Exploring the substrate specificity of OxyB, a phenol coupling P450 enzyme involved in vancomycin biosynthesis

Article abstract of DOI:10.1039/b805956j

OxyB is a cytochrome P450 enzyme that catalyzes the first oxidative phenol coupling reaction during vancomycin biosynthesis. The preferred substrate is a linear peptide linked as a C-terminal thioester to a peptide carrier protein (PCP) domain of the glycopeptide antibiotic non-ribosomal peptide synthetase. Previous studies have shown that OxyB can efficiently oxidize a model hexapeptide-PCP conjugate (R-Leu¹-R-Tyr²-S-Asn ³-R-Hpg⁴-R-Hpg⁵-S-Tyr⁶-S-PCP) (Hpg = 4-hydroxyphenylglycine) into a macrocyclic product by phenolic coupling of the aromatic rings in residues-4 and -6. In this work, the substrate specificity of OxyB has been explored using a series of N-terminally truncated peptides related in sequence to this model hexapeptide-PCP conjugate. Deletion of one or three residues from the N-terminus afforded a penta- (Ac-Tyr-Asn-Hpg-Hpg-Tyr-S-PCP) and a tri- (Ac-Hpg-Hpg-Tyr-S-PCP) peptide that were also efficiently transformed into the corresponding macrocyclic cross-linked product by OxyB. The tripeptide, representing the core of the macrocycle in vancomycin created by OxyB, is thus sufficient, as a thioester with the PCP domain, for phenol coupling to occur. The related tetrapeptide-PCP thioester was not cyclized by OxyB, neither was a related model hexapeptide containing tryptophan in place of tyrosine-6, nor were tripeptides (related to the natural product K-13) with the sequence Ac-Tyr-Tyr-Tyr-S-PCP cross-linked by OxyB. The Royal Society of Chemistry.

Full text of DOI:10.1039/b805956j

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Exploring the substrate specificity of OxyB, a phenol coupling P450 enzyme
involved in vancomycin biosynthesis†
Katharina Woithe, Nina Geib, Odile Meyer, Tanja Wo¨rtz, Katja Zerbe and John A. Robinson*
Received 8th April 2008, Accepted 30th May 2008
First published as an Advance Article on the web 26th June 2008
DOI: 10.1039/b805956j
OxyB is a cytochrome P450 enzyme that catalyzes the first oxidative phenol coupling reaction during
vancomycin biosynthesis. The preferred substrate is a linear peptide linked as a C-terminal thioester to
a peptide carrier protein (PCP) domain of the glycopeptide antibiotic non-ribosomal peptide
synthetase. Previous studies have shown that OxyB can efficiently oxidize a model hexapeptide–PCP
conjugate (R-Leu1-R-Tyr2-S-Asn3-R-Hpg4-R-Hpg5-S-Tyr6-S-PCP) (Hpg = 4-hydroxyphenylglycine)
into a macrocyclic product by phenolic coupling of the aromatic rings in residues-4 and -6. In this work,
the substrate specificity of OxyB has been explored using a series of N-terminally truncated peptides
related in sequence to this model hexapeptide–PCP conjugate. Deletion of one or three residues
from the N-terminus afforded a penta- (Ac-Tyr-Asn-Hpg-Hpg-Tyr-S-PCP) and a tri-
(Ac-Hpg-Hpg-Tyr-S-PCP) peptide that were also efficiently transformed into the corresponding
macrocyclic cross-linked product by OxyB. The tripeptide, representing the core of the macrocycle in
vancomycin created by OxyB, is thus sufficient, as a thioester with the PCP domain, for phenol
coupling to occur. The related tetrapeptide–PCP thioester was not cyclized by OxyB, neither was a
related model hexapeptide containing tryptophan in place of tyrosine-6, nor were tripeptides (related to
the natural product K-13) with the sequence Ac-Tyr-Tyr-Tyr-S-PCP cross-linked by OxyB.
