Pathway from N-Alkylglycine to Alkylisonitrile Catalyzed by Iron(II) and 2-Oxoglutarate-Dependent Oxygenases

Article abstract of DOI:10.1002/anie.201914896

N-alkylisonitrile, a precursor to isonitrile-containing lipopeptides, is biosynthesized by decarboxylation-assisted -N≡C group (isonitrile) formation by using N-alkylglycine as the substrate. This reaction is catalyzed by iron(II) and 2-oxoglutarate (Fe/2OG) dependent enzymes. Distinct from typical oxygenation or halogenation reactions catalyzed by this class of enzymes, installation of the isonitrile group represents a novel reaction type for Fe/2OG enzymes that involves a four-electron oxidative process. Reported here is a plausible mechanism of three Fe/2OG enzymes, Sav607, ScoE and SfaA, which catalyze isonitrile formation. The X-ray structures of iron-loaded ScoE in complex with its substrate and the intermediate, along with biochemical and biophysical data reveal that -N≡C bond formation involves two cycles of Fe/2OG enzyme catalysis. The reaction starts with an Fe^IV-oxo-catalyzed hydroxylation. It is likely followed by decarboxylation-assisted desaturation to complete isonitrile installation.

Full text of DOI:10.1002/anie.201914896

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
Chemie
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Accepted Article
Title: Pathway from N-alkylglycine to alkylisonitrile catalyzed by iron(II)
and 2-oxoglutarate dependent oxygenases
Authors: Wei-Chen Chang, Tzu-Yu Chen, Jinfeng Chen, Yijie Tang,
Jiahai Zhou, and Yisong Guo
This manuscript has been accepted after peer review and appears as an
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201914896
Angew. Chem. 10.1002/ange.201914896
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http://dx.doi.org/10.1002/ange.201914896

10.1002/anie.201914896
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Pathway from N-alkylglycine to alkylisonitrile catalyzed by iron(II)
and 2-oxoglutarate dependent oxygenases
[b],†
[c]
[b]
[c]
[a]
Tzu-Yu Chen,[a], Jinfeng Chen, Yijie Tang, Jiahai Zhou,* Yisong Guo,* and Wei-chen Chang*
†
We dedicate this paper to Professor Youli Xiao
[a]
[b]
[c]
Tzu-Yu Chen, Dr. Wei-chen Chang
Department of Chemistry
North Carolina State University
Raleigh, NC 27695, U.S.A.
E-mail: wchang6@ncsu.edu
Jinfeng Chen, Dr. Jiahai Zhou
State Key Laboratory of Bio-organic and Natural Products Chemistry, Center for Excellence in Molecular Synthesis
Shanghai Institute of Organic Chemistry, University of Chinese Academy of Sciences
Shanghai 200032, China
Email: jiahai@mail.sioc.ac.cn
Yijie Tang, Dr. Yisong Guo
Department of Chemistry
Carnegie Mellon University
Pittsburgh, PA 15213, U.S.A.
Email: ysguo@andrew.cmu.edu
Supporting information for this article is given via a link at the end of the document.
Abstract: N-alkylisonitrile,
a
precursor to isonitrile-containing
species which is used to activate the targeted C-H bond or acts
as an oxygen atom donor to add onto the olefin moiety.[5] For
those involving C-H activation, subsequent to hydrogen atom (H•)
abstraction, an iron bound ligand transfers to the substrate radical
to form the product and regenerates the Fe(II) (Scheme 1B).[4d, 6]
lipopeptides, is biosynthesized via decarboxylation-assisted -N≡C
group (isonitrile) formation by using N-alkylglycine as the substrate.
