Isonitrile Formation by a Non-Heme Iron(II)-Dependent Oxidase/Decarboxylase

Article abstract of DOI:10.1002/anie.201804307

The electron-rich isonitrile is an important functionality in bioactive natural products, but its biosynthesis has been restricted to the IsnA family of isonitrile synthases. We herein provide the first structural and biochemical evidence of an alternative mechanism for isonitrile formation. ScoE, a putative non-heme iron(II)-dependent enzyme from Streptomyces coeruleorubidus, was shown to catalyze the conversion of (R)-3-((carboxymethyl)amino)butanoic acid to (R)-3-isocyanobutanoic acid through an oxidative decarboxylation mechanism. This work further provides a revised scheme for the biosynthesis of a unique class of isonitrile lipopeptides, of which several members are critical for the virulence of pathogenic mycobacteria.

Full text of DOI:10.1002/anie.201804307

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Accepted Article
Title: Isonitrile Formation by a Non-heme Iron(II)-Dependent Oxidase/
Decarboxylase
Authors: Nicholas Harris, David Born, Wenlong Cai, Yaobing Huang,
Joelle Martin, Ryan Khalaf, Catherine Drennan, and Wenjun
Zhang
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201804307
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10.1002/anie.201804307
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Isonitrile Formation by a Non-heme Iron(II)-dependent
Oxidase/Decarboxylase
Nicholas C. Harris,[c] David A. Born,[d][e] Wenlong Cai,[a] Yaobing Huang,[a] Joelle Martin,[f] Ryan Khalaf,[f]
Catherine L. Drennan,[d][g][h] Wenjun Zhang*[a][b]
Abstract: The electron-rich isonitrile is an important functionality in
bioactive natural products, but its biosynthesis has been restricted to
the IsnA family of isonitrile synthases. We here provide the first
structural and biochemical evidence of an alternative mechanism for
isonitrile formation. ScoE, a putative non-heme iron(II)-dependent
enzyme from Streptomyces coeruleorubidus, was shown to catalyze
the conversion of (R)-3-((carboxymethyl)amino)butanoic acid to (R)-
3-isocyanobutanoic acid through an oxidative decarboxylation
mechanism. This work further provides a revised scheme for the
biosynthesis of a unique class of isonitrile lipopeptides, of which
several members are critical for the virulence of pathogenic
mycobacteria.
S1). Despite the widespread utility of isonitrile in nature, its
biosynthesis has long been considered endemic to the IsnA
family of isonitrile synthases, which typically convert an α-amino
group to isonitrile on an amino acid and require ribulose-5-
phosphate as a co-substrate (Figure S2).1,5–8
Our recent genome mining of a conserved gene cluster
widely present in Actinobacteria indicated an alternative route for
isonitrile formation.9 In particular, we identified and proposed the
function of five genes required for the biosynthesis of a unique
class of isonitrile lipopeptides (INLPs) that are critical for the
virulence of pathogenic mycobacteria. Taking the pathway from
Streptomyces coeruleorubidus as an example, the biosynthesis
was proposed to start with the activation and loading of crotonic
acid onto ScoB, an acyl carrier protein (ACP) by ScoC, an acyl-
ACP ligase. A Michael addition of Gly to the β-position of
crotonyl-ScoB is then promoted by ScoD, a thioesterase to form
a Gly adduct 1, followed by oxidation and decarboxylation,
presumably catalyzed by ScoE, a non-heme iron(II)-dependent
oxygenase, to generate a β-isonitrile fatty acyl-ACP intermediate
2. This β-isonitrile acyl moiety is then condensed to both amino
The electron-rich functionality of the isonitrile lends itself as a
biologically active warhead for naturally derived products. Due to
its ability to coordinate transition metals, it is often exploited for
metal acquisition, detoxification, and virulence.1–3 Indeed the
resume of potent biologically active isonitrile containing natural
products is vast, and examples include xanthocillin, an antiviral
agent;4 rhabduscin,
inhibitor;2 and many marine sponge derived metabolites (Figure
a virulence associated phenoloxidase
groups of Lys promoted by ScoA,
a single-module non-
ribosomal peptide synthetase (NRPS), and reductively released
to form a terminal alcohol product 3 (Figure 1). ScoE thus
represents a new family of enzymes distinct from isonitrile
synthases that promote the transfer of one carbon from ribulose-
5-phosphate to an amino group to form isonitrile. Although the
function of ScoE was reconstituted in E. coli for 3 biosynthesis,
in vitro reconstitution of its activity based on the proposed
biosynthetic pathway repeatedly failed.
