In Vitro Stepwise Reconstitution of Amino Acid Derived Vinyl Isocyanide Biosynthesis: Detection of an Elusive Intermediate

Article abstract of DOI:10.1021/acs.orglett.7b00258

In vitro reconstitution of a newly discovered isonitrile synthase (AmbI1 and AmbI2) and the detection of an elusive intermediate (S)-3-(1H-indol-3-yl)-2-isocyanopropanoic acid 1 in indolyl vinyl isocyanide biogenesis are reported. The characterization of iron/2-oxoglutarate (Fe/2OG) dependent desaturases IsnB and AmbI3 sheds light on the possible mechanism underlying stereoselective alkene installation to complete the biosynthesis of (E)- and (Z)-3-(2-isocyanovinyl)-1H-indole 2 and 5. Establishment of a tractable isonitrile synthase system (AmbI1 and AmbI2) paves the way to elucidate the enigmatic enzyme mechanism for isocyanide formation.

Full text of DOI:10.1021/acs.orglett.7b00258

Letter
pubs.acs.org/OrgLett
In Vitro Stepwise Reconstitution of Amino Acid Derived Vinyl
Isocyanide Biosynthesis: Detection of an Elusive Intermediate
Wei-chen Chang,*,† Dev Sanyal,† Jhih-Liang Huang,† Kuljira Ittiamornkul,‡ Qin Zhu,‡ and Xinyu Liu*,‡
†Department of Chemistry, North Carolina State University, Raleigh, North Carolina 27695, United States, and
‡Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, United States
S
* Supporting Information
ABSTRACT: In vitro reconstitution of a newly discovered isonitrile synthase (AmbI1 and AmbI2) and the detection of an
elusive intermediate (S)-3-(1H-indol-3-yl)-2-isocyanopropanoic acid 1 in indolyl vinyl isocyanide biogenesis are reported. The
characterization of iron/2-oxoglutarate (Fe/2OG) dependent desaturases IsnB and AmbI3 sheds light on the possible
mechanism underlying stereoselective alkene installation to complete the biosynthesis of (E)- and (Z)-3-(2-isocyanovinyl)-1H-
indole 2 and 5. Establishment of a tractable isonitrile synthase system (AmbI1 and AmbI2) paves the way to elucidate the
enigmatic enzyme mechanism for isocyanide formation.
atural products containing vinyl isonitrile (isocyanide)
functionality have been isolated from a diverse array of
N
microbial organisms and possess distinct structural complexities
and biological activities.1,2 As isocyanide represents a unique
class of reactive functional groups,3,4 its presence in natural
products has raised considerable interest in delineating its
biosynthetic origin.5 The genetic basis for vinyl isocyanide
biogenesis was initially disclosed by the analysis of environ-
mental DNAs.6 A putative isonitrile synthase (IsnA) and an
Fe(II)/2-oxoglutarate(2OG)-dependent oxygenase (IsnB) de-
rived from isnA and isnB genes were shown to collectively
convert L-tryptophan (L-Trp) and ribulose-5-phosphate (Ru-5-
P) to (E)-3-(2-isocyanovinyl)-1H-indole (2).7 Heavy-isotope
feeding studies established that the terminal sp-hybridized
isocyanide carbon appended to the L-Trp α-amine is derived
from the carbonyl carbon of Ru-5-P.7 In addition, feeding the
methyl ester derivative of (S)-3-(1H-indol-3-yl)-2-isocyanopro-
panoic acid 1 to E. coli cells overexpressing isnB led to the
production of 2.7 These observations led to a proposal on the
molecular basis for vinyl isocyanide biogenesis that proceeds via
intermediate 1 by the sole action of IsnA on L-Trp, followed by
hydroxylation and decarboxylative desaturation catalyzed by
IsnB (Figure 1, eq 1).7 In addition to the isn operon, two vinyl
isocyanide biogenetic pathways were identified. XnPvcA and
XnPvcB were shown to produce (E)-4-(2-isocyanovinyl)phenol
(4) from L-tyrosine (L-Tyr) via the presumptive intermediate
(S)-3-(4-hydroxyphenyl)-2-isocyanopropanoic acid 3 (Figure 1,
eq 3).8,9 More recently, enzymes for the biogenesis of (Z)-3-(2-
isocyanovinyl)-1H-indole 5 were identified in the biosynthetic
machinery for the hapalindole-type alkaloids,10,11 typified by
Figure 1. Proposed biosynthetic pathways for vinyl isocyanide
containing 2, 5, and 4 by (1) IsnA and IsnB; (2) AmbI1, AmbI2,
and AmbI3; and (3) XnPvcA and XnPvcB proteins. Equations 1−3
show those proposals outlined in the literature.7,8,10
AmbI1, AmbI2, and AmbI3 proteins and their close
homologues WelI1, WelI2, and WelI3. AmbI1 and AmbI2 are
protein homologues of IsnA.10 It was proposed they act in
complex to produce 1 as an intermediate, analogous to the
proposal for IsnA, which is stereoselectively converted to 5 by
AmbI3, an IsnB homologue (Figure 1 eq 2).