This reaction is catalyzed by iron(II) and 2-oxoglutarate (Fe/2OG)
dependent enzymes. Distinct from typical oxygenation or
halogenation reactions catalyzed by this class of enzymes, installation
of the isonitrile group represents a novel reaction type for Fe/2OG
enzymes that involves a four-electron oxidative process. Here we
report the plausible mechanism of three Fe/2OG enzymes, Sav607,
ScoE and SfaA, catalyzed isonitrile formation. The X-ray structures of
iron loaded ScoE in complex with its substrate and the intermediate,
along with biochemical and biophysical data reveal that -N≡C bond
formation involves two cycles of Fe/2OG enzyme catalysis. The
reaction starts with an Fe(IV)-oxo catalyzed hydroxylation. It is likely
followed by decarboxylation-assisted desaturation to complete
isonitrile installation.
Isonitrile-containing natural products have shown diverse
biological activities.[1] Notably, two types of enzymes are involved
in isonitrile formation (Scheme 1A). The first type employs an
isonitrilase catalyzed condensation of the amine from the L-
tyrosine/tryptophan with the carbonyl group of ribulose-5-
phoshate.[2] The second type involves an iron(II) and 2-
Scheme 1 A) Two different approaches used to install isonitrile. B) Fe/2OG
enzyme catalyzed hydroxylation.
Differently, Fe/2OG enzyme catalyzed isonitrile formation is a
four-electron oxidation and formally involves two C-H bond
cleavage steps and a decarboxylation. Initial biochemical and
structural characterizations of ScoE suggested that it is
responsible for isonitrile formation.[3] The structure of zinc-bound
ScoE indicates that it belongs to the cupin superfamily and has
the 2-His-1-carboxylate triad as the iron-binding ligands.[3a]
However, in this structure, the potential substrate-binding site was
occupied by choline and the putative 2OG binding site was
resided by acetate. Based on this structure, the reaction
mechanism has been postulated to involve a hydroxylated imine
species (5) that is likely generated via hydroxylation of the imine
intermediate (4). Subsequent dehydration/decarboxylation of 5
oxoglutarate
(Fe/2OG)
dependent
enzyme
catalyzed
decarboxylation-assisted -N≡C group installation.[3] Analysis of
the biosynthetic gene clusters responsible for the Fe/2OG
enzyme catalyzed isonitrile formation revealed that the pathway
involved in isonitrile-containing peptides production is conserved
in
pathogenic
mycobacteria
including
Mycobacterium
tuberculosis, and M. marinum. Related operons are also found in
the phylum of Actinobacteria including Streptomyces.[1a, 3b]
The majority of Fe/2OG enzymes catalyze two-electron oxidative
modifications
that
include hydroxylation,
desaturation,
epoxidation, halogenation and endoperoxide installation.[4]
Addition of O2 to the 2OG bound Fe(II) generates an Fe(IV)-oxo
1
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produces 2 (Scheme 2, pathway i). To date, this is the only
experimentally characterized Fe/2OG isonitrile forming enzyme.
To demonstrate a conserved approach may be utilized in
introducing isonitrile functionality among isonitrile-containing
peptides production, we use crystallographic, biochemical and
biophysical methods to study three enzymes, Sav607, ScoE and
from the iron with one of its hydrogens pointing toward to the iron.
Compared to other Fe/2OG enzyme structures including TauD
(PDB ID: 1os7), CarC (PDB ID: 4oj8), and PvcB (PDB ID: 3eat),
a tartrate molecule is most likely positioned at the 2OG binding
site. A putative quaternary complex structure of ScoE•Fe•2OG•1
is modeled by docking the 2OG molecule into the substrate
structure (Figure S20). In addition, a presumptive intermediate (3)
bound ScoE structure with resolution of 2.17 Å was obtained
(Figure 1B-C, S21, S22, S23 and Table S4). The electron density
map of the structure clearly shows that this compound resembles
the possible C5 hydroxylated product (3). Besides, the apo form
of SfaA is highly similar to the structure of ScoE with rmsd of ~0.37
Å (Figure S24 and Table S4).