[a]
W. Zhang, W. Cai, Y. Huang
Department of Chemical and Biomolecular Engineering
University of California Berkeley
Berkeley, California 94720 United States
E-mail: wjzhang@berkeley.edu
W. Zhang
Chan Zuckerberg Biohub
San Francisco, California, 94158, United States
N. C. Harris,
[b]
[c]
Department of Plant and Microbial Biology
University of California Berkeley
Berkeley, California 94720 United States
D. A. Born, C. L. Drennan
We previously proposed that ScoE functions on an ACP-
bound intermediate because biochemical analysis of pathway
enzymes showed that the formation of 1 requires ScoB, and the
[d]
[e]
[f]
action of the NRPS, ScoA, also requires
a ScoB-bound
Department of Biology
Massachusetts Institute of Technology
Cambridge, Massachusetts 02139 United States
D. A. Born
Graduate Program in Biophysics
Harvard University
Cambridge, Massachusetts 02138 United States
J. Martin, R. Khalaf
Department of Chemistry
University of California Berkeley
Berkeley, California 94720 United States
C. L. Drennan
Howard Hughes Medical Institute
Massachusetts Institute of Technology
Cambridge, Massachusetts 02139 United States
C. L. Drennan
Department of Chemistry
Massachusetts Institute of Technology
Cambridge, Massachusetts 02139 United States
substrate. The proposed pathway thus accounts for the
necessity and sufficiency of these five core biosynthetic
enzymes for INLP synthesis (Figure 1). However the recent
biochemical and structural characterization of three ScoD
homologues indicated that these thioesterases have dual
functions with enzymatic hydrolysis occurring immediately after
the Michael addition, both steps mediated by a single Gly
residue in the active site.10–12 These results raised the question
of what the true substrate of ScoE is. We thus initiated an effort
to reconstitute the in vitro activity of ScoE using various
substrates that were chemically or chemoenzymatically
synthesized. In particular, we chemically synthesized (R)-3-
((carboxymethyl)amino)butanoic acid (CABA) (4) and CABA-
CoA that was used alone or with the phosphopantetheinyl
transferase from Bacillus subtilis (Sfp) to form CABA-ScoB (1)
(Figure S3-6). Initial attempts to reconstitute the activity of ScoE
were unsuccessful regardless of the substrate used.
[g]
[h]
Supporting information for this article is given via a link at the end of
the document.
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10.1002/anie.201804307
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Figure 1. Schematic of isonitrile lipopeptide biosynthetic pathway. This revised pathway shows that ScoE utilizes a free acid substrate, 4, and after isonitrile
formation, the product 5 is reactivated and loaded onto ScoB by ScoC.
Meanwhile, we obtained an X-ray crystal structure of ScoE
to 1.8 Å-resolution (Figure 2, S7; SI Table 1). The structure of
ScoE is similar to the TauD family of non-heme iron(II) enzymes
with root mean square deviation (RMSD) values of 2.1-2.7 Å (for
C atoms) to structurally characterized TauD enzymes, although
the sequence identity is only 20-28% to these same enzymes
(Figure S8).13 The 2-His-1-Asp facial triad (H132, D134, and
H295) and an Arg (R310) within the substrate binding site are
conserved with TauD family members. Putative ScoE substrate
and alpha-ketoglutarate (KG) binding sites appear occupied by
exogenous ligands (Figure 2, A and C). Particularly, a Zn(II) is
bound by the 2-His-1-Asp facial triad (H132, D134, and H295).
The fourth coordination position on the Zn(II) is occupied by an
acetate molecule that is acquired from the crystallization
condition and is bound analogously to KG in the structure of
TauD (Figure 2, C and D).14,15 In the putative substrate binding
pocket, a Cl- is bound in a position similar to that of the sulfonate
moiety of taurine in a substrate-bound structure of TauD (Figure
2, C and D).14,15 Adjacent to the Cl- is electron density that is
consistent with a molecule of choline, oriented such that the
Figure 2. Structure of ScoE (PDB 6DCH) compared to TauD (PDB 1OS7).15
A) Overall structure of a ScoE protomer shown in yellow ribbon representation.
