Insights into the Desaturation of Cyclopeptin and its C3 Epimer Catalyzed by a non-Heme Iron Enzyme: Structural Characterization and Mechanism Elucidation

Article abstract of DOI:10.1002/anie.201710567

AsqJ, an iron(II)- and 2-oxoglutarate-dependent enzyme found in viridicatin-type alkaloid biosynthetic pathways, catalyzes sequential desaturation and epoxidation to produce cyclopenins. Crystal structures of AsqJ bound to cyclopeptin and its C3 epimer ar

Full text of DOI:10.1002/anie.201710567

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Accepted Article
Title: Insights into desaturation of cyclopeptin and its C3-epimer
catalyzed by a non-heme iron enzyme: structural characterization
and mechanism elucidation
Authors: WEI-CHEN CHANG, Hsuan-Jen Liao, Jikun Li, Jhih-Liang
Huang, Madison Davidson, Igor Kurnikov, Te-Sheng Lin,
Justin L. Lee, Maria Kurnikova, Yisong Guo, and Nei-Li Chan
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201710567
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10.1002/anie.201710567
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Insights into desaturation of cyclopeptin and its C3-epimer
catalyzed by a non-heme iron enzyme: structural characterization
and mechanism elucidation
Hsuan-Jen Liao,[c]† Jikun Li,[b]† Jhih-Liang Huang,[a] Madison Davidson,[a] Igor Kurnikov,[b] Te-Sheng
Lin,[c] Justin L. Lee,[b] Maria Kurnikova,[b]* Yisong Guo,[b]* Nei-Li Chan,[c]* and Wei-chen Chang[a]*
Abstract: AsqJ, an iron(II) and 2-oxoglutarate dependent enzyme
found in viridicatin-type alkaloid biosynthetic pathways, catalyzes
sequential desaturation and epoxidation to produce cyclopenins.
Herein, the crystal structures of AsqJ bound to cyclopeptin and its C3-
epimer are reported. Meanwhile, a detailed mechanistic study was
carried out to provide compelling evidence in deciphering the
desaturation mechanism. These findings suggest that a pathway
involving hydrogen atom abstraction at the C10 position of the
substrate by a short-lived Fe(IV)-oxo species and the subsequent
formation of a carbocation or a hydroxylated intermediate is likely to
be utilized during AsqJ catalyzed desaturation.
hypothesized to serve as the second hydrogen atom (H•)
abstractor to produce a di-radical species prior to C=C bond
formation. In both cases, the initial reactive species stores two
potent oxidizing equivalents for sequential C-H bond activation. It
should be noted that an alternative mechanism involving a cation
intermediate has also been proposed in P-450 desaturases[3d, 3g]
.
Similarly, an analogous 2-HAT mechanism has been suggested
in Fe/2OG enzyme catalyzed desaturation, such as in flavone
synthase I, carbapenem synthase, and clavaminate synthase[3f, 3h,
3i, 3k]. Compared to the well-studied hydroxylation mechanism[4]
where the reaction proceeds through H• abstraction by an
Fe(IV)=O species followed by formation of a C-O bond between
the substrate radical and the Fe(III)-OH intermediate, during the
desaturation, the resulting Fe(III)-OH intermediate is used as the
second H• abstractor to produce a di-radical intermediate
(Scheme 2B). Inspired by recent discovery of Fe/2OG enzyme
catalysis[4], pathways involving a carbocation or a hydroxylated
intermediate generated after the incipient C-H bond activation
also need to be considered (Scheme 2B). Herein, we use a
complementary approach consisting of probe design, X-ray
crystallography, stopped-flow optical absorption, freeze quench
coupled Mössbauer spectroscopy, MD simulations and MS-based
product analysis to elucidate desaturation mechanism. Our
results suggest that AsqJ catalyzed desaturation is likely to initiate
from C10-H abstraction by a short-lived Fe(IV)-oxo species. The
reaction is likely followed by formation of a carbocation or a
hydroxylated intermediate to complete installation of C=C bond.
AsqJ, an iron(II) and 2-oxogluatrate (Fe/2OG) enzyme, has
recently been discovered in viridicatin-type alkaloid biosynthesis
in Aspergillus nidulans. It catalyzes two consecutive reactions: a
C=C bond formation and an epoxidation to enable formation of
the 6,6-bicyclic core of the viridicatin scaffold (Scheme 1A)[1].
