Engineering a cleavable disulfide bond into a natural product siderophore using precursor-directed biosynthesis

Article abstract of DOI:10.1039/c8cc04981e

An analogue of the bacterial siderophore desferrioxamine B (DFOB) containing a disulfide motif in the backbone was produced from Streptomyces pilosus cultures supplemented with cystamine. Cystamine competed against native 1,5-diaminopentane during assembly. DFOB-(SS)₁[001] and its complexes with Fe(iii) or Ga(iii) were cleaved upon incubation with dithiothreitol. Compounds such as DFOB-(SS)₁[001] and its thiol-containing cleavage products could expand antibiotic strategies and Au-S-based nanotechnologies.

Full text of DOI:10.1039/c8cc04981e

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Engineering a cleavable disulfide bond into a natural product
siderophore using precursor-directed biosynthesis
Tomas Richardson-Sanchez and Rachel Codd*a
a
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
6
An analogue of the bacterial siderophore desferrioxamine B (DFOB) produces
1
as its native siderophore, and is one of three agents
containing a disulfide motif in the backbone was produced from used clinically to reduce excess Fe(III) that accumulates in
Streptomyces pilosus cultures supplemented with cystamine. patients with transfusion-dependent blood disorders.7
Cystamine competed against native 1,5-diaminopentane during
assembly. DFOB-(SS) [001] and its complexes with Fe(III) or Ga(III) DesABCD, with the first step involving the decarboxylation of
were cleaved upon incubation with dithiothreitol. Compounds such lysine (DesA) to produce 1,5-diaminopentane (DP) as the major
as DFOB-(SS) [001] and its thiol-containing cleavage products could diamine substrate. Mono-N-hydroxylation of DP (DesB)
The biosynthesis of 1 is directed by the enzyme cluster
1
L-
1
expand antibiotic strategies and Au-S-based nanotechnologies.
produces N-hydroxy-DP (HDP), which is subject to N-acetylation
(
DesC) or N-succinylation (DesC) to give N-acetyl-N-hydroxy-DP
The small class of non-protein natural product metabolites that
contain a disulfide motif display unique physicochemical and
biological properties, with selected members (eg, romidepsin)
showing clinical utility. Methods to engineer S–S bonds into
natural product metabolites could deliver analogues with
broader applications in medicine and self-assembled monolayer
8
(AHDP) or N-succinyl-N-hydroxy-DP (SHDP), respectively. One
unit of SHDP is then activated and condensed with AHDP (DesD,
round 1) to form AHDP-SHDP. Activation and condensation of a
second unit of SHDP to AHDP-SHDP (DesD, round 2) produces
1
9
AHDP-SHDP-SHDP (1) (Fig. 1a).
PDB has been used in recent work to produce analogues of
in S. pilosus cultures supplemented with non-native diamines
(SAM)-based Au-S nanotechnologies, such as bio-sensing and
1
bio-surveillance. Precursor-directed biosynthesis (PDB),
whereby a producing organism is cultured in medium
supplemented with non-native substrates, could have potential
in this endeavour. This approach would require knowledge of
the biosynthetic pathway of the target metabolite, and access
to appropriate S–S-containing substrates.
Here, we demonstrate the use of PDB to engineer a disulfide
motif into the backbone of the bacterial siderophore
desferrioxamine B (1). The simplicity of the PDB approach
that compete against DP. Viable non-native substrates include
9
10
1
,4-diamino-2(E)-butene and oxybis(ethanamine). Here, we
were interested in exploring the capacity of DesBCD to process
substrates that were more structurally deviant from DP than
this group. We selected cystamine (CS) (disulfanebis
(
ethanamine)), which compared to DP, is bent (C–S–S–C
3
dihedral angle 100.1), has a larger molecular volume (149 Å vs
3
1
28 Å ) and a greater dipole moment (2.83 D vs 1.57 D) (Fig. 2).
