Oxidative phenol coupling reactions catalyzed by OxyB: A cytochrome P450 from the vancomycin producing organism. Implications for vancomycin biosynthesis

Article abstract of DOI:10.1021/ja071038f

OxyB is a cytochrome P450 enzyme that catalyzes the first phenol coupling reaction during the biosynthesis of vancomycin-like glycopeptide antibiotics. The phenol coupling reaction occurs on a linear peptide intermediate linked as a C-terminal thioester t

Full text of DOI:10.1021/ja071038f

Published on Web 05/04/2007
Oxidative Phenol Coupling Reactions Catalyzed by OxyB: A
Cytochrome P450 from the Vancomycin Producing Organism.
Implications for Vancomycin Biosynthesis
Katharina Woithe, Nina Geib, Katja Zerbe, Dong Bo Li, Markus Heck,
Severine Fournier-Rousset, Odile Meyer, Francesca Vitali, Nobuatsu Matoba,
Khaled Abou-Hadeed, and John A. Robinson*
Contribution from the Department of Chemistry, UniVersity of Zurich, Winterthurerstrasse 190,
8057 Zurich, Switzerland
Received February 13, 2007; E-mail: robinson@oci.unizh.ch
Abstract: OxyB is a cytochrome P450 enzyme that catalyzes the first phenol coupling reaction during the
biosynthesis of vancomycin-like glycopeptide antibiotics. The phenol coupling reaction occurs on a linear
peptide intermediate linked as a C-terminal thioester to a peptide carrier protein (PCP) domain within the
multidomain glycopeptide nonribosomal peptide synthetase (NRPS). Using model peptides with the
sequence (R)(NMe)Leu-(R)Tyr-(S)Asn-(R)Hpg-(R)Hpg-(S)Tyr-S-PCP and (R)(NMe)Leu-(R)Tyr-(S)Asn-(R)-
Hpg-(R)Hpg-(S)Tyr-(S)Dpg-S-PCP (where Hpg ) 4-hydroxyphenylglycine, and Dpg ) 3,5-dihydroxyphe-
nylglycine), or containing (R)Leu instead of (R)(NMe)Leu, attached to recombinant PCPs derived from
modules-6 and -7 in the vancomycin NRPS, we show that cross-linking of Hpg4 and Tyr6 by OxyB can
occur in both hexapeptide- and heptapeptide-PCP conjugates. Thus, whereas OxyB may act preferentially
on a hexapeptide still linked to the PCP-6 in NRPS subunit-2, it is possible that a linear heptapeptide
intermediate linked to PCP-7 in NRPS subunit-3 may also be transformed into monocyclic product. For
turnover, OxyB requires electrons, which in vitro can be supplied by spinach ferredoxin and E. coli flavodoxin
reductase. Turnover is also dependent upon the presence of molecular oxygen. The model substrate (R)-
(NMe)Leu-(R)Tyr-(S)Asn-(R)Hpg-(R)Hpg-(S)Tyr-S-PCP is transformed into cross-linked product by OxyB
with a kcat of 0.1 s-1 and Km in the range 4-13 µM. Equilibrium binding of this substrate to OxyB, monitored
by UV-vis, is accompanied by a typical low-to-high spin state change in the heme, characterized with a
Kd of 17 ( 5 µM.
thesis, as well as the order of the coupling steps.5-8 The first
coupling occurs between the phenol rings in residues-4 and -6,
Introduction
The glycopeptide antibiotics vancomycin, balhimycin, and
chloroeremomycin comprise a common heptapeptide aglycone
backbone (Figure 1). The biosynthesis of this heptapeptide takes
place according to the thiotemplate model of peptide assembly,1
catalyzed by three large nonribosomal peptide synthetase
(NRPS) subunits.2,3 The first two subunits contain six modules
that should generate a linear hexapeptide intermediate (1), still
linked as a thioester to a peptide carrier protein (PCP) domain
of the NRPS (Figure 2). This hexapeptide should then be
transferred to the third NRPS subunit, where the seventh amino
acid is added, to afford (assuming no further modification) the
heptapeptide 2.
catalyzed by OxyB, the next is formed between the side chains
of residues-2 and -4, catalyzed by OxyA, and the last coupling
is between the phenol groups in residues-5 and -7, catalyzed
by OxyC. The first coupling reaction, however, does not occur
on a free linear heptapeptide.9 Rather, in Vitro assays only
demonstrated turnover by OxyB with a linear peptide attached
as a thioester to a PCP domain of the NRPS.10 Thus, at least
the first oxidative phenol-coupling reaction during vancomycin
biosynthesis occurs on a peptide while it is still attached to the
(5) Bischoff, D.; Bister, B.; Bertazzo, M.; Pfeifer, V.; Stegmann, E.; Nicholson,
G.; Keller, S.; Pelzer, S.; Wohlleben, W.; Su¨ssmuth, R. ChemBioChem
2005, 6, 267-272.
Vancomycin aglycone also contains three cross-links between
aromatic amino acid side chains.4 Gene knock-out experiments
identified the three gene products (OxyA, OxyB, and OxyC)
responsible for these coupling reactions in balhimycin biosyn-
(6) Bischoff, D.; Pelzer, S.; Bister, B.; Nicholson, G. J.; Stockert, S.; Schirle,
M.; Wohlleben, W.; Jung, G.; Su¨ssmuth, R. D. Angew. Chem., Int. Ed.
2001, 40, 4688-4691.
(7) Bischoff, D.; Pelzer, S.; Ho¨ltzel, A.; Nicholson, G. J.; Stockert, S.;
Wohlleben, W.; Jung, G.; Sussmuth, R. D. Angew. Chem., Int. Ed. 2001,
40, 1693-1696.
(8) Sussmuth, R. D.; Pelzer, S.; Nicholson, G.; Walk, T.; Wohlleben, W.; Jung,
G. Angew. Chem., Int. Ed. 1999, 38, 1976-1979.
(1) Sieber, S. A.; Marahiel, M. A. Chem. ReV. 2005, 105, 715-738.
(2) Pelzer, S.; Su¨ssmuth, R.; Heckmann, D.; Recktenwald, J.; Huber, P.; Jung,
G.; Wohlleben, W. Antimicrob. Agents Chemother. 1999, 43, 1565-1573.
(3) van Wageningen, A. M. A.; Kirkpatrick, P. N.; Williams, D. H.; Harris, B.