(3,5-dihydroxyphenylglycine, Dpg) (D-dpgA), or for its incor-
poration into the linear heptapeptide intermediate (D-bpsC),
afforded mutants of the balhimycin producer still able to produce
hexapeptides containing the C–O–D ring, suggesting indirectly
Introduction
Vancomycin, a glycopeptide produced by Amycolatopsis orientalis,
is a clinically important antibiotic, which acts by inhibiting
peptidoglycan biosynthesis in Gram-positive bacteria.1 The van-
comycin heptapeptide aglycone, which is also found in the related
natural products balhimycin and chloroeremomycin (Fig. 1),
contains several aromatic amino acid side chains oxidatively linked
through biaryl or biaryl-ether bridges. The heptapeptide backbone
itself is constructed by the actions of three non-ribosomal peptide
synthetase (NRPS) subunits, whereas the oxidative cross-linking
reactions are catalyzed in a defined order by three cytochrome
P450 enzymes OxyA, OxyB and OxyC.2–4 The first coupling
occurs between the phenol rings in residues-4 and -6 (the C–
O–D ring), catalyzed by OxyB, the second aryl-ether bridge is
formed between side chains of residues-2 and -4 (D–O–E ring)
by OxyA, and the last biaryl coupling between the aromatic
side chains of residues-5 and -7 is carried out by OxyC (AB
ring, Fig. 1). The crystal structures of OxyB (CYP165B1) and
OxyC (CYP165C1) from the vancomycin producer A. orientalis
have been reported, in substrate free form, confirming that these
proteins contain a fold and heme environment typical of P450
enzymes.5,6
that a linear hexapeptide may act as a substrate for OxyB.7
Complementary in vitro studies, however, demonstrated that the
first cross-linking reaction actually occurs when the linear peptide
is attached as a thioester to a peptide carrier protein (PCP)
domain in the NRPS assembly line.8,9 Thus model linear hexa-
and heptapeptides (1 and 2), comprising residues 1–6 and 1–7 of
vancomycin aglycone but containing tyrosine at positions-2 and -6
instead of b-hydroxytyrosine derivatives, and linked as thioesters
to a recombinant PCP domain from the NRPS, were transformed
by OxyB into monocyclic cross-linked products (3 and 4) (Fig. 2).
Catalytic activity with the remaining two coupling enzymes OxyA
and OxyC has not yet been reported, but both are also thought
to act upon peptidic intermediates attached as thioesters to the
NRPS assembly line.
There is now great interest in exploring in more detail the
substrate specificity of OxyB. In recent work, it was reported that
OxyB can catalyze efficiently a phenol coupling reaction on an
epimeric form of hexapeptide–PCP conjugate 1, which contains
an (R)-Tyr at position-6 instead of (S)-Tyr.10 The product of this
phenol coupling reaction, the corresponding epimeric form of
monocyclic peptide 3, had been observed in small amounts in
earlier work,9 after work-up of assays with peptide 1; the partial
epimerization of residue-6 in 1 is difficult to avoid completely
and may occur during its synthesis and/or during work-up of
enzyme assays (cleavage of the thioester with hydrazine), but it
was a surprise to discover that the (R)-Tyr6-epimer is converted
In molecular genetic studies, the inactivation of genes re-
sponsible for the biosynthesis of the seventh amino acid
Chemistry Department, University of Zurich, Winterthurerstrasse 190, 8057,
Zurich, Switzerland. E-mail: robinson@oci.uzh.ch
† Electronic supplementary information (ESI) available: Analytical data
including 1H-NMR data. See DOI: 10.1039/b805956j
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Fig. 1 Structures of vancomycin, chloroeremomycin, balhimycin, teicoplanin (alkyl chain varies), A40926 (alkyl chain varies) and A47934.
Fig. 2 Conversion of the model hexa- and heptapeptides 1 and 2 into the monocyclic products (3 and 4) by OxyB and ancillary electron transfer
proteins.9
rapidly into monocyclic product by OxyB under the in vitro assay
conditions.
which are not transformed by OxyB under the in vitro assay
conditions.
Here we report more extensive studies of the substrate specificity
of OxyB, with peptide analogues having stepwise deletions of
residues from the N-terminus of hexapeptide 1 (Fig. 3). We
show that several of the derived penta-, tetra-, and tripeptides
(all as PCP thioester conjugates) are still accepted and trans-
formed efficiently into monocyclic products by OxyB. On the
other hand, we also describe other peptide–PCP conjugates,
with a substantially different backbone, either containing tryp-
tophan, or being related to that in the natural product K-13,11
Results
Peptide synthesis
The linear peptides 5A–10A were synthesized using an optimized
solid-phase methodology reported earlier.12 This involved the use
of N(a)-Alloc protection for each amino acid, but avoided as far
as possible any side chain protecting groups, in order to avoid
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Fig. 3 Structures of complestatin and K-13 as well as the substrates (5–12) used in this study and their corresponding thioesters. Reagents and conditions:
i) thiophenol, PyBOP, DIEA in DMF, 15 min, rt; ii) coenzyme-A, phosphate buffer, pH 8.5, 2 h, rt; iii) apo-PCP-7S, B. subtilis Sfp, MgCl2, HEPES buffer,
pH 7.0, 1 h, rt.