Although 1 and 3 have been proposed as the direct
enzymatic product of IsnA (AmbI1 and AmbI2) and XnPvcA
respectively,7−11 they have not been detected in any vinyl
isocyanide producing system either in vivo or in vitro. Thus, one
cannot completely rule out the involvement of an additional
protein, such as IsnB or AmbI3, in isocyanide formation. The
Received: January 24, 2017
© XXXX American Chemical Society
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conversion of the carbon atom from the carbonyl group in Ru-
5-P to that in an isocyanide moiety requires a 2-e oxidation. It is
conceivable that IsnA/IsnB, AmbI1/AmbI2/AmbI3 or
XnPvcA/XnPvcB act cooperatively, where the Fe/2OG-
dependent oxygenases IsnB/AmbI3/XnPvcB catalyze the
required 2-e oxidation to elevate the oxidation state of the
carbonyl carbon via unknown intermediate(s). The observed in
vivo or in vitro conversions of 1 to 2/5 or 3 to 4 by IsnB/AmbI3
or XnPvcB can be attributed to the dual functionality of IsnB/
AmbI3/XnPvcB. Similar observations have been made for
several members in this enzyme family, such as H6H, AsqJ, and
IPNS in scopolamine, methoxyviridicatin, and isopenicillin
pathways, respectively.12−14
To unambiguously distinguish and define the roles of each
protein component in vinyl isocyanide biogenesis and to
elucidate the enigmatic enzymatic mechanism for isocyanide
formation, the establishment of a tractable in vitro or in vivo
system(s) to detect the intermediacy of 1 or 3 will be required.
In this work, we demonstrate that the newly discovered AmbI1,
AmbI2, and AmbI3 system fulfills this requirement, and for the
first time, the in vitro enzymatic generation of 1 from L-Trp and
Ru-5-P was achieved using AmbI1 and AmbI2. We further
demonstrate the production of 2 using a mixture of purified
AmbI1, AmbI2, and IsnB in vitro, confirming the functional
complementation of IsnA by AmbI1 and AmbI2 and supporting
the hypothesis that 1 is a common intermediate for the
biogenesis of 2 and 5. Finally, we show that IsnB is E-specific,
solely producing 2 from 1, while AmbI3 generates 5
stereoselectively. Collectively, this study provides conclusive
evidence that IsnA or AmbI1 and AmbI2 are the default stand-
alone isonitrile synthases that can covert L-Trp with Ru-5-P to
1.
We initially chose to work on isn and ambI pathways as they
generate 2 and 5, a pair of diastereomers, in a stereospecific
manner via a postulated common intermediate 1. This
constitutes a complementary system that is deemed suitable
for the detection of 1 and for understanding how stereo-
selectivity arises from 1 by protein homologues IsnB and
AmbI3. While heterologous overexpressions in E. coli and
affinity column purifications led to sufficient quantities of N-
His6-tagged AmbI1, AmbI2, AmbI3, and IsnB (SI Methods), we
were unable to procure IsnA after numerous attempts. The
putative intermediate 1 was procured by chemical synthesis
from L-Trp. Esterification of L-Trp was followed by the
introduction of a formyl group upon the α-amine.15,16 The
formyl moiety was then dehydrated to generate the isocyanide
group, and the deprotection of the carboxylate yielded 1 in
circa. 40% overall yield (SI Methods). This sequence is
different from the published route where the formylation was
carried out prior to methylation.9 In the reported sequence, due
to reactivity of the formyl group, the methyl group was
introduced using trimethylsilyl diazomethane. Installation of
the methyl ester prior to formylation allows for the use of acidic
methanol and avoiding diazomethane. Using this modified
synthetic sequence, compound 1 was procured in a gram scale.
Compound 1 was stable as a solid at −78 °C and has a half-life
>4 days in water (4 °C) that enables us to use it both as a
standard and as a substrate for subsequent investigation.