R.; Kershaw, J. K.; Lennard, N. J.; Jones, M.; Jones, S. J. M.; Solenberg,
P. J. Chem. Biol. 1998, 5, 155-162.
(9) Zerbe, K.; Pylypenko, O.; Vitali, F.; Zhang, W. W.; Rouse, S.; Heck, M.;
Vrijbloed, J. W.; Bischoff, D.; Bister, B.; Su¨ssmuth, R. D.; Pelzer, S.;
Wohlleben, W.; Robinson, J. A.; Schlichting, I. J. Biol. Chem. 2002, 277,
47476-47485.
(10) Zerbe, K.; Woithe, K.; Li, D. B.; Vitali, F.; Bigler, L.; Robinson, J. A.
Angew. Chem., Int. Ed. 2004, 43, 6709-6713.
(4) Hubbard, B. K.; Walsh, C. T. Angew. Chem., Int. Ed. 2003, 42, 730-765.
9
10.1021/ja071038f CCC: $37.00 © 2007 American Chemical Society
J. AM. CHEM. SOC. 2007, 129, 6887-6895
6887

A R T I C L E S
Woithe et al.
after cleavage from the PCP with hydrazine (Figure 3). The
hexapeptide conjugate 3 was used as a substrate in these assays,
since it is synthetically easier to produce than is the hexa- or
heptapeptide-PCP conjugates 1 and 2. In this work, a more
detailed study of the cross-linking reaction catalyzed by OxyB
is reported. We show that OxyB can also turnover model
heptapeptides linked as thioesters to a PCP, which has important
implications for the timing of this cross-linking step during
vancomycin biosynthesis. We report on the reduction system
required to provide electrons to the P450, as well as kinetic
studies with the model substrate 3. Finally, we also show that
substrate turnover by OxyB requires not only electron donors,
but also molecular oxygen.
Results
Peptide Synthesis. The free peptides (9-12) were synthe-
sized using an optimized solid-phase methodology reported
earlier,14 which makes use of temporary allyloxycarbonyl (Alloc)
protection for the N(R)-amino groups, but avoids as far as
possible the use of any acids or bases as well as side chain
protecting groups (Figure 4). After assembly, the peptides were
cleaved from 2-chlorotrityl chloride resin and purified by reverse
phase HPLC (see Supporting Information).
Figure 1. Structures of glycopeptides.
For the synthesis of heptapeptides 11 and 12 racemic N(R)-
Fmoc-3,5-dihydroxyphenylglycine (Dpg) was also used in
peptide assembly, which leads in each case to a product
containing a mixture of epimeric peptides, since in the course
of loading onto the PCP, and also later during cleavage from
the PCP with hydrazine (Figure 3), an epimerization of residue-7
could in any case not be avoided (Vide infra). Each pair of
epimers of 11 and 12 (as free C-terminal carboxylic acids) could
not be resolved by analytical HPLC under the standard
conditions used.
In order to couple each peptide (9-12) to a PCP, the
C-terminus was activated and converted first into a S-phenyl
thioester (Figure 5). Note, that at the stage of the C-terminal
S-phenyl thioesters, it was possible to separate by HPLC and
characterize by MS and NMR, each pair of epimers derived
from 11 and 12. At this stage the Boc group could be removed
from 10 and 12 by careful treatment with TFA. Then by thioester
exchange, the S-coenzyme-A (SCoA) thioesters were produced
and characterized by MS and NMR (see Supporting Informa-
tion). The SCoA thioesters were then used with recombinant
phosphopantetheinyl transferase Sfp from B. subtilis to load the
peptide-pantetheinyl portion onto the active site Ser residue of
the apo-PCP domain.
PCP Domains. A PCP domain from module-6 of the
vancomycin NRPS, comprising 30 residues on the N-terminal
side and 52 residues on the C-terminal side of the active site
Ser (i.e., 30-Ser-52 corresponding to residues 3955-4037 in
VpsB), with an additional Cys-to-Ser mutation of residue 3960
near the N-terminus (called here apo-PCP-6S) was produced
in the apo form as a His6-tagged protein in E. coli as described
earlier10 (see Supporting Information).
The PCP domain from NRPS module-7 (residues 967-1043
in VpsC, or 37-Ser-39, called here apo-PCP-7S) was produced
as a soluble N-terminal His6-tagged fusion protein. An unpaired
Cys residue near the N-terminus (residue 979) was also mutated
to Ser, in order to avoid problems arising from disulfide-bridged
NRPS assembly line (Figure 2). No in Vitro turnover has so far
been reported with OxyA and OxyC, although it seems likely
that the substrates for these cross-linking enzymes may also
comprise NRPS-bound peptides.
A single halogenase introduces chlorine substituents in
residues-2 and -6 during the process of peptide assembly.11
Further gene knock-out studies suggest that the halogenase
reactions also occur on peptidic intermediates still attached to
the NRPS, although in Vitro activity with the halogenase has
so far not been reported. Conceivably, therefore, it might well
be the finished vancomycin aglycone that is released from the
NRPS through the action of a thioesterase (TE) domain situated
at the C-terminus of NRPS subunit-3 (Figure 2). However, this
remains to be proven.
The three cross-linking enzymes, OxyA-C, belong to the
cytochrome P450 family of hemoproteins. The X-ray crystal
structures of OxyB and OxyC from Amycolatopsis orientalis
have been solved at 1.7 Å and 2.0 Å resolution, respectively.9,12
Both exhibit the typical P450-fold, with a cysteine residue acting
as proximal axial thiolate ligand for the heme. Heterologous
complementation of Oxy-knockout mutants of the balhimycin
producer Amycolatopsis balhimycina with the corresponding
oxygenase genes (oxyA/B/C) from the vancomycin producer
A. orientalis restored production of balhimycin, thus proving
the functional equivalence of the vancomycin oxygenase genes
used for in Vitro assays, with those analyzed genetically in the
balhimycin pathway.13
In Vitro activity was demonstrated for OxyB using a model
hexapeptide linked to a recombinant PCP domain (3) from
module-6 in NRPS subunit-2 (Figure 2).10 The product of this
OxyB reaction (5) was isolated as the peptide hydrazide (7),
(11) Puk, O.; Bischoff, D.; Kittel, C.; Pelzer, S.; Weist, S.; Stegmann, E.;
Su¨ssmuth, R. D.; Wohlleben, W. J. Bacteriol. 2004, 186, 6093-6100.
(12) Pylypenko, O.; Vitali, F.; Zerbe, K.; Robinson, J. A.; Schlichting, I. J.
Biol. Chem. 2003, 278, 46727-46733.