The metal-coordinating 2-His-1-Asp facial triad and a conserved active site
positive charge of the trimethylamine moiety is pointing toward
Cl- (Figure 2C).
Arg are shown in yellow ball-and-stick representation. Acetate and choline
ligands within the active site are shown in green ball-and-stick. Only one of the
two observed conformations of choline is shown for clarity. Zn(II) and Cl- are
shown as gray and green spheres, respectively. B) Overall structure of a TauD
protomer shown in teal ribbon representation and oriented similarly to ScoE in
A). The metal-coordinating 2-His-1-Asp facial triad and a conserved active site
Arg are shown in teal ball-and-stick representation. Taurine and KG within
the active site are shown in tan ball-and-stick. Fe(II) is shown as an orange
sphere. C) Zoomed in view of the active site of ScoE. Composite omit density
is shown in Figure S7. D) Active site of TauD displayed similarly to C).
Since choline and Zn(II) were identified within the crystal
structure of ScoE but absent from the crystallization condition,
we hypothesized that choline and Zn(II) were co-purified with
ScoE during protein purification and they could potentially
interfere with substrate binding and Fe(II) reconstitution of the
holo-enzyme. To mitigate the problem of unwanted choline that
may be abundant in LB media used for protein purification,16 we
then used M9 defined medium for future purifications of ScoE
(Figure S9). ScoE was reconstituted immediately before each
assay with fresh Fe(II) to form the holo-protein.
We then incubated holo-ScoE with KG, ascorbate, and
either CABA-ScoB (1), CABA (4), or CABA-CoA. Upon
incubation of ScoE with 4 and subsequent LC-HRMS analysis,
we observed a mass spectrum associated with the formation of
(R)-3-isocyanobutanoic acid (5) (Figure 3A; S10). This product
was not observed when either 4, KG, or ScoE was omitted, or
when boiled ScoE was added to the reaction. To further confirm
this
result,
we
synthesized
(R)-3-
substrate
((carboxymethyl)amino)butanoic-5-13C-acid as
a
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tolerance was used. The deconvoluted mass spectrum of 2 is displayed on the
right. C) Extracted ion chromatograms showing the formation of 3 in the total
enzymatic synthesis using ScoA, ScoB, ScoC, ScoE and 4. The calculated
mass of 3 with a 10-ppm mass error tolerance was used.
(Figure S11). When ScoE was incubated with this labeled
substrate, the expected mass spectral shift of the product was
observed (Figure S10). This mass spectrum was observed only
when all necessary components of the ScoE reaction were
included and only when the labeled substrate was utilized. We
also confirmed the identity of the product 5 from the enzymatic
reaction by comparing to a chemically synthesized standard
(Figure 3A). In addition, the presence of the unique isonitrile
functionality in 5 was confirmed by click reactions with tetrazines
(Figure S12). The absolute configuration of the C3 of the
enzymatic product 5 was determined to be R, demonstrating that
the chirality at this position was retained during the ScoE-
catalyzed reaction (Figure S13). We further determined the
The reconstitution of ScoE activity using the free acid
substrate, 4, raised additional questions about the function of
enzymes in INLP biosynthesis. We have previously shown that
the NRPS, ScoA, requires a ScoB-bound substrate for the
subsequent amide bond forming condensation reactions. Our in
vitro ScoE system however utilized only 4, which after forming
isonitrile product 5, would need to be activated and loaded onto
ScoB again for ScoA to function (Figure 1). We then tested
whether the acyl-ACP ligase, ScoC, could function again after
ScoE to activate 5. We first conducted the ScoE in vitro reaction
and after a short incubation period, added ScoB, ScoC, and ATP.
The LC-HRMS analysis showed a strong signal for the formation
of 2, which was absent when 1 was directly used as a substrate
for the reaction of ScoE (Figure 3B). This result indicated that
ScoC functions twice in the pathway, to first activate and load
crotonic acid, and subsequently to activate and load 5 onto
ScoB to provide a preferred isonitrile substrate for ScoA (Figure
1). To further support the proposed functions of enzymes in
INLP biosynthesis, we next performed an in vitro total enzymatic
synthesis of INLP using purified enzymes including ScoA, ScoB,
ScoC and ScoE. After incubating enzymes with 4, ATP, Lys, and
NADPH, the expected product 3 was successfully produced as
compared to a chemical standard based on LC-HRMS analysis
(Figure 3C; S16).
kinetic parameters of ScoE toward 4 (Km = 286 ± 93 µM, kcat
=
21.9 ± 2.1 min-1) and KG (Km = 20.9 ± 3.2 µM, kcat = 0.50 ±
0.02 min-1) using LC-MS to monitor the formation of 5 and the
succinate formation assay to monitor NADH oxidation,
respectively (Figure S14). No activity of ScoE was observed
when 1 or CABA-CoA was used as a substrate.