Recently, we and other research groups have presented a
plausible mechanism for the epoxidation catalyzed by AsqJ[1-2]
.
However, mechanistic understanding of AsqJ-catalyzed
desaturation has not been elucidated.
In general, two approaches have been utilized by biological
systems to construct C=C bonds. One involves introduction of a
leaving group followed by -elimination. The other approach
involves a direct C-H bond activation process where metal-
cofactors, such as heme-iron centers, non-heme mono-iron
centers, or di-iron centers, are required to trigger the reaction[3].
For two mechanistically characterized desaturases, fatty acyl
desaturases and cytochrome P-450 enzymes (Scheme 1B),
enzymatic and biomimetic model studies support a mechanism
involving two consecutive hydrogen atom transfer processes (2-
HAT) to construct a C=C bond[3a-c, 3e, 3j]. Specifically, a di-(µ-oxo)-
di-iron(IV/IV) intermediate or a ferryl porphyrin radical cation
species, compound I, functions as the key intermediate to trigger
the first C-H bond cleavage, and
a proposed Fe2(III/IV)
intermediate or an Fe(IV)-hydroxo complex, compound II, is
[a] J.-L. Huang, M. Davidson, Prof. W.-c. Chang
Department of Chemistry, North Carolina State University
Raleigh, NC 27695, U.S.A.
Scheme 1. (A). AsqJ catalyzed consecutive desaturation and epoxidation. (B)
Examples of desaturation catalyzed by cytochrome P-450 and di-iron cofactors
containing enzymes.
E-mail: wchang6@ncsu.edu
[b] J. Li, I. Kurnikov, J. L. Lee, Prof. M. Kurnikova, Prof. Y. Guo
Department of Chemistry, Carnegie Mellon University
Pittsburgh, PA 15213, U.S.A.
E-mail: ysguo@andrew.cmu.edu & kurnikova@cmu.edu
[c] H.-J. Liao. T.-S. Lin, Prof. N.-L. Chan
Institute of Biochemistry and Molecular Biology, College of Medicine,
National Taiwan University, Taipei 100, Taiwan
E-mail: nlchan@ntu.edu.tw
Supporting information for this article is given via a link at the end of the
document.
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10.1002/anie.201710567
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abstraction. Subsequently, if a second H• abstraction is required
to complete desaturation, the use of 2 will abolish the C3-H•
abstraction and may lead to the acculumation of the substrate
radical species or redirect the reaction outcome from desaturation
to hydroxylation. In contrast, if 2 can be converted to 3, other
posssible pathway(s) which involve either a carbocation or a
hydroxylated intermediate would need to be considered.
To support that the substrate binding modes obtained from our
crystallographic data are energetically stable in solution, we
conducted molecular dynamics (MD) simulations on the available
crystal structures of AsqJ. MD simulations in combination of
QM/MM calculations have been previously used to elucidate
Fe/2OG enzyme catalyzed chlorination, desaturation and
epoxidation
mechanisms[6].
Using
the
published
AsqJ•Ni(II)•1•2OG structure (PDB: 5DAW) as the starting point
with the replacement of Ni(II) to Fe(II), MD equilibrated structure
of the AsqJ•Fe(II)•2OG•1 complex resembles closely to the
crystal structure described above (Fig. S8). Furthermore, in
accordance with the crystal structure of AsqJ•Fe•2OG•2 complex,
the MD simulation predicted binding configuration of 2 is similar
to that of 1, except that the C3-H points away from the iron center
(Fig. S8 and S10). In addition, MBAR computed the binding free
energy difference between 1 and 2 as ~0.4 kcal/mol with 1 having
slightly tighter binding to AsqJ (Fig. S9). Thus, both
experimentally determined and computationally simulated
structures reveal the highly similar binding configurations of 1 and
2 to AsqJ.
Scheme 2. (A) Mechanistic probes and AsqJ reaction products used in this
study. (B) Proposed pathways accounting for AsqJ catalzyed desaturation. After
C10-H activation, the reaction can proceed through (i) second hydrogen atom
abstraction (2-HAT) and followed by di-radical recombination, (ii) hydroxylation
and dehydration, or (iii) a carbocation formation and subsequent H+ removal to
complete the C=C bond installation.
