Tandem Hydration of Diisonitriles Triggered by Isonitrile Hydratase in Streptomyces thioluteus

Article abstract of DOI:10.1021/acs.orglett.8b01341

The biosynthetic pathway of diisonitrile chalkophore SF2768 was identified in Streptomyces thioluteus through heterologous expression recently. Isolation and structure elucidation of the N-substituted formamides that coexisted with the diisonitriles impli

Full text of DOI:10.1021/acs.orglett.8b01341

Letter
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
pubs.acs.org/OrgLett
Tandem Hydration of Diisonitriles Triggered by Isonitrile Hydratase
in Streptomyces thioluteus
†
,∥
†,‡,∥
‡
§,†
†
†
Mengyi Zhu,
Lijuan Wang,
Qingbo Zhang, Muhammad Ali, Siqi Zhu, Peiqing Yu,
†
‡
‡
,†
Xiaofei Gu, Haibo Zhang, Yiguang Zhu, and Jing He*
†State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University,
Wuhan 430070, China
‡
CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, RNAM Center for Marine Microbiology, Guangdong Key
Laboratory of Marine Materia Medica, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang
Road, Guangzhou 510301, P. R. China
§
Biotechnology Program, Department of Environmental Sciences, COMSATS University Islamabad, Abbottabad campus,
Abbottabad, Pakistan
*
S Supporting Information
ABSTRACT: The biosynthetic pathway of diisonitrile chalkophore SF2768 was identified in Streptomyces thioluteus through
heterologous expression recently. Isolation and structure elucidation of the N-substituted formamides that coexisted with the
diisonitriles implied that a hydration event was involved. In vitro enzymatic assays of an endogenous isonitrile hydratase
suggested a rare sequential-hydration of the diisonitriles. Additionally, the results of Cu-CAS assays indicate that both partial and
complete hydration led to the loss of the copper-chelating ability of SF2768.
6
sonitrile natural products, a class of generally toxic and
hydrolysis in Arthrobacter pascens by Kobayashi’s group. These
investigations brought new insights into microbial isonitrile
assimilation and detoxification.
I
pungent substances, are usually discovered along with the
1
corresponding N-substituted formamides. Researchers once
speculated that formamide was the biogenetic precursor of
isonitrile when they initially observed the co-occurrence of an2
isonitrile−formamide pair in marine sponge Halichondria.
Afterward, the results of a series of preliminary assays implied
that formamide, on the contrary, was biosynthesized from
Diisonitrile chalkophore SF2768 (compound 1, Figure 1)
was originally isolated from Streptomyces spp., and its structure
was elucidated two decades ago. Structurally, SF2768
possesses a pyran ring connected to two isonitrile moieties
with amide bonds. One very intriguing and function-related,
but to date rarely encountered structural feature of SF2768 is
the diisonitrile functional group, which was observed only in a
7
3
isonitrile.
In 2001, Kobayashi and his colleagues discovered a novel
isonitrile hydratase InhA (Q8G9F9.1) that hydrated isonitrile4
to the corresponding formamide from Pseudomonas putida,
offering the first enzymatic evidence for the hypothesis that
formamide could be derived from isonitrile through bio-
transformation. Kobayashi also predicted that isonitrile
hydratase could serve as a probe to clone the isonitrile
biosynthetic gene since they are expected to be closely located
8
handful of bioactive natural products, including xanthocillin,
9
10
hazimycins, and kalihinols. In our previous study, the
biosynthetic gene cluster (BGC) of SF2768 (sfa, genbank
KY427327, Figure 2) was identified in Streptomyces thioluteus
DSM40027, and the biosynthetic pathway was proposed based
on metabolic profiles of the heterologous expression strains.
Herein, the formamides coproduced with diisonitriles were
11
4
in the genome in isonitrile-producing organisms. Moreover, a
1
01
highly conserved cysteine residue (Cys ) in InhA family
isolated and identified, leading to the discovery of a rare
isonitrile hydratases was verified to play an essential role for
5
catalysis. In 2010, another isonitrile hydratase InhB
(BAJ17399.1), which had no sequential similarity with InhA,
Received: April 27, 2018
was found to catalyze both isonitrile hydration and nitrile
©
XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.8b01341
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
the substrates in vitro. The enzymatic assays of SfaF were
performed in a volume of 50 μL (50 mM Tris-HCl, 0.1 mM
substrate, 35 μM SfaF, pH = 8.0) at 30 °C, and the boiled
protein was utilized as the control. The reactions were
terminated at different time points by adding 50 μL of cold
acetonitrile and analyzed by LC-MS after filtration. The
extracted ion chromatogram (EIC) traces of compounds in
the time-course analyses illustrated that 1 and 4 were converted
to the corresponding hydrated final products (Figure 3, blue
Figure 1. Structures of SF2768 (1) and its congeners.
Figure 2. Isonitrile hydratase SfaF adjacent to the biosynthetic gene
cluster sfa in S. thioluteus catalyzes the isonitrile groups of SF2768 (1)
and its analog 4 to the corresponding N-substituted formamides.
isonitrile hydratase SfaF (previously Orf23, Genbank
ATY72534) that catalyzed a sequential hydration.
