margins

Please do not adjust margins
Organic & Biomolecular Chemistry
Page 6 of 7
COMMUNICATION
Organic & Biomolecular Chemistry
TOC graphic and text:
View Article Online
DOI: 10.1039/D0OB01470B
The cyclisation mechanism of ophiobolin F synthase AcldOS and
the absolute configuration of its product were investigated by
isotopic labelling experiments.
6 | Org. Biomol. Chem., 2020, 00, 1-3
This journal is © The Royal Society of Chemistry 2020
Please do not adjust margins

Page 7 of 7
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/D0OB01470B