To probe the potential mechanism of AsqJ catalyzed desaturation,
cyclopeptin (1), its C3-stereoisomer (2), and products (3) and (4)
were prepared (Scheme 2A)[2b, 5]. To examine the suitability of 2
as a mechanistic probe, we solved the crystal structures of
AsqJ•Fe•2OG•1 and AsqJ•Fe•2OG•2 complexes at 1.96 and 2.05
Å resolution by molecular replacement using the published
substrate bound AsqJ structure (AsqJ•Ni•2OG•1 complex)[2a] as
the search model (PDB: 5Y7R, 5Y7T) (Table S1). In contrast to
the published structures where the Fe was replaced by Ni during
protein purification[2a]
, the two structures reported herein
represent the first visualization of AsqJ in its native Fe-bound
forms. An XANES spectrum collected from an AsqJ•Fe•2OG•1
crystal unambiguously shows the presence of iron in the crystal
(Fig. S4). The structure of AsqJ•Fe•2OG•1 closely resembles the
structure of AsqJ•Ni•2OG•1 (Fig. S5)[2a]. Importantly, 1 and 2
bound AsqJ structures are also highly similar. In both structures,
the benzodiazepinedione, the phenyl group, and both C3, and
C10 centers are well defined in the electron density maps (Fig. 1,
S6 and S7). The phenyl ring of 1 or 2 is stabilized by van der
Waals interactions with residues V72, L79, M118, and F139,
along with plausible - interaction with H134 (Figure S6). The
change of chirality at the C3 position does however cause
structural flipping of C10 and a subtle reposition of the phenyl
group (Fig. 1C). Compared to the distances of C10 and C3 of 1 to
the iron center (4.8 and 4.5 Å), the corresponding distances in 2
are 3.7 and 4.9 Å, respectively (Fig. 1D). More importantly,
different from the binding configuration of 1 where the C3-H and
one of the C10-Hs are poised toward the iron center, in the
structure of AsqJ•Fe•2OG•2, the C3-H points away from the iron
center and only one of the C10-Hs is poised toward it. These
structures clearly demonstrate that 1 and its C3-epimer (2) bind
analogously to the active site of AsqJ. Furthermore, It implicates
the plausibility of using 2 to probe the desaturation mechanism.
We hypothesize that the reaction is likely to initiate from C10-H
Figure 1. Crystal structures of the AsqJ•Fe•2OG•1 and AsqJ•Fe•2OG•2. (A)
and (B) Close-up views of the AsqJ active site. The Fo-Fc electron density maps
are shown in grey mesh and contoured at 2.8 σ. The bound 2OG, 1 (panel A)
or 2 (panel B), and the Fe-ligating residues are shown in stick form. The Fe is
shown as orange sphere. The carbon atoms of 2OG, substrate, and selected
amino acid side chains are colored green (for AsqJ•Fe•2OG•1) or cyan (for
AsqJ•Fe•2OG•2). The remaining atoms (nitrogen, oxygen, sulfur) in both
structures are colored according to the CPK scheme. (C) Alteration of the
stereochemistry at the C3 (red asterisk) repositions the C3-hydrogen, C10 (red
shaded), and the phenyl group (brown shaded) of 2 (cyan) relative to 1 (green).
(D) The Fe-C3 and Fe-C10 distances observed in the 1 (left) and 2 (right) bound
AsqJ structures. (PDB: 5Y7R, 5Y7T).
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Next, we conducted stopped-flow optical absorption (SF-Abs)
measurements to reveal the effect of 1 and 2 on AsqJ reaction
kinetics. The SF-Abs experiment was carried out by rapidly mixing
an anaerobic solution containing the AsqJ•Fe(II)•2OG•subtrate
complex (AsqJ 0.5 mM, Fe(II) 0.4 mM, 2OG 4.0 mM, and 1 or 2
0.4 mM) with air-saturated buffer (~0.4 mM of O2). The SF-Abs
spectra showed a very similar kinetic behavior of the absorption
feature centered ~470 nm when either 1 or 2 was used (Fig. 2).