Two heterologous expression strains, Streptomyces livid-
ans::p13C and ΔsfaE, were constructed in the previous study,
11
and their scaled-up fermentation was performed as described.
Figure 3. LC-MS analysis of the in vitro enzymatic assay of SfaF. Time-
course analyses of the conversions of 1 into 3 (left) and 4 into 8
(right) by SfaF are illustrated by EIC overlays. The splitting peak
patterns of 1, 2, and 3 likely reflect corresponding two anomers,
respectively.
LC-MS metabolite analysis revealed that the peaks with [M +
+
H] at m/z 355 and m/z 341 were observed along with those of
1
(m/z 337) and 4 (m/z 323) in the fermentation broth of S.
lividans::p13C and ΔsfaE, respectively, suggesting that a
possible hydration event of 1 and 4 was concomitant with
the diisonitriles biosynthesis (Figure S1). Thereafter, two
hydrated analogs of 4, compound 6 (obsd m/z 341.2190 [M +
lines, m/z 373.2082 and 359.2289) through the partially
hydrated intermediates (Figure 3, red lines, m/z 355.1976 and
341.2183). Subsequently, SfaF-catalyzed enzymatic reactions
were carried out at large scale and the resultant products 2, 3, 6,
and 8 were used for structural elucidation after purification. The
chemical structures and retention time of the metabolites
unambiguously indicated that although SfaF eventually
hydrated two isonitrile groups of the substrates (1 or 4) to
N-substituted formamides, only one preferred intermediate (2
or 6) occurred during the corresponding catalytic process.
Compound 7 cannot be yielded by SfaF, implying that a
chemical transformation might be involved. To some extent,
these findings coincide with Kobayashi’s plausible hypothesis
that isonitrile hydrating and synthesizing enzymes are expected
+
H] , calcd for C H N O ) and 7 (obsd m/z 341.2188 [M +
16
28
4
4
+
H] , calcd for C H N O ), were isolated from ΔsfaE via a
16
28
4
4
high-resolution mass spectrometry (HRMS) based fractiona-
tion, allowing the elucidation of their structures (Figure 1 and
Figure S2). The putative hydrated 1 (obsd m/z 355.1972 [M +
+
H] , calcd for C H N O , Figure S1) in S. lividans::p13C was
16
26
4
5
not structurally characterized due to its low yield.
Notably, a putative isonitrile hydratase encoding gene sfaF
was found to be located downstream of sfaE, an essential gene
for the biosynthesis of SF2768, through a BLASTp search
against the UniprotKB/Swiss-Prot database. SfaF showed a
3
2% identity to the previously verified isonitrile hydratase InhA,
4
implying an enzymatic origin of those hydrated diisonitriles. To
investigate whether the formamides were enzymatically
generated by SfaF or converted spontaneously in an acidic
to be coupled in isonitrile-producing organisms.
Previous work exhibited the substrate promiscuity of InhA
(Pseudomonas putida) and the dual function of InhB
5
a,12
4,6
environment,
SF2768 (1), the final product of the
(Arthrobacter pascens). To further characterize the substrate
biosynthetic cluster sfa, and its analog 4 were employed as
specificity of SfaF, two commercially available chemicals,
B
DOI: 10.1021/acs.orglett.8b01341
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Organic Letters
Letter
cyclohexyl isocyanide (9) and cyclohexanecarbonitrile (11),
were recruited as substrates in vitro in a volume of 500 μL
consisting of 50 mM Tris-HCl (pH = 8.0), 1 mM substrate
(dissolved in 20 μL acetonitrile and then added to the aqueous
reaction), and 3 μM SfaF at 30 °C, respectively. The reactions
were quenched by adding 500 μL of cold acetonitrile and
analyzed by LC-MS after filtration as before. A time-dependent
accumulation of the peak at m/z 128.1070 (the expected
product hydrated 9, namely N-cyclohexylformamide 10) was
observed (Figure 4A). Comparison based on retention time
Figure 5. (A) Kinetic parameters of SfaF-catalyzed reactions. Error
bars are exhibited as SEM. (B) Sequence alignment of three isonitrile
hydratases from P. putida (InhA), Pseudomonas fluorescens (PDB ID:
3
NON), and S. thioluteus (SfaF). (C) Catalytic activities comparison
between SfaF and the mutants against three isonitrile substrates. Error
bars represent SEM from six replicate trials.
roles of the conserved residues in SfaF were thus surveyed via
site-directed mutagenesis and subsequent in vitro analysis. The
hydration activities toward 1, 4, and 9 of four constructed
mutants, D67A, D67E, C161A, C161S, were measured (Figure
5
C). Consequently, compared to SfaF, the activities of D67A,
D67E, and C161A were obviously reduced, which was in
agreement with the previous work,
4
,5b
whereas C161S
Figure 4. In vitro validation of hydration activity of SfaF against
cyclohexyl isocyanide and cyclohexanecarbonitrile. (A) Time-course
analysis of the conversion of cyclohexyl isocyanide 9 into N-
cyclohexylformamide 10. (B) GC-MS spectral comparison of the
enzymatic product and N-cyclohexylformamide standard. (C)
Substrate preference of SfaF for cyclohexyl isocyanide 10 over
cyclohexanecarbonitrile 11.