(13) Stegmann, E.; Pelzer, S.; Bischoff, D.; Puk, O.; Stockert, S.; Zerbe, K.;
Robinson, J. A.; Su¨ssmuth, R. D.; Wohlleben, W. J. Biotechnol. 2006, 124,
640-653.
(14) Li, D. B.; Robinson, J. A. Org. Biomol. Chem. 2005, 3, 1233-1239.
9
6888 J. AM. CHEM. SOC. VOL. 129, NO. 21, 2007

Oxidative Phenol Coupling Reactions
A R T I C L E S
Figure 2. Schematic representation of the three subunits of the vancomycin NRPS. Illustrated are the activation (A), condensation (C), epimerization (E),
peptide carrier protein (PCP), and thioesterase (TE) domains, as well as a further domain of unknown function (X). The PCPs are post-translationally
modified by phosphopantetheinylation. Peptide intermediates likely to be bound to the PCPs at various stages of assembly are also shown.
Figure 3. Conversion of a PCP-bound hexapeptide into a monocyclic
product by OxyB.10
dimer formation. The CD spectra of apo-PCP-6S and apo-PCP-
7S, as well as the peptide-PCP-7S conjugate (3), are shown in
Figure S8 (Supporting Information).
Both apo-PCP-6S and apo-PCP-7S could be loaded efficiently
with a peptide-phosphopantetheinyl group after incubation with
a peptide-SCoA thioester and the recombinant phosphopanteth-
einyl transferase Sfp from B. subtilis.15 Each peptide-loaded PCP
(3, 4, 13, or 14) was isolated in ca. 95% purity, as analyzed by
reverse phase HPLC, and each gave a molecular ion by MS
consistent with the expected mass (see Experimental Section).
Reduction System. Spinach ferredoxin (sp-ferredoxin) was
produced as an N-terminal His6-tagged protein in E. coli and
Figure 4. Model hexapeptides and heptapeptides studied in this work.
purified using metal ion affinity chromatography. The molecular
weight of the protein determined by ESI-MS corresponded to
the calculated mass of the His6-tagged protein with one [2Fe-
2S] cluster, and showed UV-vis absorption maxima at 328,
420, and 463 nm (see Supporting Information). The spinach
ferredoxin reductase used was of commercial origin (Sigma).
The E. coli flavodoxin (eco-Flv) and flavodoxin reductase (eco-
FlvR) were also prepared as soluble N-terminal His6-tagged
proteins in E coli and were purified and characterized in the
same way (see Supporting Information).
In assays with the peptide-PCP-7S construct 3, conversion
to monocyclic product (5) was consistently more efficient using
sp-ferredoxin and spinach ferredoxin reductase than with eco-
Flv/eco-FlvR. It was also observed that excellent turnover could
be achieved using sp-ferredoxin and eco-FlvR, with the added
advantage that both these proteins could be easily produced,
purified, and characterized (see above). All assays described
below, therefore, used the sp-ferredoxin/eco-FlvR pair.
Assays with OxyB. Assay conditions were first established
using as substrate the hexapeptide-PCP-6S conjugate (3). After
incubation of 3 with OxyB, sp-ferredoxin, eco-FlvR, and
(15) Quadri, L. E. N.; Weinreb, P. H.; Lei, M.; Nakano, M. M.; Zuber, P.; Walsh,
C. T. Biochemistry 1998, 37, 1585-1595.
9
J. AM. CHEM. SOC. VOL. 129, NO. 21, 2007 6889

A R T I C L E S
Woithe et al.
no monocyclic product was formed, showing that oxygen is
required for turnover (see Figure S6, Supporting Information).
Substrate Specificity of OxyB. We then tested the hexapep-
tide-PCP-7S conjugate 4 having an unmethylated N-terminus.
Under standard conditions with OxyB, complete conversion of
the peptide to monocyclic products was observed (see
Supporting Information for analytical data) again with the major
product being 8 and a second minor cyclic product (ca. 10%)
corresponding to an artefactual epimeric form, as seen during
assays with 3. We conclude that both 3 and 4 are converted
into monocyclic products (5 and 6), with comparable rates,
indicating that the presence or absence of an N-methyl group
in (R)Leu-1 has no significant influence on these OxyB
reactions.
Figure 5. Activation of peptides as thioesters.
NADPH with glucose-6-phosphate and glucose-6-phosphate
dehydrogenase, the peptidic products were cleaved from the
PCP domain by treatment with hydrazine, and the cor-
responding peptide hydrazides were purified by solid-phase
extraction and reverse-phase HPLC. In this way, the monocyclic
product 7 could be isolated and fully characterized by MS and
NMR, as described earlier.10 In order to confirm the proposed
structure, which so far had been deduced by spectroscopic
methods, the monocyclic peptide (7) was also synthesized
using established methods, as described in the Supporting
Information. The synthetic material and product from the
enzymatic assays were then compared by high field (600 MHz)
1H NMR and ESI-MS and MS/MS analysis, which confirmed
both the connectivity and the configuration of the enzymatic
product.
We next tested the two heptapeptides 13 and 14, each linked
to PCP-7S, as substrates for OxyB. These assays were performed
with a peptide comprising a mixture of two epimers at the Dpg-7
C(R) position. This was unavoidable, since during the conversion
of each C-terminal S-phenyl thioester to the SCoA thioester
(Figure 5), an epimerization in Dpg-7 occurred, and the resulting
epimers could not be resolved by HPLC. After incubation with
OxyB and the redox proteins and cofactors (see above), the
peptidic products were again cleaved from the PCP with
hydrazine and then analyzed by HPLC. Typically, starting from
13 or 14, four peaks could be identified, corresponding to two
epimeric linear peptides having the same mass by ESI-MS but
differing 1H NMR spectra, and two epimeric monocyclic
peptides (15 and 16) (Figure S4, Supporting Information). These
cross-linked products were purified and characterized by MS
and NMR, which confirmed the formation of the macrocycle
linking the aromatic rings in Hpg-4 and Tyr-6. This shows that
OxyB is also able to transform heptapeptides (13 and 14) linked
to PCP-7S into the expected cross-linked products.
Closer analysis of the products isolated from the enzymatic
assays, however, consistently revealed in HPLC chromatograms,
apart from unreacted linear peptide and the expected monocyclic
product 7, a second minor peak (ca. 10-20% of the major
product) with a shorter retention time (Figure S3, Supporting
Information), which by MS exhibited the same mass as the
monocyclic product (7), and by 1D and 2D 1H NMR and ESI-
MS/MS fragmentation analysis the same overall connectivity.