Viability of CS as a substrate would produce a group of disulfide-
containing analogues of containing one ( ), two ( ) or a
complete ( ) DP-for-CS substrate exchange (Fig. 1b,c).
Constitutional isomers within or arise from the insert
makes it an attractive means to generate unusual analogues of
structurally complex natural products, including siderophores.2
Siderophores are low-molecular-weight organic compounds
1
2
–4
5–7
8
2
–4
5–7
3
secreted by bacteria to acquire essential Fe(III) for growth. The
position of CS, with the binary naming system (0, native; 1, non-
Fe(III)-siderophore complex is recognised by receptors at the
9
native), as defined previously. Additional merit of CS as a
4
cell surface of the source bacterium. The selectivity of
substrate was its potential to introduce cleavable disulfide
bonds into a natural product siderophore. This could present
new applications of siderophores as prodrugs, with constructs
containing variably positioned disulfide motifs in the
siderophore scaffold having potential to deliver conjugated
antibiotics and/or toxic metal ions upon exposure to reducing
conditions.
recognition provides a platform to develop siderophore-based
5
antibiotic strategies. The soil bacterium Streptomyces pilosus
a.The University of Sydney, School of Medical Sciences (Pharmacology) and Bosch
Institute, Camperdown New South Wales 2006, Australia. Rachel Codd:
rachel.codd@sydney.edu.au
Electronic Supplementary Information (ESI) available: [HRMS (
2); MS isotope
patterns ( , Fe(III) or Ga(III) complexes)]. See DOI: 10.1039/x0xx00000x
1, 2, 9
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Cultures of S. pilosus were grown in base medium (control provisionally attributed to an analogue of
Journal Name
from the subset 2–
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1
culture) or in medium supplemented with CS (10 mM).‡11 The
production of Fe(III)-coordinating species was monitored over detected that corresponded with
0 d by the absorbance value (500 nm) of an aliquot of
4
containing one DP-for-CS substrate exchange. No peaks were
(Fig. 2e) or (Fig. 2f).
DOI: 10.1039/C8CC04981E
5
–7
8
1
12
supernatant with added Fe(III) (Fig. 2a).‡ The similarity
between the data for the control and CS-supplemented cultures
indicated that CS at 10 mM was not attenuating the production
of Fe(III)-responsive compounds. At 10 d after inoculation, the
cultures were harvested by centrifugation and the supernatant
purified by XAD and IMAC chromatography and desalted for
analysis by liquid chromatography-mass spectrometry (LC-MS).
Fig. 2 Space-filling models of DP and CS. (a) Visible absorption ( 500 nm) from aliquots
of S. pilosus supernatant (0–10 d) analysed with added Fe(III), from control (black) or CS-
supplemented cultures (grey). LC-MS traces from semi-purified S. pilosus supernatant
(
10 d) from control (black) or CS-supplemented cultures (grey), detected as (b) TIC, or
+
EIC with values corresponding with the [M+H] adducts of (c) 1, (d) 2–4, (e) 5–7, or (f) 8.
LC-MS/MS analysis was undertaken on the CS-
supplemented sample (Fig. 3). The peak identified as
1 using
selected ion monitoring (SIM) was subject to fragmentation,
with the data in agreement with the predicted fragmentation
9
-10
pattern for
assigned to
1
2–4
(Fig. 3a-c).
gave a MS fragmentation pattern that matched
(Fig. 3d-f), in which the CS-derived
substrate was inserted at the amine-containing region of
Fragments observed at m/z 169.0, 251.1, 369.2 and 451.2 were
not present in the pattern for and were consistent with an
analogue of with one DP-for-CS substrate exchange.
Fragments at m/z 169.0, 361.2 and 443.3 confirmed the isomer
Analogous processing of the peak
the predicted pattern of
2
1
.