SfaA,
originated
from
Streptomyces
avermitilis,
S.
coeruleorubidus and S. thioluteus, that are known, or are
predicted, to catalyze isonitrile formation.[1a, 3b, 7]
To establish the reaction pathway, the stoichiometry between
2OG and 1 was established. During Fe/2OG enzyme reaction, the
Fe(II) is converted to the Fe(IV)-oxo species by reacting with O2
and 2OG where 2OG is converted to succinate and CO2 (Scheme
1B). Thus, the stoichiometry of 2OG and 1 can be established by
quantifying succinate, and the isonitrile (2). Reactions with
different amount of 2OG were carried out and analyzed by LC-MS
(detailed in SI). As depicted in Figure S16, the peak with the same
retention time and mass/charge (m/z) as the synthetic 2
accumulated when 2OG and O2 were introduced. By quantifying
succinate and 2, the stoichiometry between succinate and 2 is
determined as 2.3, 3.2 and 4.5 for SfaA, Sav607 and ScoE
(Figure S17). Compared to SfaA, the higher uncoupling ratio
observed in Sav607 and ScoE possibly arise from unproductive
pathways. Similar uncoupling reactions have been observed in
other Fe/2OG enzymes.[8] These results suggest that for one
equivalent of 2 produced, two equivalents of 2OG are required.
Thus, isonitrile group installation likely involves two sequential
reactions.
Scheme 2 Possible pathways for isonitrile formation. Starting from C5-
hydroxylation, two pathways involving either second hydroxylation (pathway i)
or decarboxylation-assisted desaturation (pathway ii) are proposed.
To overcome the reported issues of co-purification of zinc and
choline with the protein, and the instability of the product that
hamper direct mechanistic studies and structural analysis,[3a] we
expressed and purified Sav607, ScoE and SfaA as metal-free
proteins by chelating the co-purified metal using EDTA and then
introduced Fe(II) in all experiments. A synthetic approach was
developed to afford substrate (1), its isotope-labeled analogs ([5-
2H2-1] and [5-13C-1]), product (2), and mechanistic probes (cis-4
and 6) (detailed in Supporting Information (SI)).
We first carried out structural analysis. All three proteins were
subjected to crystallization conditions, the ScoE and SfaA
samples yielded diffraction quality crystals. Co-crystals of the
substrate (1) and presumptive intermediate (3) bound ScoE were
obtained by the sitting drop vapor diffusion technique (detailed in
the SI). The structure of 1 and iron bound ScoE (ScoE•Fe•1) was
then solved at a resolution of 2.18 Å. The overall structure
contains 15 strands of β-sheet and 8 α-helices, eight of which
forming a jellyroll motif (β3, β13, β5, β15, β4 and β12, β6, β14)
(Figure 1A-B, S20 and Table S4). Dali search suggested the
structure of ScoE resembles those of other Fe/2OG-enzymes
such as CarC (PDB ID: 4oj8) and a putative dioxygenases (PDB
ID: 4y0e), with Z-score of 20.0 and 26.7, respectively. In the
structure of ScoE, the octahedron geometry of iron is bound by a
2-His-1-Asp facial triad (H132, D134, and H295). The fourth
coordination site is occupied by a tartrate (TAR) molecule that is
acquired during crystallization. The remaining two coordination
positions are occupied by water molecules (W1 and W2).
Significant electron density corresponding to 1 is visualized in the
active site which has hydrogen-bonding interactions with Y101,
K193, G128, Y96, Y135 and R310. Importantly, this structure
implies the plausible reaction site where the C5 of 1 is 4.9 Å away
2
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Figure 1. Crystal structures of ScoE. A) The overall structure of ScoE is shown
in cartoon model, with the jellyroll motif colored in cyan. 2Fo-Fc (light gray mesh,
contoured at 1.0 σ) electron density map for 1. Dashed lines illustrate hydrogen-
bonding interactions involving 1 or bonds between the iron and its ligands. B)
Active site of ScoE in the presence of 1 or 3. Putative 2OG binding sites appear
to be occupied by exogenous ligands colored in forest. The 2-His-1-Asp triad is
shown in wheat stick format. Molecules 1 and 3 are colored in cyan, the electron
density maps for 1 and 3 are shown in dark-gray mesh and contoured at 1.0 σ.