Our in vitro biochemical analysis provided direct evidence
for a second mechanism of isonitrile formation by a non-heme
iron(II) and KG dependent oxidase/decarboxylase. We propose
that ScoE functions similarly to TauD and TfdA,14,17 utilizing an
enzyme-bound iron-oxo species for oxidation of 4 which likely
goes through two sequential steps with an imine intermediate
(Figure S15). The position of the choline hydroxyl group and Cl-
in our structure of ScoE may indicate the binding mode of the
two carboxylate groups of 4. However,
a high-resolution
structure of ScoE with substrate bound is necessary to
determine the precise substrate binding arrangement and
provide insight in substrate activation, which is currently under
way.
In summary, with the aid of a high-resolution crystal
structure, we were able to circumnavigate inhibitory
circumstances and biochemically reconstitute the activity of
ScoE, a non-heme iron(II)-dependent enzyme, for isonitrile
synthesis for the first time. We demonstrated that ScoE
catalyzes the formation of isonitrile via the oxidative
decarboxylation of the free acid substrate, 4. We also provided
evidence for a revised pathway for INLP synthesis with the
second role of a promiscuous acyl-ACP ligase, ScoC, which
activates the isonitrile product of ScoE before the NRPS-
promoted INLP formation. This revision is expected to be
applicable to other homologous INLP biosynthetic pathways
found in Actinobacteria (Figure S17). This work paves the way
to elucidate the enigmatic enzymatic mechanism for isonitrile
formation using a non-heme iron(II)-dependent enzyme, of
which homologues are conserved and critical for the virulence of
pathogenic mycobacteria, including M. tuberculosis.
Experimental Section
Detailed experimental methods can be found in the Supporting
Information.
Figure 3. In vitro characterization of ScoE. A) Extracted ion chromatograms
showing the conversion of 4 to 5 catalyzed by ScoE. For simplicity, only the no
KG assay is shown as a representative negative control. The calculated
mass of 5 with a 10-ppm mass error tolerance was used. B) Extracted ion
chromatograms showing the production of 2. Bottom trace displays the assay
with 1 and ScoE. Top trace shows the coupled reaction containing ScoE,
ScoB, ScoC and 4. The calculated mass of 2 with a 10-ppm mass error
Acknowledgements
N.C.H. is supported by the National Science Foundation
Graduate Research Fellowship Program and D.A.B. by a
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10.1002/anie.201804307
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National Institutes of Health (NIH) Molecular Biophysics Training
Grant T32 GM008313. This research was financially supported
by grants to W.Z. from the NIH (DP2AT009148), Alfred P. Sloan
Foundation, and the Chan Zuckerberg Biohub Investigator
Program. C.L.D. is a Howard Hughes Medical Institute
Investigator. This work used Northeastern Collaborative Access
Team beamlines (GM103403) and a Pilatus 6M detector
(RR029205) at the Advanced Photon Source (DE-AC02-
06CH11357).
Keywords: Biosynthesis • Acyl-acyl carrier protein ligase •
Oxidoreductase • Protein Structures • Isocyanide
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Entry for the Table of Contents (Please choose one layout)
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The electron-rich isonitrile is an
important functionality in
Nicholas C. Harris, David A. Born,
Wenlong Cai, Yaobing Huang,
Joelle Martin, Ryan Khalaf,
Catherine L. Drennan, Wenjun
Zhang*
bioactive natural products, but its
biosynthesis has been restricted
to the IsnA family of isonitrile
synthases. We here provide the
first biochemical and structural
evidence for a novel mechanism
of isonitrile formation using a
non-heme iron(II)-dependent
oxidase/decarboxylase.
Page No. – Page No.
Isonitrile Formation by a Non-
heme Iron(II)-dependent
Oxidase/Decarboxylase
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