any deprotection steps requiring strong acid (as is usual in the
Boc and Fmoc solid-phase methods) at the end of each synthesis.
However, the tripeptides 11A and 12A were made using standard
solid-phase Fmoc-chemistry, since these do not contain sensitive
phenylglycine derivatives.
NADPH regeneration system. After reaction for 60 min at 30 ◦C,
the peptidic products were cleaved from the PCP by hydrazine
treatment and the corresponding peptide hydrazides were iso-
lated by solid-phase extraction and purified by reverse phase
HPLC.
In order to couple each peptide to a PCP, the peptide was
first converted into a S-phenyl thioester (5B–12B) and then trans-
formed by thioester exchange into the corresponding coenzyme-A
thioester (5C–12C), using methods reported earlier.8,9,13 Each pep-
tide thioester was characterized by MS and NMR spectroscopy.
The peptide-SCoA thioesters were then used together with the
pantetheinyl transferase Sfp from Bacillus subtilis,14 and the
recombinant apo-PCP-7S described earlier,9 derived from module-
7 of the vancomycin NRPS, to prepare the PCP conjugates 5D–
12D (Fig. 3). These peptide–PCP-7S conjugates were isolated in
>95% purity, as analyzed by reverse phase HPLC, and gave the
expected molecular ion by MALDI-MS.
Influence of the N-terminal residues
Under the standard assay conditions, the pentapeptide–PCP
conjugate 5D, lacking the N-terminal Leu1 compared to the model
hexapeptide (1), showed rapid turnover to the corresponding
monocyclic peptide hydrazide (13) (Fig. 4). However, by HPLC
analysis of the product, two epimers were detected, each with the
expected mass of the monocyclic peptide (m/z = 811.3 ([M +
H]+)). The major product was the expected macrocyclic peptide
hydrazide (13) and the minor product (ca. 10%) is most likely
an epimeric form having (R)-Tyr in place of (S)-Tyr as the C-
terminal amino acid, in analogy to earlier results reported for
the hexapeptide 1.9,10 Residual unchanged linear pentapeptide
hydrazide 5E was not seen within the limits of detection, indicating
essentially complete turnover of the linear peptide. The identity of
the major product 13 was further confirmed by high resolution
ESI-MS (exact mass calcd. for C40H42N8O11Na: 833.2871 ([M +
Na]+), m/z found 833.2873). The fragmentation pattern of the
Assays with OxyB
OxyB assays were carried out under the standard conditions
reported earlier.9 Present in the assay are spinach ferredoxin (sp-
Fd), E. coli flavodoxin reductase (eco-FlvR) and NADPH, with
glucose-6-phosphate and glucose-6-phosphate dehydrogenase as
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Fig. 4 Assays with OxyB. Reagents and conditions: i) OxyB, sp-Fd, eco-FlvR, NADPH, O2, 30 ◦C. ii) NH2-NH2.
major product 13 by MS/MS, together with the full chemical
shift assignment and analysis of NOEs by 1D and 2D H-NMR
spectroscopy confirmed that macrocycle formation had occurred
between the side chains of 4-hydroxyphenylglycine-3 (Hpg3) and
tyrosine-5 (Tyr5).
10–20% into the corresponding monocyclic product (15 and 16).