This feature represents the metal-to-ligand charge transfer
(MLCT) band of the Fe(II)-2OG complex[7] and similar decay
kinetics suggests that both 1 and 2 can trigger 2OG consumption
with similar efficacy. In addition, a simultaneous increase of
absorption at ~310 nm was observed (Fig. 2, S15). In
characterized Fe/2OG enzymes[8], the absorbance change at
~310 nm is attributed to the accumulation of an Fe(IV)-oxo
intermediate. However, based on Mössbauer and LC-MS results
(see below), the absorbance change at ~310nm likely
represented formation of 3. The highly similar kinetic behavior of
1 and 2 implies that the C3 chirality is not critical for the reaction.
originated from a high-spin ferrous species (see Figure S13 for
detailed analysis) and can be tentatively assigned as an
AsqJ•Fe(II)•succinate•3 complex. This assignment was
corroborated by the observation of an identical quadrupole
doublet obtained by
a sample containing an anaerobic
AsqJ•Fe(II)•3•succinate complex (Fig. S11, S13, Table S5).
However, this product complex is distinct from the product
complex observed in the AsqJ-catalyzed epoxidation reaction[2b]
and the product bound TauD complex[9] (Table S4). At 1.5 s, the
same enzyme-product complex decreased to ~20%. The lack of
accumulation of the Fe(IV)-oxo intermediate suggests that the
benzylic H• abstraction is probably not the rate-limiting step under
the current experimental conditions.
0.0
1.5 s
1.0
0.0
0.2 s
0.0
0.01 s
1.0
0.0
0 s
1.0
4.2 K, 0 T
-6 -4 -2
0
2
4
6
Velocity (mm/s)
Figure 3. 4.2
K
zero-field Mössbauer spectra of the reaction of
Figure 2. SF-Abs kinetics of AsqJ catalyzed reactions by using 1 or 2. Left:
Absorption changes at the indicated reactions times after mixing anaerobic
AsqJ•Fe(II)•2OG•1 complex with O2 (inset: absorption changes centered at 470
nm). Right: The decay of Fe(II)-2OG MLCT band at 470 nm (left vertical axis)
and the increase of the absorption at ~310 nm (right vertical axis) in the reaction
of the AsqJ•Fe(II)•2OG•substrate complex (1, black open circle or 2, blue full
circle) with O2.
AsqJ•Fe(II)•2OG•1 with O2 at different times. Black vertical bars: experimental
data; black lines: overall spectral simulations (see Table S2); blue lines:
AsqJ•Fe(II)•2OG•1 complex; green lines: AsqJ•Fe(II)•succinate•3 complex;
grey line: the inactive enzyme (< 10% of the total enzyme). The arrows point to
the portion of the spectra at ~0 mm/s that is likely due to the presence of high-
spin ferric species by enzyme auto-oxidation (Fig. S12) and is not properly
represented by the simulations that only include quadrupole doublets.
To reveal plausible changes to the iron center during the reaction,
freeze-quench coupled Mössbauer experiments were performed.
The anaerobic AsqJ•Fe(II)•2OG•1 complex showed a quadrupole
doublet with parameters of a high-spin ferrous species (isomer
shift δ = 1.25 mm/s and quadrupole splitting |ΔEQ| = 2.54 mm/s
(Fig. 3, Table S2 and S3). This assignment was validated by the
variable field/temperature measurements (Fig. S14, Table S5).
The parameters of the AsqJ•Fe(II)•2OG•1 complex are identical
to those of the AsqJ•Fe(II)•2OG•3 complex (Fig. S11), but are
distinct from those of the TauD•Fe(II)•2OG•taurine complex (δ =
1.16 mm/s, |ΔEQ| = 2.76 mm/s)[8a]. Compared to the anaerobic
sample, the sample quenched at 0.01 s showed ~ 20%
consumption of the substrate-AsqJ complex. However, no
obvious spectral component representing an Fe(IV)-oxo species
was observed. Instead, a new quadrupole doublet, with δ = 1.28
mm/s and |ΔEQ| = 1.83 mm/s, developed to ~15% of the total iron
(Fig. 3 and Table S2, S3). This species increased to ~30% at 0.2
s with the corresponding decrease of the AsqJ•Fe(II)•2OG•1
complex to ~40%. This newly formed quadrupole doublet is
Finally, LC-MS analysis was employed to determine the outcome
of the AsqJ-catalyzed reaction when 2 was used as a substrate.