To assess the proposed function of AmbI1 and AmbI2 on the
biogenesis of 1, deuterium-enriched d5-L-Trp was incubated in a
reaction mixture containing Ru-5-P, AmbI1, and AmbI2
(Figure 2). The reaction was monitored using liquid
chromatography−mass spectrometry (LC-MS). After a 20
Figure 2. (a) Detection of the elusive intermediate 1 from in vitro
assay using AmbI1 and AmbI2. The LC-MS EIC chromatograms of
synthetic 1 (m/z = 213.1) and d5-1 (m/z = 218.1) from enzymatic
assay of AmbI1 and AmbI2 in the presence/absence of substrates Ru-
5-P and d5-L-Trp. (b and c) Formation of the imine intermediate by
reacting the amine of L-Trp with the ketone of Ru-5-P and a plausible
mechanism accounts for the formation of 1 from the imine
intermediate.
min incubation at ambient temperature, a new product peak
with the identical retention time as synthetic 1 was observed
(Figure 2a, lanes 1/4). The peak associated with the enzymatic
reaction product exhibits the expected mass shift of +5,
establishing that this product arises from the isotopically
labeled L-Trp. This peak was not detected in chromatographs of
control experiments where proteins (AmbI1 and AmbI2) or
substrates (d5-L-Trp and Ru-5-P) were absent (Figure 2a, lanes
2/3). The peak was also not seen when Ambl3 and 2OG/
Fe(II) were included in the reaction with molecular oxygen
(Figure 2a, lane 5), suggesting that 1 was rapidly utilized by
AmbI3. The enzymatic conversion of L-Trp and Ru-5-P to 1 by
AmbI1 and AmbI2 is a single turnover event under our assay
conditions (SI Methods), implying it is likely the rate-limiting
step in the vinyl isocyanide formation. Based on the isotope
tracer experiment result,7 where the terminal carbon of
isocyanide 1 originates from the keto-center of Ru-5-P, we
speculate the enzymatic reaction by AmbI1 and AmbI2 is
initiated by forming an imine intermediate to connect two
substrates (L-Trp and Ru-5-P) (Figure 2b). While how this
postulated imine intermediate is transformed to 1 is a subject
for future studies, one plausible route is outlined in Figure 2c.
After formation of the imine intermediate, the reaction may
proceed through β-keto imine formation generated through
loss of the phosphate group and followed by enol−keto
tautomerization. Carbon−carbon bond cleavage is triggered by
nucleophilic attack of the nitrogen lone pair to generate the
future isocyanide fragment and hydroxyl acetone. A subsequent
“retro aldol type” reaction would give 1 with the concomitant
formation of formaldehyde.
Having confirmed 1 is the enzymatic product of AmbI1 and
AmbI2, we proceeded to validate the functional roles of AmbI3
and its homologue IsnB in the stereodivergent generation of 2
and 5 (Figure 3). First, the products of a reaction containing all
three AmbI proteins, d5-L-Trp, Ru-5-P, and 2OG/Fe(II), were
characterized (Figure 3a, lane 4). The peak that corresponds to
5, but not its diastereomer 2, was readily detected on an LC-
B
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2
2
The Csp −Csp bond formation is then triggered by a
decarboxylation via an E2 pathway where the carboxylate and
the expelling hydroxyl group are in an anti-coplanar
conformation (6 → 2). Analogously, the formation of 5 will
go through pro-S C−H cleavage to form intermediate 7 prior to
decarboxylation (7 → 5).
The partial erosion of stereoselectivity by AmbI3 when using
1 as the substrate can be attributed to the cooperative nature of
isonitrile synthase systems discovered to date. That is, they
likely act as a complex to catalyze the stereospecific vinyl
isocyanide formation from the corresponding amino acids and
Ru-5-P. This is consistent with the observation that while the
generation of diffusible 1 by AmbI1 and AmbI2 is merely a
single turnover event, when coupled to AmbI3, the system can
generate ca. 30 turnovers (unoptimized) to give 5 within 25
min when the enzyme substrate ratio is set at 1/100 (SI
Methods), suggesting protein−protein interactions between
AmbI1 and/or AmbI2 with AmbI3 is likely operant to shuttle
the intermediate 1 efficiently for the following stereospecific
alkene installation. When IsnB is coupled with AmbI1 and
AmbI2 under the same conditions, ca. 10 turnovers
(unoptimized) that led to the production of 2 were observed.