In both cases, unreacted linear peptides could be re-isolated as the
corresponding peptide hydrazides. As minor products, epimeric
forms of the linear tetrapeptide hydrazide and the monocyclic
tetrapeptide hydrazide (15 and 16) were also identified. The
identities of the major macrocyclic products were supported
by ESI-MS (15: m/z = 605.4 ([M + H]+), 16: m/z = 683.4
([M + H]+)), high resolution ESI-MS (15: exact mass calcd. for
C30H32N6O8Na: 627.2179 ([M + Na]+), m/z found 627.2194, 16:
exact mass calcd. for C35H34N6O9Na: 705.2285 ([M + Na]+), m/z
found 705.2285) and ESI-MS/MS analysis. The poor resolution
of the monocyclic and linear products in HPLC chromatograms
prevented isolation of pure compounds in sufficient quantities for
1
No turnover by OxyB was detected in assays with the
tetrapeptide–PCP-7S conjugate (6D). Only the corresponding
linear tetrapeptide hydrazide was detected in the product by
HPLC and by ESI-MS analysis (m/z = 650.2 ([M + H]+)). To
confirm this assignment, the linear tetrapeptide hydrazide was
isolated and fully characterized by high resolution ESI-MS (exact
mass calcd. for C31H35N7O9Na: 672.2394 ([M + Na]+), m/z found
1
672.2407), and by 1D and 2D H-NMR spectroscopy. A second
1
minor epimer (ca. 5%) with the same mass was also detected by
HPLC.
a full NMR analysis. However, 1D and 2D H-NMR analyses
of the product mixtures enabled a full chemical shift assign-
ment of the linear and monocyclic tetrapeptide hydrazides 15
and 16.
In contrast, under standard assay conditions the tripeptide
7D was transformed by OxyB into a macrocyclic peptide (14)
(Fig. 4). HPLC analysis of the reaction products indicated ca. 80%
conversion into the tripeptide hydrazide 14, which was identified
by high resolution ESI-MS (exact mass calcd. for C27H27N5O7Na:
556.1808 ([M + Na]+), m/z found 556.1806) and ESI-MS/MS.
1D and 2D-NMR spectroscopy also confirmed the formation
and the connectivity of the expected macrocycle. A second minor
component (ca. 15%) in the product mixture was assigned to
the corresponding linear tripeptide hydrazide 7E, derived from
unreacted 7D. A further minor peak (<5%) detected by HPLC,
with a mass also corresponding to macrocyclic product, was
assigned again as an epimer of 14.
Other peptides
The hexapeptide (10D) containing (S)-Trp at position-6 was
assayed under standard conditions, but afforded no monocyclic
product. The corresponding linear peptide hydrazide and a puta-
tive C-terminal epimer (≈20%) were recovered, and characterized
by ESI-MS (m/z = 921.6 ([M + H]+)), high resolution ESI-MS
(exact mass calcd. for C47H57N10O10: 921.4259 ([M + Na]+), m/z
found 921.4278), ESI-MS/MS and NMR spectroscopy. The two
tripeptides related to K-13, 11D (configuration (S)(S)(S) as in
K-13) and 12D (configuration (R)(R)(S) in analogy to 7) were
also assayed under standard conditions. Again, no monocyclic
products were detected by HPLC, only unreacted linear tripeptide
was recovered, isolated as the hydrazide, and confirmed by ESI-MS
(11E: m/z = 564.4 ([M + H]+), 12E: m/z = 564.4 ([M + H]+)), high
resolution ESI-MS (11E: exact mass calcd. for C29H33N5O7Na:
586.2278 ([M + Na]+), m/z found 586.2282, 12E: exact mass calcd.
Substituted tetrapeptides
Two additional tetrapeptides containing N-terminal (S)-Ala (8D)
or (S)-Hpg (9D), instead of (S)-Asn (compare 6D), were also tested
as substrates. Under standard assay conditions, each peptide–
PCP-7S conjugate (8D and 9D) was converted to the extent of
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for C29H33N5O7Na: 586.2278 ([M + Na]+), m/z found 586.2285),
incorporation of the respective b-HT residue into the growing
peptide chain by the NRPS, catalyzed by the single halogenase
encoded within the biosynthetic gene cluster.15 This implies that
OxyB should never encounter an NRPS-bound peptide substrate
containing tyrosine at positions-2 and -6, only b-HT or Cht.
The b-HT itself is thought to be formed by a pathway similar
to that demonstrated for novobiocin biosynthesis,16 in which a
cytochrome P450 is used to hydroxylate a PCP-thioester bound
form of tyrosine, which is then released as free b-HT by hydrolysis.
Interestingly, the biosynthesis of glycopeptide antibiotics of the
teicoplanin/A40926/A47934 family, which possess an additional
side chain cross-link between amino acids 1 and 3 (an F–O–G
link) and a Cht only at position-6, appears to proceed differently.