It seems most likely that this minor product is an epimer of
peptide 7. It is noteable from the NMR assignments of the major
and minor products (see Supporting Information), that the
chemical shifts of corresponding protons within each residue
in each peptide are the same to within (0.05 ppm, with the
exception only of protons in Hpg-4 and Tyr-6, where significant
differences (0.3-1.1 ppm) are seen between the major and minor
forms. Hence, it seems most likely that the minor isomer arises
due to an epimerization at either Tyr-6 C(R) or at Hpg-4 C(R),
at some point during the assay procedure.
The model hexapeptide (9) was also assayed without modi-
fication, as a free acid, as an N-acetylcysteamine S-thioester
(SNAC), and as the SCoA thioester. Under standard conditions,
where the hexapeptide loaded on PCP-7S (3) would be
converted to the extent of 90% into monocyclic product within
10 min, it was possible to detect formation of small amounts
of monocyclic products from these other derivatives after 60
min incubation. Thus, the SCoA thioester was converted to the
extent of ca. 40-50% into the monocyclic product, the SNAC
thioester to about 15-20%, and even the free carboxylic acid
form 9 was transformed to the extent of 5-10% into a
monocyclic product identified by HPLC and showing the
expected high-resolution mass by ESI-MS (data not shown).
Insufficient amounts of this last material were isolated for a
full NMR analysis, but fragmentation patterns in MS/MS
provide strong support for the proposed formation of a cross-
link between rings in Hpg-4 and Tyr-6. These results confirm
that the PCP domain in the substrate 3 contributes substantially
to the efficiency of the phenol coupling reaction catalyzed by
OxyB, although small amounts of cross-linked products are
detectable, under these optimized conditions, with the SCoA
and SNAC thioesters, and even with the peptide 9 as a
C-terminal free acid.
The time-course of the OxyB reaction was followed by HPLC
analysis of peptide hydrazide formation. Thus, it was consis-
tently found that peptides loaded onto PCP-7S were significantly
better substrates for OxyB than the corresponding PCP-6S
conjugates; for example, under conditions where 9 loaded on
PCP-6S was typically converted 50% into monocyclic product,
9 loaded on PCP-7S would be converted 90% into the same
monocyclic product. Therefore, more detailed analyses were
performed with peptides loaded on PCP-7S.
Using the hexapeptide-PCP-7S (3) as substrate, when any
one of the sp-ferredoxin, eco-FlvR, NADPH and regeneration
system, or OxyB was absent from the assay, no conversion
of the substrate into monocyclic product was observed (see
Figure S5, Supporting Information). Hence, we conclude that
the OxyB reaction is strictly dependent upon the presence of
all four components. We also performed assays with all these
components present, but under deoxygenated conditions. Again,
Kinetic Studies. The steady-state kinetics of the OxyB
reaction with peptide-PCP-7S 3 were investigated to determine
Km and kcat. To avoid effects due to uncoupling of NADPH
consumption from product formation, NADPH consumption was
9
6890 J. AM. CHEM. SOC. VOL. 129, NO. 21, 2007

Oxidative Phenol Coupling Reactions
A R T I C L E S
Figure 6. (A) Monitoring the rate of substrate (3) turnover. HPLC
chromatograms obtained after 30, 60, 90, 120, 150 s and 60 min (see
Experimental Section). (B) Rate vs substrate concentration with hexapeptide
PCP-7S (3).
not used to monitor reaction rates, but rather a direct measure-
ment of turnover rate was established based on HPLC quanti-
tation of product formation (Figure 6A). The dependence of
sp-ferredoxin and eco-FvR concentrations on the reaction
rate under standard conditions were also investigated, to
ensure that the concentrations of each used were high enough
to saturate OxyB during the first few minutes of the reaction.
In this way, an apparent Km in the range 4-13 µM (three
independent determinations) and kcat ) 0.1 ( 0.02 s-1 were
found (Figure 6B).
Binding Studies by UV-vis. The binding of hexapeptide-
PCP-7S conjugate (3) to OxyB was monitored by UV-vis
spectroscopy. As reported earlier, OxyB displays a Soret band
at 419 nm and â and R peaks at 537 and 571 nm, respectively,
typical for those of a low spin cytochrome P450. Upon
addition of hexapeptide loaded on PCP-7S (3) to OxyB, the
Soret band is shifted to lower wavelength, as detected in a UV-
difference spectrum by a positive band at 391 nm and a trough
at 426 nm (Figure 7A). The concentration dependence of the
peak intensities fitted well a binding equation involving a 1:1
interaction, with a Kd of 17 ( 5 µM. These data are consistent
with a typical type-I ligand (substrate), which upon binding
displaces the water molecule as axial Fe ligand and causes a
shift in the equilibrium spin state from hexacoordinate low spin
toward pentacoordinate high spin. The hexapeptide 10 loaded
on PCP-7S behaved in a similar way, with a Kd of 18 ( 5 µM
(data not shown).
Figure 7. UV-Difference spectra of (A) hexapeptide PCP-7S (3) binding
to OxyB, and (B) hexapeptide PCP-6S (3) binding to OxyB. The
concentration dependence of the spectral changes and the wavelengths of
the minima and maxima are shown.
to 3 on PCP-7S, the relative magnitude of the trough is much
reduced, and the concentration dependence of the changes did
not show saturation behavior in the range studied, suggesting
that the Kd may be much higher. Similar UV/vis spectra were
obtained when OxyB was titrated with the heptapeptide 14
loaded on PCP-7S (data not shown), although here the peptide
comprised a mixture of two diastereomers (see above).
Discussion
The OxyB reaction belongs to one of only a handful of
oxidative phenol couplings catalyzed by specific cytochrome
P450 hemoproteins, which to date have been characterized
biochemically. Other examples include P450s involved in plant
alkaloid biosynthesis,16-19 a Streptomyces coelicolor flaviolin
oxidase,20 a Streptomyces griseus tetrahydroxynaphthalene oxi-
dase,21 and an aryl-aryl coupling enzyme involved in stauro-
sporine biosynthesis.22
(16) Gerardy, R.; Zenk, M. H. Phytochemistry 1993, 32, 79-86.
(17) Kraus, P. F. X.; Kutchan, T. M. Proc. Natl. Acad. Sci. U.S.A. 1995, 92,
2071-2075.
(18) Stadler, R.; Zenk, M. H. J. Biol. Chem. 1993, 268, 823-831.
(19) Nasreen, A.; Rueffer, M.; Zenk, M. H. Tetrahedron Lett. 1996, 37, 8161-
8164.