Iron and waters are shown in orange and red sphere. Putative 2OG binding sites
appear to be occupied by exogenous ligands (TAR or FMT) colored in forest.
C) Superposition of ScoE•Fe•1 and ScoE•Fe•3.
Figure 2. 13C-NMR of SfaA catalyzed 2 formation under C-H decoupling and C-
H coupling conditions. The top, middle and bottom traces represent the 13C-
NMR recorded with 5-13C-1 to 2OG ratio at 1:0, 1:1.5 and 1:2.5, respectively.
The requirement for the Fe(IV)-oxo, a.k.a. ferryl, species to cleave
C5-H bond is verified by the Mössbauer analysis. Samples were
prepared by mixing the SfaA•Fe(II)•2OG•1 or 5-2H2-1 complex
(the quaternary complex) with oxygenated buffer and freeze
quenching at various time points. The Mössbauer spectra of
SfaA•Fe(II)•2OG•1 or 5-2H2-1 complex exhibit a quadrupole
doublet with an isomer shift (δ) of 1.23 mm/s and quadrupole
splitting (ΔEQ) of 2.74 mm/s, which represents high-spin ferrous
species (Figure 3, S19 and Table S1-S3). When 5-2H2-1 was used,
the sample quenched at 0.03 s showed ~30% decrease of the
SfaA•Fe(II)•2OG•5-2H2-1 complex with the simultaneous
accumulation of a species with parameters of δ = 0.31 mm/s and
ΔEQ = 0.59 mm/s, representing ~20% of the total iron. This new
species is similar to the ferryl intermediate observed in other
Fe/2OG enzymes.[5b, 9] In addition, a minor ferrous species with
parameters of δ = 1.25 mm/s and ΔEQ = 1.90 mm/s (~13% of the
iron in the sample) was also observed. This ferrous species was
more readily accumulated in the sample quenched at 0.03 s when
1 was used, which represented ~ 25% of the total iron. We
tentatively assign this ferrous species as a potential product
complex. Importantly, compared to the reaction using 5-2H2-1, the
sample quenched at 0.03 s using 1 did not show a significant
accumulation of the ferryl intermediate (< 5%). Thus,
accumulation of the ferryl species using 5-2H2-1 is likely due to the
kinetic isotope effect from the replacement of the targeted C5-H
bond with the C5-D bond. Overall, Mössbauer analysis suggests
that the initial C-H activation by the ferryl intermediate occurs at
the C5 position of 1.
The presence of the C5-hydroxyalted compound (3) was
established by the analysis of the 13C-NMR spectrum of a sample
prepared by reacting SfaA with 5-13C-1, O2 and 2OG (with the
molar ratio of 5-13C-1 to 2OG of 1:1.5). The spectrum recorded
under C-H decoupling mode exhibits a new peak at ~91 ppm that
is not present in the absence of 2OG. The spectrum collected
under C-H coupling mode reveals a doublet with a coupling
constant of ~163 Hz (Figure 2 and S15). The chemical shift and
the coupling pattern indicate that this carbon is connected to one
proton, one carbon and two heteroatoms. This result is consistent
with the ScoE•Fe•1 structure where the C5-H of 1 points toward
the iron and observation of the ScoE•Fe•3 structure. When the
ratio of 5-13C-1 to 2OG changed from 1:1.5 to 1:2.5, we observed
a decrease of this species (3) and the appearance of the new
peak centered at ~150 ppm. The NMR spectrum obtained under
C-H coupling mode indicates that no proton is connected to this
newly formed carbon. This newly formed peak has the same
chemical shift as of the synthetic 2 (Figure S11 and S15). These
results suggest that the isonitrile formation is at the expense of
2OG and 3 serves as the possible intermediate during isonitrile
formation.