Gene knock-out studies in the A40926 producer suggest that
tyrosine, rather than b-HT, is recognized and incorporated by the
NRPS at positions-2 and -6, and that subsequently only residue-
6 is b-hydroxylated (exact timing uncertain), but now catalyzed
by a non-heme iron oxygenase (Dbv28).17 However, a dbv28
knock-out mutant does produce small amounts of a glycopeptide
related to A40926, lacking the b-hydroxy group in residue-6.
The timing of chlorination in the biosynthesis of this family of
glycopeptides is presently also unknown. One other remarkable
observation relating to the specificity of vancomycin OxyB, is that
this vanOxyB was able to cross-complement a staH mutant of the
A47934 producer.18 StaH catalyzes the analogous cross-coupling
of the C–O–D rings in A47934 biosynthesis, but apparently this
function can also be fulfilled by vanOxyB, although the peptide
backbone of A47934 is different to that of vancomycin (Fig. 1).
Deletion of halogenase genes in the balhimycin producer
(bhaA) abolished halogenation, and afforded the correspond-
ing dechloro-balhimycin.19 This shows that OxyB can cat-
alyze cross-linking in the absence of chlorine substituents, in
agreement with the in vitro studies. In related work, feed-
ing various isomers of fluoro-b-hydroxytyrosine to a D-bhp
mutant provided novel fluorobalhimycins,20 and growth of
the balhimycin producer on media containing bromide (but
no chloride) afforded bromobalhimycins.21 Remarkably, a D-
dpgA mutant of the balhimycin producer was able to yield
very small amounts of a tricyclic glycopeptide aglycone with
the usual 3,5-dihydroxyphenylglycine (Dpg) replaced by 4-
hydroxyphenylglycine (Hpg),7 although in related work it was
reported that feeding Hpg to this mutant led to no (detectable) gly-
copeptide production.22 Similarly, feeding the dpgA mutant with
Dpg analogues afforded tricyclic aglycones containing methoxy
groups in amino acid residue-7.22 These knock-out studies do
not directly inform about the substrate specificity of OxyB, not
least due to uncertainties in the timing of several key steps in
the biosynthetic pathway. It should also be borne in mind that
fermentations of gene knock-outs may lead to the isolation of
intermediates that have been processed in ways that do not reflect
the preferred pathway in the wild-type strain. Moreover, some
intermediates might be modified slowly by enzymes unrelated to
glycopeptide biosynthesis (e.g. by proteolysis) and accumulate only
during the course of long fermentations. It is, therefore, important
to complement such in vivo studies with direct enzymic studies
in vitro.
ESI-MS/MS and 1D and 2D 1H-NMR spectroscopy.
Binding studies by UV-vis
The binding of the pentapeptide 5D, the tetrapeptide 6D and
tripeptide 7D to OxyB was monitored by UV-vis difference
spectroscopy. Upon titration of 5D with OxyB, the expected type-I
binding spectrum was obtained (Fig. 5) with a peak at 393 nm,
a trough at 426 nm and an isosbestic point at 413 nm. The
concentration dependence of the absorbance fitted well a binding
equation involving a 1 : 1 interaction, with a Kd of 28 4 lM for
the interaction of 5D with OxyB.
Fig. 5 UV-vis difference spectra of the pentapeptide 5–PCP-7S binding
to OxyB. The concentration dependence of the spectral changes and the
wavelengths of the minima and maxima are shown.
Binding studies using the tetrapeptide 6D and tripeptide 7D
produced significantly different changes in UV-vis difference
spectra, with a shift of the positions of the maximum (to 405–
414 nm) and the trough (to 435–441 nm). Additionally, the relative
magnitude of the trough was much reduced and the concentration
dependence of the changes was approximately linear in the range
that could be tested (not shown). A similar behaviour was observed
in earlier work for the interaction of OxyB with a heptapeptide–
PCP-7S conjugate, and a hexapeptide–PCP-6S conjugate.9 The
results indicate that the binding affinity of 7D to OxyB is much
lower (Kd >100 lM) than that measured here for 5D, and for 1 in
earlier work (Kd 17 5 lM).9
Discussion
In earlier work,8,9 model hexa- and heptapeptide–PCP conjugates
(1 and 2), related to putative intermediates in vancomycin aglycone
assembly were tested in vitro as substrates for OxyB. These model
substrates contain tyrosine at positions-2 and -6.