(20) Zhao, B.; et al. J. Biol. Chem. 2005, 280, 11599-11607.
(21) Funa, N.; Funabashi, M.; Ohnishi, Y.; Horinouchi, S. J. Bacteriol. 2005,
187, 8149-8155.
(22) Howard-Jones, A. R.; Walsh, C. T. J. Am. Chem. Soc. 2006, 128, 12289-
12298.
Similar studies using hexapeptide loaded on PCP-6S (3)
produced changes in the UV difference spectra, which were
significantly different (Figure 7B). Thus the positions of the
maximum (410 nm) and trough (435 nm) are shifted compared
9
J. AM. CHEM. SOC. VOL. 129, NO. 21, 2007 6891

A R T I C L E S
Woithe et al.
At the outset of our work, the preferred substrate of OxyB
was unknown, although attempts to detect turnover using the
free heptapeptides 17 (derived from 2, Figure 2) had proven
unsuccessful.9 In more recent gene knock-out experiments, a
bpsC mutant of the balhimycin producer A. balhimycina (i.e., a
mutated condensing domain in module-7 of NRPS subunit-3
(BpsC)) was found to produce the monocyclic hexapeptide 18,5
showing that the first cross-link could be introduced in ViVo at
the hexapeptide stage. Hence, attention turned to assays with
hexa- and heptapeptides loaded as thioesters on PCP domains
from the NRPS. For reasons of their easier synthesis, however,
we chose to focus first on peptide analogues containing tyrosine
at positions-2 and -6, i.e., hexapeptide-PCP conjugates 3 and
4, and heptapeptide-PCP conjugates 13 and 14.
The free linear peptides required for this work comprised the
model hexapeptides 9 and 10, and heptapeptides 11 and 12
(Figures 3 and 4) that could each be assayed as the correspond-
ing peptide-PCP thioesters 3, 4, 13, and 14. The N-terminus
of 3 and 9 contains (R)-N-methylleucine. N-Methylation actually
occurs at a later stage in glycopeptide biosynthesis, after peptide
assembly and cross-linking.23 Our first experiments were
performed with these N-methylated peptides, since it was
expected that a secondary amine might be more stable in the
presence of the C-terminal thioester group,24 yet the peptides
might nevertheless be acceptable substrates for OxyB. These
assumptions were shown to be valid in the course of this work.
Subsequently, a method for the preparation of the non-N-
methylated peptides (4 and 14) was established, employing a
temporary Boc-N-protecting group (peptides 10 and 12), which
could be removed with TFA after activation of the C-terminus
as a S-phenyl thioester (Figure 5). Conversion into the PCP-
derivatives was then achieved using the SCoA derivatives
and the phosphopantetheinyl transferase Sfp from Bacillus
subtilis.15,24
Interestingly, the CD spectrum of the apo-PCP-7S showed
minima at 208 and 222 nm, with a shape suggesting that the
protein was folded with significant helical content, consistent
with the expected four-helix bundle fold (Figure S8, Supporting
Information).26 The CD spectrum of apo-PCP-6S, however,
showed a much more intense minimum at 202 nm, suggesting
either that a significant proportion of the protein may be
unfolded, or that the global fold includes a much larger
proportion of disordered regions.27,28 Certainly, the CD signa-
tures suggest a significant difference between the structures of
apo-PCP-6S and apo-PCP-7S, which is of interest here in the
light of binding studies with the peptide-loaded PCPs and OxyB
(see later).
Bacterial class-I P450s typically require the presence of a
ferredoxin-like [Fe-S]-protein and a flavin-containing ferre-
doxin reductase for substrate turnover, which receive reducing
equivalents from NADPH. The class-II P450s, on the other hand,
usually receive electrons from NADPH through a flavin-
containing reductase.29-31 Within the published glycopeptide
biosynthetic gene clusters no ferredoxin or ferredoxin reductase
has been identified,3,32-36 with the exception of a single
ferredoxin in the complestatin cluster.32 So the natural redox
partners for the biosynthetic cross-linking enzymes are mostly
unknown. In assays with the peptide 9 loaded on PCP-7S (3),
the sp-ferredoxin/eco-FlvR pair was used as electron-transfer
proteins.
The conversion of hexapeptide-PCP-6S (3) into monocyclic
product (5), isolated as the peptide hydrazide 7, was reported
earlier.10 We could show here for the first time that the same
hexapeptide loaded on PCP-7S is converted at a significantly
faster rate into the monocyclic product 5. This observation is
interesting, on the one hand, because in UV-vis binding assays
we could also show that the interaction of hexapeptide-PCP-
6S (3) with OxyB is substantially weaker than that of hexapep-
tide-PCP-7S (3) with OxyB (Figure 7). The binding data
suggest a dissociation constant of hexapeptide-PCP-7S (3) with
OxyB of about 17 µM. On the other hand, we have also seen
that PCP-6S and PCP-7S have different CD signatures (Figure
S8), with that of PCP-7S resembling the spectrum expected of
a four-helix bundle PCP or acyl carrier protein (ACP),26 and
that of PCP-6S resembling more closely those of ACP domains
The PCP domains used here were derived from the vanco-
mycin NRPS modules-6 and -7. So far no structural information
is available on the vancomycin NRPS. Nevertheless, the 3D
structure of a typical NRPS PCP domain, e.g., TycC3 from the
tyrocidine NRPS (PDB files 2GDW/2GDX/2GDY), comprises
a compact four-helix bundle,25 with the phosphopantetheinylated
active site Ser residue located in a loop connecting helix-1 and
helix-2. The minimal four-helix bundle includes about 32
residues on the N-terminal side and 38 residues on the
C-terminal side of the active site Ser (denoted here 32-Ser-38).
We, therefore, first attempted to produce recombinant N-terminal
His6-tagged apo-PCP domains encompassing residues 30-Ser-
40, as well as 37-Ser-40, from module-6 of the vancomycin
NRPS (Figure 2). However, both were insoluble when produced
in the cytoplasm of E. coli. On the other hand, a 30-Ser-52
construct (PCP-6S) could be produced in a soluble form and
was easily purified as described earlier.10 In addition, the PCP
domain from NRPS module-7 (37-Ser-39, called PCP-7S) could
be produced in the same way as a soluble N-terminal His6-
tagged fusion protein.
(26) Mofid, M. R.; Finking, R.; Marahiel, M. A. J. Biol. Chem. 2002, 277,
17023-17031.
(27) Flaman, A. S.; Chen, J. M.; Van Iderstine, S. C.; Byers, D. M. J. Biol.