1
1
as
2
, since among
2
–
3
4
–
, these fragments are unique to
, based on the absence of characteristic
), 194.1 ( ), 319.2 ( ), 411.2 ( ) or
). The peak containing was collected by semi-
2. There
was no evidence of
fragments at m/z 119.1 (
4
3
,4
4
2
4
3,4
4
93.3 (
3
,4
preparative HPLC, with HRMS analysis (found [M+H]+
Fig. 1 Biosynthesis of (a) DFOB (1), (b) the disulfide analogue of DFOB from complete DP-
+
for-CS substrate exchange (8), or (c) disulfide analogues of DFOB from partial DP-for-CS 611.28927, [C24
H
47
N
6
O
S
8 2
] requires 611.28913) consistent with
substrate exchange (2–7).
2
(Fig. S1, ESI†). The yield of 2 was approximately 0.5 mg per 50
mL culture. Based on the relative peak areas (88%:12%) of
100% DP) and (66.6% DP, 33.3% CS), the rate of CS
incorporation was 4%. This was similar to the incorporation of
,6-diaminohexane in macrocyclic desferrioxamine E.2a The
integrity of as a siderophore was shown by MS measurements
from solutions of Fe(III) and , which gave MS isotope patterns
1
The LC-MS trace from the control culture (Fig. 2b, black)
showed a major peak at 43.4 min, which corresponded with
This peak from total ion current (TIC) detection was co-incident
(
2
1
.
1
with a peak using the extracted ion chromatogram (EIC) mode
2
+
set to the [M+H] adduct of
1
(Fig. 2c). The LC-MS trace from the
as the major species (Fig.
b,c, grey), with an additional low-intensity peak present in the
2
+
CS-supplemented culture showed
1
2
+
+
consistent with the [M+2H] , [M+H] and [M+Na] (where M is
Fe(III)- (3 )]) (Fig. S2, ESI†).
The biosynthesis of demonstrates that CS is a viable
2
[
2 
TIC at 46.6 min. The LC-MS traces in the CS-supplemented
2
+
system with EIC values set to the [M+H] adducts of
2
–4
,
5
–7
or
substrate for DesB to form N-hydroxy-CS (HCS) and that HCS
remains a viable substrate for DesC-mediated N-succinylation
8
gave a peak at 46.7 min at EIC 611 (Fig. 2d, grey). This peak
was not observed in the control culture (Fig. 2d, black) and was
to form N-succinyl-N-hydroxy-CS (SHCS). The final product
2
2
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DOI: 10.1039/C8CC04981E
Fig 3 LC-MS trace (left) and experimental (middle) or predicted (right) MS/MS fragmentation of 1 (a–c), 2 (d–f) or 9 (g–i). Fragments unique to 1, 2 or 9 are in bold (middle, right).
would be formed following the second cycle of DesD catalysed
condensation of SHCS with AHDP-SHDP, which is the native
9
dimer precursor of
1
and common to
2
.The absence of
3
and
4
could be attributed to one or a combination of the following
steps being unviable: (i) N-acetylation of HCS (DesC); first round
condensation (DesD) of (ii) SHCS with AHDP (dimer precursor of
3
); and/or (iii) SHDP with AHCS (dimer precursor of
round condensation (DesD) of SHDP with (iv) AHDP-SHCS to
form ; and/or (v) AHCS-SHDP to form . While the AHDP-SHDP
dimer precursor common to and was detected (m/z 361.2),
4); second
3
4
1
2
no peaks were detected that corresponded with AHDP-SHCS,
AHCS-SHDP or AHCS-SHCS, which suggests that the biosynthetic
pathway for
3 and 4, and for 5–8, is more likely halted at
upstream steps (i)–(iii) than downstream steps (iv)–(v).
Fig. 4 Coordination between 2 and Fe(III) or Ga(III) to form Fe(III)-2 or Ga(III)-2,
respectively. Cleavage of the disulfide bond in 2, Fe(III)-2 or Ga(III)-2 with dithiothreitol
(
DTT) to form 2-aminoethanethiol and 9, Fe(III)-9 or Ga(III)-9, respectively.