Glycopeptide antibiotics of the vancomycin/balhimycin/
chloroeremomycin family, however, all contain m-chloro-b-
hydroxytyrosine (Cht) at positions-2 and -6. Gene knock-out
studies have indicated that b-hydroxytyrosine (b-HT), but not
tyrosine nor Cht, are recognized and incorporated into the
peptide backbone at positions-2 and -6 by the NRPS during
balhimycin biosynthesis.15 The timing of chlorination of both
aromatic rings is not yet known, but gene knock-out studies
again suggest that each chlorination may occur directly after
The work described here extends our earlier in vitro studies
using peptide 1, with the aim of exploring further the substrate
tolerance of OxyB using firstly the series of peptides 5–7, which
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represent stepwise deletions of residues from the N-terminus of 1.
The pentapeptide 5D lacking the N-terminal Leu was converted
into the monocyclic hydrazide 13 almost quantitatively under the
assay conditions. Indeed, the dissociation constant (Kd ≈ 28 lM)
for this interaction, determined by UV-vis difference spectroscopy
(Fig. 5), is only slightly lower than that observed for the model
hexapeptide 1 (Kd ≈ 17 lM). Hence, the Leu1 residue does not
contribute significantly to substrate recognition by OxyB. Kinetic
studies of this enzymic reaction, using methods described earlier,9
were prevented by the fact that the linear and cyclic peptide
products could not be sufficiently well resolved by HPLC.
When peptide 6D was tested, however, no turnover to cross-
linked tetrapeptide product was detected under the standard assay
conditions. This result would not be so surprising if it were not
for the fact that the tripeptide 7D was converted, quite efficiently
(80% conversion) under the standard assay conditions, into cross-
linked product 14. The turnover of 7D was slower than for
pentapeptide 5D, and this was also reflected in binding studies
to OxyB by UV-vis-difference spectroscopy, which now suggested
a much weaker interaction of 7D with the enzyme (Kd >100 lM).
It was technically not possible to determine kinetic parameters
(kcat, Km) for this reaction, due to the high concentrations of
substrate required. We conclude, therefore, that the tripeptide
7D, representing the core of the macrocycle created by OxyB is
sufficient for phenol coupling to occur. These observations also
explain why vanOxyB is able to cross-complement a staH mutant
of the A47934 producer, as discussed above.
more detailed studies of these cross-linking enzymes are warranted
to understand the requirements for Trp oxidation.
Peptides with structural similarities to vancomycin aglycone,
in particular with similar aryl-ether bridged macrocycles, are
found in many natural products, including K-13, an inhibitor of
angiotensin I converting enzyme (Fig. 3).11 This, together with the
observation made here that OxyB can cross-link the tripeptide 7D,
suggested it might be interesting to examine the two tripeptides
11D and 12D as potential substrates for OxyB. The absolute
configurations of 11 and 12 are related to those in K-13 and 7,
respectively. Each tripeptide was assayed as a thioester conjugate
to PCP-7S from the vancomycin NRPS (11D and 12D), however,
under standard conditions no conversion to a macrocyclic product
could be detected. Hence, we conclude that the configuration and
identity of the building blocks in the minimal tripeptide core, in
particular Hpg, are important for recognition and turnover by
OxyB.
The results described here, with those reported earlier, help
to define more clearly the substrate specificity of OxyB. One
remaining aspect of this specificity that should now be addressed in
more detail, is the influence of the b-hydroxylation of tyrosine and
the chlorination of the aromatic rings on these oxidative phenol
coupling reactions.
Experimental section
¨
Reverse phase HPLC was performed using a Pharmacia AKTA
The reasons for the failure to detect cross-linking with the
tetrapeptide 6D are unclear. At the end of the assay, the
tetrapeptide could be re-isolated as the hydrazide in good yield. To
explore this further, two additional tetrapeptides 8D and 9D were
prepared, and shown to be converted into macrocyclic products
15 and 16, respectively, albeit with significantly lower overall
turnover (10–20%) under the standard assay conditions. With
these substrates at least some cross-linking occurs, but again not as
efficiently as with the tri-, penta- and hexa-peptides. We speculate
that the reason for the lack of turnover seen with 6D, and the poor
turnover seen with 8D and 9D, might be linked to the adoption
of non-productive binding conformations in the corresponding
enzyme–substrate complexes, but this remains to be proven.