Chem. 2001, 276, 35934-35939.
(28) Schulz, H. J. Biol. Chem. 1975, 250, 2299-2304.
(29) Ortiz de Montellano, P. R. Cytochrome P450. Structure, mechanism, and
Biochemistry, 3rd ed.; Kluwer Academic/Plenum Publishers: New York,
2005.
(30) Pylypenko, O.; Schlichting, I. Annu. ReV. Biochem. 2004, 73, 991-1018.
(31) Paine, M. J. I.; Scrutton, N. S.; Munro, A. W.; Gutierrez, A.; Roberts, G.
C. K.; Wolf, C. R. Electron transfer partners of cytochrome P450. In
Cytoschrome P450 Structure, Mechanism, and Biochemistry; de Montellano,
P. R. O., Ed.; Kluwer Academic/Plenum Publishers: New York, 2005; pp
115-148.
(32) Chiu, H. T.; Hubbard, B. K.; Shah, A. N.; Eide, J.; Fredenburg, R. A.;
Walsh, C. T.; Khosla, C. Proc. Nat. Acad. Sci. U.S.A. 2001, 98, 8548-
8553.
(33) Li, T. L.; Huang, F. L.; Haydock, S. F.; Mironenko, T.; Leadlay, P. F.;
Spencer, J. B. Chem. Biol. 2004, 11, 107-119.
(23) O’Brien, D. P.; Kirkpatrick, P. N.; O’Brien, S. W.; Staroske, T.; Richardson,
T. I.; Evans, D. A.; Hopkinson, A.; Spencer, J. B.; Williams, D. H. Chem.
Commun. 2000, 103-104.
(34) Pootoolal, J.; Thomas, M. G.; Marshall, C. G.; Neu, J. M.; Hubbard, B.
K.; Walsh, C. T.; Wright, G. D. Proc. Nat. Acad. Sci. U.S.A. 2002, 99,
8962-8967.
(24) Vitali, F.; Zerbe, K.; Robinson, J. A. Chem. Commun. 2003, 2718-
2719.
(35) Sosio, M.; Kloosterman, H.; Bianchi, A.; deVreugd, P.; Dijkhuizen, L.;
Donadio, S. Microbiol. 2004, 150, 95-102.
(36) Sosio, M.; Stinchi, S.; Beltrametti, F.; Lazzarini, A.; Donadio, S. Chem.
Biol. 2003, 10, 541-549.
(25) Koglin, A.; Mofid, M. R.; Lohr, F.; Schafer, B.; Rogov, V. V.; Blum, M.-
M.; Mittag, T.; Marahiel, M. A.; Bernhard, F.; Dotsch, V. Science 2006,
312, 273-276.
9
6892 J. AM. CHEM. SOC. VOL. 129, NO. 21, 2007

Oxidative Phenol Coupling Reactions
A R T I C L E S
in a partially unfolded state.27,37 These differences between PCP-
6S and PCP-7S suggest the importance of having a correctly
folded PCP domain for optimum substrate recognition by OxyB.
This conclusion is reinforced by the large decrease in turnover
seen when the hexapeptide-SCoA and -SNAC thioesters, and
even more pronounced, the hexapeptide free carboxylic acid,
were tested as substrates. However, it is also relevant to note
that during the biosynthesis of vancomycin in ViVo a hexapeptide
intermediate should only ever be bound to PCP-6 and not to
PCP-7. At this time it is unclear which residues in the PCP are
important for recognition by OxyB.
The phenol coupling reaction catalyzed by OxyB was shown
here to depend upon the presence of the complete reduction
system (ferredoxin, ferredoxin reductase (or equivalent), and
NADPH) and molecular oxygen. When any one component was
omitted, no significant turnover of substrate was observed.
OxyB assays (turnover and UV-binding) were also carried
out with model heptapeptides bound to PCP-7S (13 and 14).
These assays were complicated by the unavoidable use of
heptapeptides that comprised a mixture of two diastereomers,
epimeric at the Dpg-7 C(R) position. The results, however,
clearly show that OxyB can catalyze a phenol coupling reaction
on heptapeptide-PCP as well as on hexapeptide-PCP conju-
gates. The use of epimeric heptapeptides in these assays
prevented more detailed kinetic studies, but the rates of
conversion for hexa- and hepta-peptides appeared not to be
vastly different.
These results have important implications in defining the
timing of the phenol coupling reaction during vancomycin
biosynthesis. Thus, whereas OxyB may function on a hexapep-
tide still linked to the PCP-6 in NRPS subunit-2, it seems
possible that a linear heptapeptide intermediate linked to PCP-7
in NRPS subunit-3 may also be transformed into monocyclic
product. Additional studies, in particular with the correct
â-hydroxytyrosine-containing hexa- and heptapeptides, perhaps
linked to larger, natively folded fragments of NRPS subunits-2
and -3, will be required to confirm or refine these conclusions.
Peptides containing â-hydroxytyrosine residues will also be of
interest, since in at least two other P450-catalyzed reactions,38,39
hyroxyl groups in the substrate have been show to interact with
and participate in the breakdown of iron-bound dioxygen during
the P450 catalytic cycle.
Finally, with hexapeptide-PCP-7S (3) the steady-state kinetic
parameters kcat and Km were measured, with the assumption that
OxyB exhibits Michaelis-Menten kinetics under the conditions
used. The kcat of 0.1 s-1 found is comparable to turnover
numbers determined for other cytochrome P450 enzymes,29 and
the Km value (in the range 4-13 µM) is close to the Kd (17
µM) of hexapeptide-PCP-7S (3) binding to resting OxyB
determined here by UV-vis spectroscopy (Figure 7A). These
data should provide a useful benchmark for more detailed studies
in the future with other substrate analogues.
enzymes. The crystal structure of substrate-free OxyB revealed
a typical P450-fold, with the L-helix containing the Cys347
residue that provides the axial thiolate ligand of the heme iron.