Fig 5 LC-MS traces from semi-purified S. pilosus supernatant from control (a–h) or CS-
supplemented (i–p) cultures, in the absence (a, b, i, j) or presence of Fe(III) (c–f, k–n) or
Ga(III) (g, h, o, p); and in the absence (a, c, e, g, i, k, m, o) or presence of DTT at 10 mM
Cleavage of the disulfide bond of
2
was predicted to release
with
2
-aminoethanethiol and (Fig. 4). The potential to use 2
9
an antibiotic covalently bound at the amine terminus as a (b, d, h, j, l, p) or 2 mM (f, n). SIM values (single or multiple) corresponded with 1 (561),
2
(611), 9 (536), Fe(III)-1 (614), Ga(III)-1 (627), Fe(III)-2 (664), Ga(III)-2 (677), Fe(III)-9
prodrug for antibiotic delivery could require S–S bond cleavage
of Fe(III)- or Ga(III)- , assuming that bacterial cell-surface
recognition of
rather than the free ligand. Aliquots of the semi-purified
supernatant from the control and CS-supplemented cultures, in
the absence or presence of Fe(III) or Ga(III), were incubated with
dithiothreitol (DTT) at a final concentration of 2 or 10 mM. LC-
MS analysis used SIM detection, with values set for species
relevant to each system (Fig. 5).
(
589), Ga(III)-9 (602). Selected data (e, f, m, n) were acquired at a later day, giving
2
2
different retention times for a given species, due to instrumental variability.
1
is optimised for the metal-siderophore complex
4
The LC-MS trace from the control culture in the absence (Fig.
5
5
disappeared in the presence of 10 mM DTT, with a new peak
appearing at 56.7 min that, based on the SIM detection, was
provisionally assigned to
a) or presence of 10 mM DTT (Fig. 5b) gave a peak for
1 (Fig.
b). The peak for in the CS-supplemented culture (Fig. 5i)
2
9
(Fig. 5j). Confirmation was provided
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Journal Name
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from LC-MS/MS analysis, with fragments at m/z 176.1, 294.2
and 376.2 characteristic of the predicted pattern for (Fig. 3g-
i). This demonstrated the veracity of reductive S–S bond
cleavage of to give . The intensity of the signal for Fe(III)- in
the control culture was attenuated with 10 mM DTT (Fig.
2
017, 117, 5784-5863; (b) J. Li, C. Wang, Z.-M. Zhang, Y.-Q. Cheng
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9
1
1
3
5
c,d). The signal for Fe(III)-
was diminished upon incubation with 10 mM DTT, with the
appearance of a low-intensity signal for Fe(III)- and a signal in
a higher relative intensity for (Fig. 5k,l). Subsequent analysis
of a second Fe(III)- -containing sample treated with DTT at a
lower concentration (2 mM) showed the presence of unreacted
Fe(III)- , together with Fe(III)- , and a low-intensity signal for
Fig. 5m,n). Redox-inactive Ga(III), which forms high-affinity
2 in the CS-supplemented culture
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2
9
9
(
14
complexes with siderophores, was examined as a substitute
for Fe(III). The signal for Ga(III)- in the control culture showed
minimal diminution in the presence of DTT at 10 mM (Fig. 5g,h).
In the case of Ga(III)- in the pre-DTT treated CS-supplemented
sample, incubation with DTT gave a well resolved signal for
Ga(III)- with no discernible signal for (Fig. 5o,p). This
supported the DTT-mediated reduction of the S–S bond in
redox-inactive Ga(III)- gave Ga(III)- as the major product, with
(a) C. Hennard, Q. C. Truong, J.-F. Desnottes, J.-M. Paris, N. J.
1
Moreau and M. A. Abdallah, J. Med. Chem., 2001, 44, 2139-2151; (b)
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U. Möllmann, L. Heinisch, A. Bauernfeind, T. Köhler and D. Ankel-
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T. A. Wencewicz and M. J. Miller, Top. Med. Chem., 2017, 26, 151-
2
9
9
2
9
1
84; (g) R. Liu, P. A. Miller, S. B. Vakulenko, N. K. Stewart, W. C.
no evidence of complex dissociation. Experimental MS isotope
patterns for all species agreed with calculated data (Fig. S2,
ESI†).