Next, we were encouraged to test linear hexapeptide 10, which
contains tryptophan in place of Tyr6, since a side chain cross-
linking reaction involving Trp must occur during complestatin
biosynthesis, catalyzed by a cytochrome P450-like enzyme related
to OxyB.23 It was of interest to test whether or not OxyB from
the vancomycin producer could oxidize a Trp residue within
the otherwise familiar scaffold of peptide 1. The results show
conclusively that this is not possible.
Two P450-like enzymes have been implicated in cross-coupling
reactions during complestatin biosynthesis, ComI and ComJ,
which share 47% and 54% identity, respectively, with OxyB. OxyB
normally catalyzes a C–O–C coupling and not a C–C-biaryl
coupling reaction. The structural and mechanistic basis for
selectivity between aryl ether and biaryl cross coupling in this
class of enzymes is not yet understood. However, assays were
also performed here with 10D and OxyC, which does catalyze
a C–C coupling (identity with ComI and ComJ: 46% and 42%
respectively), and OxyA (identity with ComI and ComJ: 45% and
52% respectively) again without success (data not shown). Clearly,
Purifier system with an Agilent Zorbax Eclipse XDB (RP C18,
˚
250 × 4.6 mm, 5 lm particle size, 80 A pore size) for analysis
of assay products. One dimensional and two dimensional 1H-
NMR spectra were recorded at 500 MHz on a Bruker AV2-500
or at 600/700 MHz on a Bruker DRX-600 or AV-600 or AV-700
typically at a peptide concentration of ca. 1–10 mg ml−1. The
solvent was DMSO-d6 (Cambridge Isotope Laboratories). For
complete spin system assignments DQF-COSY, TOCSY, ROESY
and NOESY experiments were carried out. Chemical shift values
are given in ppm relative to the solvent resonances or to the internal
standard tetramethylsilane. Electrospray ionisation (ESI) mass
spectrometry was performed on a Finnegan TSQ-700, MALDI-
TOF spectra were recorded on a Autoflex I MALDI-TOF with
sinapinic acid as matrix (Bruker). UV-vis (difference) absorbance
spectra were recorded using a double beam Varian Cary300Bio
spectrophotometer.
Peptide synthesis
The peptides were synthesized by the methods described in earlier
work.9 Relevant analytical data for each new compound are given
in the supplementary material.†
Synthesis of peptide–PCP-7S conjugates
The synthesis of peptide–PCP-7S conjugates used in this study was
accomplished according to the general method described earlier
(Fig. 3).9 Thioester products were analyzed by MS and 1D/2D 1H-
NMR spectroscopy. NMR assignments were made by standard
methods, using 2D DQF-COSY, TOCSY and NOESY spectra.
Relevant analytical data for the thioester intermediates is given in
the supplementary material.†
2866 | Org. Biomol. Chem., 2008, 6, 2861–2867
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OxyB activity assays
Wohlleben, J. A. Robinson and I. Schlichting, J. Biol. Chem., 2002,
277, 47476.
7 D. Bischoff, B. Bister, M. Bertazzo, V. Pfeifer, E. Stegmann, G. J.
Nicholson, S. Keller, S. Pelzer, W. Wohlleben and R. D. Sussmuth,
ChemBioChem, 2005, 6, 267.
8 K. Zerbe, K. Woithe, D. B. Li, F. Vitali, L. Bigler and J. A. Robinson,
Angew. Chem., Int. Ed., 2004, 43, 6709.
9 K. Woithe, N. Geib, K. Zerbe, D. B. Li, M. Heck, S. Fournier-Rousset,
O. Meyer, F. Vitali, N. Matoba, K. Abou-Hadeed and J. A. Robinson,
J. Am. Chem. Soc., 2007, 129, 6887.
The general OxyB assay was carried out according to the
procedure described earlier.9 The peptide hydrazide products were
purified by analytical HPLC and analyzed by MS and 1D and 2D
1H-NMR spectroscopy. NMR assignments were made by standard
methods, using 2D DQF-COSY, TOCSY and NOESY spectra,
and are given in the supplementary material.†
10 N. Geib, K. Woithe, K. Zerbe, D. B. Li and J. A. Robinson, Bioorg.
Med. Chem. Lett., 2008, 18, 3081.