Figure 3. The Mössbauer spectra of SfaA•Fe(II)•2OG•5-2H2-1 and
SfaA•Fe(II)•2OG•1 are shown in A and E; The spectra of the samples quenched
at 0.03 s are shown in B and F; The difference spectra between A and B (B-A),
E and F (F-E), and the spectral simulations (grey lines) are shown in C and G,
which indicate the decrease of the quaternary complex (upward blue
simulations), the accumulation of the ferryl intermediate (the red simulations)
and the additional ferrous species (the green simulations in D and H). See Table
S1-S3 for details simulation parameters.
In the structure of ScoE•Fe•3, the C5 of 3 moves ~0.4 Å closer to
the iron center (4.9 → 4.5 Å from ScoE•Fe•3 to ScoE•Fe•1).
However, the orientation of 3 is probably not at optimal position
for H• abstraction where the C5-H is not pointing toward the iron.
Meanwhile, the N atom located ~5.9 Å away from the iron center
may also not be properly positioned to interact with the iron
3
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10.1002/anie.201914896
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(Figure 1C, Figure S22). To reveal possible pathway involved in
the second reaction, the proposed intermediates 4 and 6 were
synthesized. As shown in Scheme 2, pathway i, followed by
hydroxylation at the C5 position of 4, 5, which likely tautomerizes
to 6 rapidly, may proceed through dehydration/decarboxylation to
produce 2. Alternatively, second hydroxylation of 3 will produce 6
through the proposed intermediate 7. After incubating enzyme, Fe,
2OG, O2 and cis-4, the reactions were analyzed by LC-MS. Under
the current conditions, no production of 2 can be detected. When
6 was tested with enzyme under anoxic conditions or in the
presence of 2OG and O2, production of 2 was not observed
(Figure S18). Although it is possible that cis-4 or 6 cannot enter
the active site, or the trans-4 serves as the actual intermediate,
these results imply the pathway involving 4 or 6 is less likely and
suggest that the alternative pathway may be utilized (pathway ii)
where isonitrile formation possibly involves a reactive iron species
assisted decarboxylation. Namely, during the second reaction,
the presumptive ferryl species is used to trigger decarboxylation
by activating the N and the reaction is then followed by
dehydration (3  8  2). Alternatively, subsequent to dehydration
of 3, an analogous decarboxylation will result in protonated
isonitrile (9). Although it is less common, desaturation via
oxidative decarboxylation has been reported in several Fe/2OG
enzymes catalyzed olefination.[10] In addition, N-hydroxylation
was reported in a non-heme iron enzyme catalyzed reaction in
streptozotocin biosynthesis.[11]
In short, in studying three Fe/2OG enzyme catalyzed isonitrile
installation, the experimental analyses establish that isonitrile
formation goes through two consecutive, but distinctive, reactions.
In the first reaction, an Fe(IV)-oxo species is utilized to generate
C5-hydroxylated product (3). Conversion of 3 to 2 likely proceeds
by decarboxylation-assisted desaturation. Although the detailed
mechanism remains to be evaluated, these observations enrich
our view in diverse strategies that Fe/2OG enzymes used to
catalyze novel reactions. Considering the conserved biosynthetic
approach used to install the isonitrile-containing peptides found in
several pathogenic bacteria, these observations may shed light
for development of new anti-virulence therapeutics target for
bacterial infection.
Keywords: Fe/2OG enzyme • isonitrile • enzyme mechanism •
decarboxylation • catalysis
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This work was supported by North Carolina State University,
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Research
and
Development
Program
of
China
(2018YFA0901900 to J.Z.), Shanghai Municipal Science and
Technology Commission (19XD1404800 to J.Z.), and the
National Institutes of Health (GM127588 to W.-c. C., and Y. G.).
We thank the staffs in beamline BL17U1 and BL19U1 of Shanghai
Synchrotron Radiation Facility for help in data collection. We also
thank Prof. Jianhua Gan for his help in structure refinement.
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Author Contributions
†These authors contributed equally.
4
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A subclass of Fe/2OG enzyme catalyzes decarboxylation-assisted desaturation of N-alkyl glycine to install the isonitrile functionality.
By using a complementary approach including structure determination, NMR, and Mössbauer analyses, the reaction intermediate and
plausible reaction mechanism is established.
5
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