Moreover, the F and G helices are rotated out of the active site
compared to other closely related P450s, such as P450nor,
creating a much more open active site, most likely for binding
the large peptide-PCP substrate.9
The UV-vis changes observed upon titrating the protein with
hexapeptide-PCP-7S (3), in particular the shift of the Soret
band to 391 nm (Figure 7), are consistent with the displacement
of the axial water ligand and a low- to high-spin conversion, as
seen after substrate binding to other P450s such as P450cam.29
Following substrate binding to OxyB, the reaction mechanism
most likely involves first electron transfer from the ferredoxin
to the heme-Fe(III), and subsequent binding of molecular oxygen
(Figure 8). After a second electron transfer from the ferredoxin,
cleavage of the Fe-bound dioxygen should occur with loss of a
water molecule and formation of compound-I, a Fe(IV)dO
porphyrin-π-radical cation.29
In the catalytic cycle of a typical P450 hydroxylase like
P450cam, this intermediate oxidizes an aliphatic substrate, first
by hydrogen atom abstraction and then by hydroxyl radical
rebound from the heme to the substrate.40 However, it now
seems clear with several P450 hydroxylases, including P450cam,
that a competing intramolecular electron transfer can occur,
from tyrosine and/or tryptophan residues to the transient Fe-
(IV)dO porphyrin-π-radical cation, to form protein-based
tyrosyl or tryptophan radicals41-43 and a new FedOH species,
called compound-II. Conceivably, a similar electron transfer
might occur during the OxyB reaction, except now the electron
is transferred from a phenol group provided by the bound
peptide-PCP substrate, and the process is not a competing side
reaction but part of the normal catalytic cycle of the enzyme.
This would leave the second electron-transfer step, from the
second phenol group in the peptide substrate to compound-II,
to afford a substrate biradical, which could collapse by radical
coupling and deprotonation to give the product. A similar
process is thought to occur during peroxidase-mediated phenol
coupling reactions.44 Presently, however, alternative mechanisms
cannot be excluded. One possibility is that compound-I might
oxidize a phenol group in the substrate to form an arene oxide,
a process that is thought to occur in P450s that catalyze the
hydroxylation of aromatic groups.45 Rather than a rearrangement
of the arene oxide to an aromatic system, a nucleophilic attack
by the second phenol ring in the substrate followed by
elimination of water could lead to the desired product (Figure
8). Clearly, more experiments will be required to distinguish
between these and other mechanisms.
Experimental Section
General Method for the Synthesis of Peptide S-Phenyl Thioesters.
The peptide (9-12) (2 mg) in freshly distilled DMF (1 mL) was stirred
with PyBOP (1.2 equiv), iPr2EtN (1.2 equiv), and thiophenol (2.4 equiv)
The results presented here show that OxyB has spectroscopic
properties and substrate requirements (electron-transfer proteins,
molecular oxygen) that mirror those seen in many other P450
(40) Altun, A.; Guallar, V.; Friesner, R. A.; Shaik, S.; Thiel, W. J. Am. Chem.
Soc. 2006, 128, 3924-3925.
(41) Jung, C.; Schunemann, V.; Lendzian, F. Biochem. Biophys. Res. Commun.
2005, 338, 355-364.
(42) Jung, C.; Schunemann, V.; Lendzian, F.; Trautwein, A. X.; Contzen, J.;
Galander, M.; Bottger, L. H.; Richter, M.; Barra, A. L. Biol. Chem. 2005,
386, 1043-1053.
(43) Schu¨nemann, V.; Lendzian, F.; Jung, C.; Contzen, J.; Barra, A. L.; Sligar,
S. G.; Trautwein, A. X. J. Biol. Chem. 2004, 279, 10919-10930.
(44) Derat, E.; Shaik, S. J. Am. Chem. Soc. 2006, 128, 13940-13949.
(45) Guengerich, F. P. Arch. Biochem. Biophys. 2003, 409, 59-71.
(37) Park, S. J.; Kim, J.-S.; Son, W.-S.; Lee, B. J. J. Biochem. (Tokyo) 2004,
135, 337-346.
(38) Nagano, S.; Cupp-Vickery, J. R.; Poulos, T. L. J. Biol. Chem. 2005, 280,
22102-22107.
(39) Zhao, B.; Guengerich, F. P.; Voehler, M.; Waterman, M. R. J. Biol. Chem.
2005, 280, 42188-42197.
9
J. AM. CHEM. SOC. VOL. 129, NO. 21, 2007 6893

A R T I C L E S
Woithe et al.
Figure 8. Putative mechanisms for the OxyB reaction (see text). After formation of compound-I, path-A leads via single electron transfers to a phenolic
biradical, whereas path-B involves formation of an arene-oxide intermediate.
under N2. The reaction was monitored by LC/MS and was typically
complete after 5-15 min. After lyophilization, the product was purified
by semipreparative reverse-phase HPLC (C18 Vydac 218TP1010, 250
× 10 mm, pore diameter 300 Å, particle size 5 µm) with a gradient of
5-100% MeCN/0.1% TFA in water/0.1% TFA in 5 column volumes
at a flow rate of 5 mL/min. The corresponding peptide S-phenyl
thioester was obtained in >90% yield. When the reaction was started
from 10 and 12, the Boc group was removed by stirring the peptide
S-phenyl thioster (2 mg) in CH2Cl2/TFA (10:1, 2 mL) at rt for 2 h.
After being dried in vacuo, the product was isolated by HPLC as above,
in near quantitative yield. For all analytical data see Supporting
Information.
7S, m/z 11730 ( 2 [M + H]+, calcd mass 11728; 14-PCP-7S, m/z
11715 ( 2 [M + H]+, calcd mass 11714.
Upon complete reaction, the mixture was dialyzed against HEPES
buffer (25 mM, pH 7.0, 50 mM NaCl), and the resulting solution
containing peptide-PCP conjugate could be used in assays without
further purification.
For kinetic and UV binding studies, the peptide-PCP conjugate was
first purified by anion exchange chromatography (MonoQ HR10/10,
20 mM diethanolamine, pH 8.5, using a gradient of 0 to 0.8 M KCl
over 10 column volumes). The eluate was dialyzed against Tris-HCl
buffer (50 mM, pH 7.5) and concentrated to 100-150 µM using an
Amicon ultrafiltration cell containing a YM-3 membrane. The con-
centration of peptide-PCP was determined by UV using the following
General Method for the Synthesis of Peptide-SCoA Thioesters.
The corresponding peptide-S-phenyl thioester (2 mg) and coenzyme A
(4 equiv) were stirred in phosphate buffer (4 mL, 50 mM, pH 8.5).
The reaction was complete after 15-120 min, as monitored by LC/
MS, and the product was purified by semipreparative HPLC on a
reverse-phase C18 column (Vydac 218TP1010, 250 × 10 mm, pore
diameter 300 Å, particle size 5 µm) using a gradient of 45-100%
MeCN in 20 mM NH4AcO in 5 column volumes, with a flow rate of
5 mL/min, desalting: 25-40% MeCN/0.1% TFA in water/0.1% TFA
in 5 column volumes at 5 mL/min. The corresponding peptide-SCoA
thioesters were recovered in 40-60% yield. For characterization see
Supporting Information.
molar extinction coefficients: 3-PCP-6S ꢀ280 ) 12810 cm-1 M-1
3-PCP-7S ꢀ280 ) 7120 cm-1 M-1; 13-PCP-7S ꢀ275 ) 8868 cm-1 M-1
;
.