In conclusion, by utilizing a newly discovered isonitrile
synthase system (AmbI1, AmbI2, and AmbI3), we achieved the
stepwise reconstitution of indolyl vinyl isocyanide biosynthesis
in vitro from L-Trp and Ru-5-P. This endeavor led to the
detection of an elusive biosynthetic intermediate 1 and
provided conclusive evidence that 1 is the enzymatic product
of standalone isonitrile synthase AmbI1 and AmbI2. Further
characterization of Fe/2OG dependent desaturases IsnB and
AmbI3 in combination with AmbI1/AmbI2 and 1 sheds light
on the origin of stereodivergent biogenesis of indolyl vinyl
isocyanide by isn and ambI pathways. The establishment of
AmbI1 and AmbI2 as a tractable isonitrile synthase paved the
way to elucidate the enigmatic enzymatic mechanism of the
isocyanide formation, a subject of continuing studies by our
groups.
Figure 3. Stereodivergent alkene formation in vinyl isocyanide
biogenesis by AmbI3 and IsnB and its potential enzymatic mechanism.
The LC-MS EIC chromatograms of 2/5 (m/z = 167.1) and d5-2/5
(m/z = 172.1) from enzymatic assays of the following: (a) AmbI1 and
AmbI2 in combination of AmbI3 or IsnB in the presence of substrates
(d5-)L-Trp, Ru-5-P, O2, and 2OG/Fe(II); (b) AmbI3 or IsnB with
Fe(II) and substrate 1 in the presence/absence of O2, and 2OG. (c)
Proposed IsnB and AmbI3 mechanism accounts for the stereoselective
alkene formation.
MS chromatogram (Figure 3a, lanes 4 vs 1/2) and exhibits the
expected +5 shift in m/z value. When unlabeled (all protium) L-
Trp was used, the enzymatic product with the +5 shift in m/z
was not seen (Figure 3a, lane 3). Instead, the peak with the
same retention time and m/z value as the synthetic standard 5
was detected (data not shown). These results are consistent
with the retention of all deuteria from d5-L-Trp. As we were
unable to obtain IsnA protein in a stable form by heterologous
expression, we proceeded to probe the function of IsnB by
coupling with AmbI1 and AmbI2 that are IsnA homologues. A
reaction containing AmbI1, AmbI2, and IsnB proteins, along
with all relevant substrates, was analyzed (Figure 3a, lane 5).
The peak having m/z = 172.1 and the same retention time of 2
was evident in the LC-MS chromatograms. In the reaction
using AmbI1, AmbI2, and IsnB, the peak for the (Z)-isomer 5
was not observed. This result is in agreement with prior reports
concerning the geometric isomers production from isn and
ambI pathways.7,10 It further suggests that 1 is a common
substrate of IsnB and Ambl3 and likely a true product of the
IsnA reaction.
To further probe the stereoselective formation of 2 and 5 by
IsnB and AmbI3, we incubated synthetic 1 with IsnB and
AmbI3 in the presence of O2 and 2OG/Fe(II) (Figure 3b). In
the case of IsnB, 2 was the only product detected (Figure 3b,
lanes 5 vs 1/2). Accumulation of 2 was not detected when O2
and 2OG were omitted (Figure 3b, lane 3). In the reaction of
AmbI3, 5 was observed as the major product. Surprisingly, a
minor amount (circa. 5%) of 2 was also detected (Figure 3b,
lane 4). These results collectively imply that the stereoselective
alkene formation in vinyl isocyanide biogenesis is dominantly
governed by the intrinsic differences between AmbI3 and IsnB.
Both proteins are homologues of Fe/2OG-dependent oxy-
genases that typically catalyze hydroxylation via aliphatic C−H
bond cleavage using an Fe(IV)-oxo species.17−19 The observed
stereodivergence can be viewed as a downstream result of
stereoselective hydroxylation as depicted in Figure 3c.
Specifically, to form 2, the hydroxylation is likely to proceed
through pro-R C−H cleavage of 1 to produce intermediate 6.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.7b00258.
Detailed experimental procedures, including the syn-
thetic preparation of 1, purifications of AmbI1, AmbI2,
AmbI3, and IsnB proteins, and subsequent enzymatic
assays (PDF)
AUTHOR INFORMATION
Corresponding Authors
■
*E-mail: wchang6@ncsu.edu.
*E-mail: xinyuliu@pitt.edu.
ORCID
Wei-chen Chang: 0000-0002-2341-9846
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This work is supported by North Carolina State University and
University of Pittsburgh.
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REFERENCES
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