Boggess and M. J. Miller, J. Med. Chem., 2018, 61, 3845-3854; (h) W.
Neumann, M. Sassone-Corsi, M. Raffatellu and E. M. Nolan, J. Am.
Chem. Soc., 2018, 140, 5193-5201.
In conclusion, a cleavable disulfide bond has been
engineered into the amine-containing region of the bacterial
6
R. Codd, T. Richardson-Sanchez, T. J. Telfer and M. P. Gotsbacher,
ACS Chem. Biol., 2018, 13, 11-25.
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7
8
siderophore
involved culturing the native
supplemented medium and is attractive in its simplicity. The
veracity of DTT-mediated S–S bond cleavage of to produce
thiol was demonstrated. DTT-mediated cleavage of the S–S
bond in Ga(III)- produced Ga(III)- in an apparent 1:1
stoichiometric conversion, with lower conversion of Fe(III)- to
Fe(III)- , likely due to Fe(III)/(II) redox chemistry. Covalent
attachment of an antibiotic to the amine terminus of
1
to produce
2
. The PDB production method
1
-producer S. pilosus in CS-
Challis, ChemBioChem, 2005,
Costales, F. Barona-Gómez and G. L. Challis, Nat. Chem. Biol., 2007,
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6, 601-611; (c) N. Kadi, D. Oves-
2
3
9
9
1
2
9
2
2
0 T. Richardson-Sanchez, W. Tieu, M. P. Gotsbacher, T. J. Telfer and R.
Codd, Org. Biomol. Chem., 2017, 15, 5719-5730.
9
2
could 11 At 40 mM CS, no Fe(III)-coordinating species were detected,
including . Cell growth was slower and less dense. Although
1
2 was
have potential as an antibiotic prodrug recognised as the Ga(III)-
or Fe(III)-complex by pathogenic bacteria, such as
observed at 20 mM CS in yields about twice observed at 10 mM, the
lower concentration was selected to maintain a conservative
approach towards production versus potential toxicity.
2 The non-homogeneous growth of S. pilosus in liquid medium
prevents the use of conventional OD measurements of cell growth.
This Fe(III)-addition assay is a surrogate measure of culture progress.
3 This could be due to Fe(III)/Fe(II) reduction and the formation of
15
Staphylococcus aureus or Vibrio furnissi, that use
xenosiderophore. Cell-surface uptake of the Fe(III)- - or Ga(III)-
-antibiotic conjugate could be followed by antibiotic release
1 as a
2
1
1
2
upon exposure to intracellular reductants. Work published
during the period that the current study was under review
neutral, MS-silent Fe(II)-
dissociation of Fe(II)- to give
detected (EIC ( ), SIM ( )), which suggested that any 1
1
(in positive-ion mode), and/or the
and/or -adducts. was not
that may have
was subject to
1
6
supports this strategy.
Disulfide- and thiol-containing
and 9,
1
1
1
1
analogues of native bacterial siderophores, such as
2
d
f
respectively, could have applications in bio-sensing and bio-
surveillance and other Au–S-dependent self-assembled
monolayer (SAM) nano-technologies or nano-medicines.17
been liberated from the dissociation of Fe(II)-
1
further Fe(III)/(II)/DTT-mediated chemistry.
14 A. Evers, R. D. Hancock, A. E. Martell and R. J. Motekaitis, Inorg.
Chem., 1989, 28, 2189-2195.
1
This work was supported by the Australian Research Council
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, 542-553; (b) T. Tanabe, T. Funahashi, K. Miyamoto, H. Tsujibo and
(DP180100785 to RC) and The University of Sydney (Australian
3
Postgraduate Award to TRS).
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Conflicts of interest
There are no conflicts to declare.
14, 5577-5583.
Notes and references
4
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