General Assay Procedure with OxyB. The assay contained the
following components at the concentrations given: peptide-PCP-
conjugate (80 µM), OxyB (8 µM), spinach ferredoxin (20 µM), E.coli
flavodoxin reductase (10 µM), NADPH (1 mM), glucose-6-phosphate
(1 mM), glucose-6-phosphate-dehydrogenase (0.5 U, Sigma) in HEPES
buffer (25 mM, pH 7.0, 50 mM NaCl), incubated at 30 °C for 60 min.
Addition of 1/10 volume of a 25% v/v aqueous hydrazine and
incubation at 30 °C for 30 min yielded the peptide hydrazides, which
were separated from proteins by solid-phase extraction (Waters OASIS
HLB cartridges). The peptidic fraction was analyzed by analytical HPLC
on a reverse-phase C18 Zorbax Eclipse XDB column (Agilent, 250 ×
4.6 mm, pore diameter 80 Å, particle size 5 µm) under the following
conditions: 5-40% acetonitrile/0.1% TFA in water/0.1% TFA in 8
column volumes with a flow rate of 1 mL/min.
Anaerobic assays were performed in a glovebox with oxygen levels
below 2 ppm. All solutions were degassed in four freeze-thaw cycles.
The His6-OxyB activity assay was initiated by addition of NADPH,
incubated at room temperature and quenched by addition of trichlo-
roacetic acid (TCA) prior to removal from the glovebox.
Kinetic Studies. Assays containing peptide-PCP-conjugate (5-100
µM), spinach ferredoxin (20 µM), E. coli flavodoxin reductase (10 µM),
glucose-6-phosphate (1 mM), and glucose-6-phosphate-dehydrogenase
General Method for the Synthesis of Peptide-PCP Conjugates.
The reaction mixture contained the following components at the
concentrations given: apo-PCP (120 µM), peptide-SCoA thioester (100
µM), B. subtilis Sfp (5 µM), and MgCl2 (50 mM) in Tris/HCl buffer
(50 mM, pH 7.5) and was incubated at 37 °C for 30 min. The reaction
was monitored by analytical HPLC (Vydac C18 218TP54 column, 250
× 4.6 mm, pore diameter 300 Å, particle size 5 µm) using a gradient
of 5-100% MeCN/0.1% TFA in water/0.1% TFA over 4 column
volumes at 1 mL/min. The conversion proceeded quantitatively. Each
peptide-PCP conjugate was collected from the HPLC column and
analyzed by MALDI-MS; 3-PCP-6S, m/z 11373 ( 2 [M + H]+, calcd
mass 11373; 3-PCP-7S, m/z 11564 ( 2 [M + H]+, calcd mass 11563;
4-PCP-7S, m/z 11551 ( 2 [M + H]+, calcd mass 11549; 13-PCP-
9
6894 J. AM. CHEM. SOC. VOL. 129, NO. 21, 2007

Oxidative Phenol Coupling Reactions
A R T I C L E S
(0.5 U, Sigma) in Tris-HCl buffer (1.2 mL, 50 mM, pH 7.5) were
incubated at 30 °C. The reaction was initiated by addition of a mixture
of His6-OxyB (0.5 µM) and NADPH (1 mM). Samples (200 µL) were
withdrawn at time intervals (t ) 15, 30, 75, 60, 75, 90 s) and added to
TCA (20 µL, 6 M). The pellet was washed with 10% (v/v) TCA in
Tris-HCl (50 mM, pH 7.5), and resuspended in 2.5% (v/v) hydrazine
in Tris-HCl (50 mM, pH 8.0). After 30 min at 30 °C, protein was
precipitated with TCA. The supernatant containing the peptide hy-
drazides was analyzed by analytical reverse-phase HPLC (Agilent C18
Zorbax Eclipse XDB column, 250 × 4.6 mm, pore diameter 80 Å,
particle size 5 µm) using a gradient of 5-40% MeCN/0.1% TFA in
water/0.1% TFA in 8 column volumes, flow rate 1 mL/min. The peaks
corresponding to the linear and monocyclic peptide hydrazides were
integrated to give initial velocities, which were used with the Michae-
lis-Menten equation to derive the steady-state kinetic parameters Km
change (Aobs) was taken from the absolute difference betwen the positive
and negative peaks in each difference spectrum. This value is related
to the total substrate concentration (S), the total enzyme concentration
(Et), and the Kd through the following eq 1 describing a 1:1 binding
interaction:46
A
Aobs
)
max[(S + E + K ) - (S + E + K )2 - (4SE )] (1)
x
t
d
t
d
t
2[Et]
Amax and Kd were obtained by nonlinear least-squares curve fitting of
data using eq 1 and Origin software (Microcal).
Acknowledgment. This work was supported by the Swiss
National Science Foundation and the European Union 6th
Framework Program.
Supporting Information Available: Complete ref 20, syn-
thesis and analytical data of the linear and cyclic peptides and
all thioesters, production and characterization of all recombinant
proteins, total synthesis of the monocyclic peptide 7. This
material is available free of charge via the Internet at
http://pubs.acs.org.
and Vmax
.
Binding Studies by UV. Absorbance spectra were recorded using
a double beam Varian Cary300 spectrophotometer. OxyB (1.6 µM) in
Tris-HCl (2.4 mL, 50 mM, pH 7.5) was divided between reference
and sample cuvette. After thermal equilibration at 30 °C, a baseline
was recorded between 600 and 350 nm, followed by sequential additions
(25 µL) of concentrated substrate solution (200 µM substrate in 50
mM Tris-HCl pH 7.5, containing 1.6 µM OxyB to maintain a constant
OxyB concentration) to the sample cuvette, with stirring, resulting in
a final substrate concentration in the range 0-60 µM. UV-difference
spectra were recorded after each addition. A maximal overall absorption
JA071038F
(46) Segel, I. H. Enzyme Kinetics: BehaVior and Analysis of Rapid Equilibrium
and Steady State Enzyme Systems; J. Wiley & Sons: New York, 1975; p
73.
9
J. AM. CHEM. SOC. VOL. 129, NO. 21, 2007 6895