When the anaerobic AsqJ•Fe(II)•2OG•2 complex was incubated
with excess amount of O2 for ~20 min, complete consumption of
2 and production of the epoxide (4) were observed (similar to the
reaction when 1 was used as a substrate). When 2OG was absent,
no production of 4 was detected (Fig. 4A and B). This observation
implies that 2 is converted to 4 through intermediate 3, which was
previously established for 1[1]. To establish the production of 3
when 2 was used, chemical quench coupled LC-MS analysis was
conducted. LC-MS analysis revealed time-dependent product
formation (Fig. 4C). The reaction quenched at 0.2 s showed that
3 was the major product. Subsequently, the sample quenched at
1.5 s showed ~25% increase of 3 production. At 20 s, the amount
of 3 decreased and 4 became the major product. Next, the
enzymatic assay conducted under limiting 2OG condition
revealed that substrate consumption and desaturated product (3)
formation were at the similar level when either 1 or 2 was tested
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10.1002/anie.201710567
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(Fig. S16). These results clearly suggest that 1 and 2 are
comparable substrates for AsqJ.
This work was supported by North Carolina State University,
Carnegie Mellon University, National Taiwan University, and
Ministry of Science and Technology grant 106-2113-M-002-021-
MY3, R.O.C., Taiwan, We thank Mr. Andrew Weitz and Prof.
Michael P. Hendrich for the use of the FQ apparatus. We also
thank Mr. Serzhan Sakipov for the MD simulations.
Conflict of Interest
The authors declare no conflict of Interest.
Keywords: desaturation • C-C bond formation • enzyme
mechanism • viridicatin • carbocation
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Figure 4. LC-MS chromatogram of AsqJ catalyzed reactions. In panel (A), 1
and 2 were consumed when 2OG was added; (B) 1 and 2 were converted to
epoxide 4; and (C) chemical quench results of 2 at 0.2, 1.5 and 20 seconds and
anaerobic control sample revealed the formation of 3 and 4 at different times.
In summary, the experimental data presented herein suggest that
both 1 and 2 can be converted to cyclopenin (4) through a
common intermediate 3 during AsqJ catalysis. Crystallographic
structures and MD simulations, together with the SF-Abs,
Mössbauer, and LC-MS analyses on the reaction of
AsqJ•Fe•2OG•2 (or 1) complex reveal that the chirality of C3 is
not critical to the desaturation reaction. Furthermore, in the case
of 2, these results suggest that the primary H• abstraction site is
at C10 and the subsequent C3-H• abstraction is unlikely due to
spatial conformation of 2 in the AsqJ active site. Thus, the AsqJ
catalyzed desaturation is likely initiated from C10-H bond
activation by a short-lived Fe(IV)-oxo species. Subsequently, a
second H• abstraction pathway that has been proposed in P450,
di-iron and non-heme iron desaturases[3] is less likely to operate.
Instead, pathways involving a carbocation or a hydroxylated
intermediate are more likely to be utilized in AsqJ catalyzed
desaturation (Scheme 2).
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Acknowledgements
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COMMUNICATION
An interesting twist in installing a
C=C bond was revealed. Desaturation
reaction mechanism catalyzed by
AsqJ, a non-heme iron enzyme, was
elucidated using a complementary
approach including probe design, X-
ray crystallography, molecular
Hsuan-Jen Liao, Jikun Li, Jhih-Liang
Huang, Madison Davidson, Igor
Kurnikov, Te-Sheng Lin, Justin L. Lee,
Maria Kurnikova,* Yisong Guo,* Nei-Li
Chan,* and Wei-chen Chang*
Page No. – Page No.
dynamics simulations, and
Insights into desaturation of
cyclopeptin and its C3-epimer
catalyzed by a non-heme iron
enzyme: structural characterization
and mechanism elucidation
spectroscopic characterizations.
Subsequent to the primary C-H bond
activation, a carbocation or a
hydroxylated intermediate is likely to
be involved in the desaturation.
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