remained active to a certain extent. Based on sequence
alignment and the previous structural study of the homologous
5b
isonitrile hydratase, the plausible reason for the retained
activity of C161S is that the hydroxyl group of serine is as
excellent a nucleophile as cysteine thiolate that attacks the
carbon of isonitrile. Future studies focused on the enzyme−
substrate cocrystallization will shed more light on this
hypothesis.
and the GC-MS spectrum of the enzymatically converted
product and authentic 10 demonstrated that SfaF hydrated not
only the native isonitriles 1 and 4 but also a chemically
synthesized substrate (Figure 4B). In addition, no product was
detected when 11 was utilized, signifying that SfaF did not
perform a nitrilase activity such as InhB (Figure 4C).
Compound 1 functions as a chalkophore that captures
environmental copper for the producer due to its copper-
11
chelating ability. The results of copper chrome azurol S (Cu-
CAS) assays revealed that copper chelating activities of 1 and 4
are approximately 30-fold higher than those of the correspond-
ing partially hydrated diisonitriles (2, 6, and 7), while the
difomamides (3 and 8) were completely inactive, indicating
that the presence of two isonitrile groups is essential for
diisonitriles to function as a chalkophore (Table 1). In addition
To probe the catalytic efficiency of SfaF, the kinetic
parameters were determined by a quantitative LC-MS-based
assay at 30 °C (Figure 5A). SfaF showed a comparable K
M
−1
4
value toward 9 to InhA (K = 16.2 mM, k = 16 s , 20 °C),
M
cat
while presenting a significantly lower kcat value. This is possibly
due to the differences in enzyme sequence (Figure S3A) or
reaction condition such as temperature or buffer. The
specificity constant (k /K ) for 1 was approximately 3-fold
Table 1. Copper Chelating Activities (EC ) of the SF2768
5
0
(
1) and Its Derivatives
compound
isonitrile group
EC50 (μM)
cat
M
a
higher than that for the chemically synthesized 9, indicating
that the native substrate 1 was better consumed by SfaF.
Previous site-directed mutagenesis experiments of two validated
isonitrile hydratases from Pseudomonas putida and Pseudomonas
fluorescens have established that the residues D17 and C101
1
2
1
0
2
2
1
1
0
−
82.2 ± 1.13
2
2335.0 ± 66.3
3
−
a
4
62.5 ± 2.8
a
5
84.6 ± 4.1
4
,5b
were critical for the enzymatic catalysis.
The conserved
6
2117.0 ± 27.7
2424.0 ± 389.1
−
amino acid residues of SfaF were analyzed by aligning the target
sequence with those of the verified isonitrile hydratases (Figure
7
8
a
5B). Although the amino sequence of SfaF is approximately
EDTA
358.3 ± 25.9
one-tenth longer than the other two, the aspartic acid and
cystine residues are conserved (Figure S3A). The mechanistic
a
11
These data are cited from the previous work.
C
DOI: 10.1021/acs.orglett.8b01341
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
to isonitrile assimilation/degradation, another possible physio-
logical function of SfaF is to inactivate the overproduced or
accumulated chalkophore 1 in cells by hydrating the functional
group to prevent an endogenous disturbance of copper
utilization, thus contributing to the copper homeostasis of S.
thioluteus. Combined with the fact that SfaF, the “diisonitrile
inactivator”, is expected under different control from other
biosynthetic enzymes in the pathway, this “self-resistance”
hypothesis is also consistent with the high Michaelis−Menten
constant and the modest catalytic efficiency of SfaF. Beyond
that, SfaF is believed to play a role in chemical defense since
isonitrile natural products secreted by microorganisms in the
environment are generally antimicrobial due to their metal
affinity that leads to enzyme inhibition.
In conclusion, a rare isonitrile hydratase that leads to a
unique tandem hydration of the diisonitrile in S. thioluteus is
reported. From this point of view, our studies complement the
knowledge of isonitrile metabolism in microorganisms and
enrich the toolbox for enzymatic reactions, thereby providing a
mild method other than general acid catalysis for isonitrile
hydration in aqueous media. Moreover, our case showed that
isonitrile hydrating and biosynthetic enzymes can be closely
located. Accordingly, more cryptic isonitrile-generating BGCs
and underlying isonitrile natural products can be explored
through targeted genome mining based on this strategy.
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The authors declare no competing financial interest.
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■
This work is supported by the National Natural Science
Foundation of China (31270136), the Natural Science
Foundation for Distinguished Young Scholars of Hubei
Province of China (No. 2018CFA069), the Fundamental
Research Funds for the Central Universities (No.
2
662018PY053), and the Open Project Program of Guangdong
Key Laboratory of Marine Materia Medica (LMM2018-4). We
thank Z. Zeng (College of Science, Huazhong Agricultural
University) for her assistance with NMR experiments.
D
DOI: 10.1021/acs.orglett.8b01341
Org. Lett. XXXX, XXX, XXX−XXX