UV-vis binding studies
11 T. Yasuzawa, K. Shirahata and S. Hiroshi, J. Antibiot., 1987, 40, 455.
12 D. B. Li and J. A. Robinson, Org. Biomol. Chem., 2005, 3, 1233.
13 F. Vitali, K. Zerbe and J. A. Robinson, Chem. Commun., 2003, 2718.
14 L. E. N. Quadri, P. H. Weinreb, M. Lei, M. M. Nakano, P. Zuber and
C. T. Walsh, Biochemistry, 1998, 37, 1585.
15 O. Puk, D. Bischoff, C. Kittel, S. Pelzer, S. Weist, E. Stegmann, R. D.
Su¨ssmuth and W. Wohlleben, J. Bacteriol., 2004, 186, 6093.
16 H. Chen and C. T. Walsh, Chem. Biol., 2001, 74, 1.
17 S. Stinchi, L. Carrano, A. Lazzarini, M. Feroggio, A. Grigoletto, M.
Sosio and S. Donadio, FEMS Microbiol. Lett., 2006, 256, 229.
18 B. Hadatsch, D. Butz, T. Schmiederer, J. Steudle, W. Wohlleben, R.
Sussmuth and E. Stegmann, Chem. Biol., 2007, 14, 1078.
19 O. Puk, P. Huber, D. Bischoff, J. Recktenwald, G. Jung, R. D. Su¨ßmuth,
K.-H. van Pe´e, W. Wohlleben and S. Pelzer, Chem. Biol., 2002, 9, 225.
20 S. Weist, B. Bister, O. Puk, D. Bischoff, S. Pelzer, G. J. Nicholson, W.
Wohlleben, G. Jung and R. D. Su¨ssmuth, Angew. Chem., Int. Ed., 2002,
41, 3383.
21 B. Bister, D. Bischoff, G. J. Nicholson, S. Stockert, J. Wink, C.
Brunati, S. Donadio, S. Pelzer, W. Wohlleben and R. D. Sussmuth,
ChemBioChem, 2003, 4, 658.
22 S. Weist, C. Kittel, D. Bischoff, B. Bister, V. Pfeifer, G. J. Nicholson, W.
Wohlleben and R. D. Sussmuth, J. Am. Chem. Soc., 2004, 126, 5942.
23 H. T. Chiu, B. K. Hubbard, A. N. Shah, J. Eide, R. A. Fredenburg,
C. T. Walsh and C. Khosla, Proc. Natl. Acad. Sci. U. S. A., 2001, 98,
8548.
To monitor substrate binding to OxyB by UV-vis difference
spectroscopy, the peptide–PCP-7S conjugate was purified by anion
exchange chromatography and concentrated as described earlier.9
The concentration of peptide–PCP-7S conjugates was determined
by UV using the following extinction coefficients: 5D e280
=
7120 cm−1 M−1; 6D e280 = 5630 cm−1 M−1; 7D e280 = 4517 cm−1 M−1.
References
1 B. K. Hubbard and C. T. Walsh, Angew. Chem., Int. Ed., 2003, 42, 730.
2 R. D. Sussmuth, S. Pelzer, G. Nicholson, T. Walk, W. Wohlleben and
G. Jung, Angew. Chem., Int. Ed., 1999, 38, 1976.
3 D. Bischoff, S. Pelzer, A. Holtzel, G. J. Nicholson, S. Stockert, W.
Wohlleben, G. Jung and R. D. Sussmuth, Angew. Chem., Int. Ed., 2001,
40, 1693.
4 D. Bischoff, S. Pelzer, B. Bister, G. J. Nicholson, S. Stockert, M. Schirle,
W. Wohlleben, G. Jung and R. D. Sussmuth, Angew. Chem., Int. Ed.,
2001, 40, 4688.
5 O. Pylypenko, F. Vitali, K. Zerbe, J. A. Robinson and I. Schlichting,
J. Biol. Chem., 2003, 278, 46727.
6 K. Zerbe, O. Pylypenko, F. Vitali, W. W. Zhang, S. Rouse, M. Heck,
J. W. Vrijbloed, D. Bischoff, B. Bister, R. D. Su¨ssmuth, S. Pelzer, W.
This journal is
The Royal Society of Chemistry 2008
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