Regiospecific chlorination of (S)-β-tyrosyl-S-carrier protein catalyzed by SgcC3 in the biosynthesis of the enediyne antitumor antibiotic C-1027

Article abstract of DOI:10.1021/ja072311g

C-1027 is a potent antitumor antibiotic composed of an apo-protein and a reactive enediyne chromophore. The chromophore consists of four different chemical subunits including an (S)-3-chloro-4,5-dihydroxy-β-phenylalanine moiety, the biosynthesis of which

Full text of DOI:10.1021/ja072311g

Published on Web 09/22/2007
Regiospecific Chlorination of (S)-â-Tyrosyl-S-Carrier Protein
Catalyzed by SgcC3 in the Biosynthesis of the Enediyne
Antitumor Antibiotic C-1027
Shuangjun Lin,† Steven G. Van Lanen,† and Ben Shen*,†,‡,§
Contribution from the DiVision of Pharmaceutical Sciences, UniVersity of Wisconsin National
CooperatiVe Drug DiscoVery Group, and Department of Chemistry, UniVersity of Wisconsins
Madison, Madison, Wisconsin 53705
Received April 2, 2007; E-mail: bshen@pharmacy.wisc.edu
Abstract: C-1027 is a potent antitumor antibiotic composed of an apo-protein and a reactive enediyne
chromophore. The chromophore consists of four different chemical subunits including an (S)-3-chloro-4,5-
dihydroxy-â-phenylalanine moiety, the biosynthesis of which from L-R-tyrosine is catalyzed by six proteins,
SgcC, SgcC1, SgcC2, SgcC3, SgcC4, and SgcC5. Biochemical characterization of SgcC3 unveiled the
following: (i) SgcC3 is a flavin adenine dinucleotide (FAD)-dependent halogenase; (ii) SgcC3 acts only on
the SgcC2 peptidyl carrier protein-tethered substrates; (iii) SgcC3-catalyzed halogenation requires O2 and
reduced FAD and either the C-1027 pathway-specific flavin reductase SgcE6 or E. coli flavin reductase
(Fre) can support the SgcC3 activity; (iv) SgcC3 also efficiently catalyzes bromination but not fluorination
or iodination; (v) SgcC3 can utilize both (S)- and (R)-â-tyrosyl-S-SgcC2 but not 3-hydroxy-â-tyrosyl-S-
SgcC2 as a substrate. These results establish that SgcC3 catalyzes the third enzymatic transformation
during the biosynthesis of the (S)-3-chloro-4,5-dihydroxy-â-phenylalanine moiety of C-1027 from L-R-tyrosine.
SgcC3 now represents the second biochemically characterized flavin-dependent halogenase that acts on
a carrier protein-tethered substrate. These findings will facilitate the engineering of new C-1027 analogs
by combinatorial biosynthesis methods.
Introduction
The induction of DSB correlates well with cytotoxicity, and
because of this novel mechanism of DNA damage, the enediynes
C-1027 is a chromoprotein antitumor antibiotic isolated from
the fermentation broth of Streptomyces globisporus.1 Similar
to other nine-membered enediynes, which includes neocarzi-
nostatin2 and maduropeptin,3 C-1027 is composed of an apo-
protein (CagA) and a reactive enediyne chromophore that is
essential for bioactivity.4 Upon release from CagA, the C-1027
chromophore readily undergoes a Bergman cycloaromatization
to form a benzenoid diradical intermediate (Figure 1). These
free radicals are capable of abstracting hydrogen atoms from
the DNA backbone, ultimately resulting in DNA double-
stranded breaks (DSB) in the presence of molecular oxygen.5-7
have attracted interest as potential cancer chemotherapeutic
agents.8,9
The nine-membered enediynes all have an enediyne core
consisting of an unsaturated nine-membered carbocycle contain-
ing two acetylenic groups conjugated to a recipient or incipient
double bond. The C-1027 chromophore (1) has three additional
chemical components that are covalently appended to the
enediyne core, namely, a deoxy aminosugar, a benzoxazolinate,
and a â-amino acid moiety (Figure 1). The C-1027 biosynthetic
gene cluster was previously identified and, based on bioinfor-
matics analysis, the biosynthetic pathway for each moiety was
predicted and a convergent pathway was hypothesized to yield
1. Of note, the â-amino acid moiety (S)-3-chloro-4,5-dihydroxy-
â-phenylalanine was predicted to be biosynthesized from L-R-
tyrosine (2) mediated by five proteins: SgcC, SgcC1, SgcC2,
SgcC3, and SgcC4, with the condensation enzyme SgcC5
catalyzing the final incorporation of 2 into 1 via a â-aminoacyl-
S-SgcC2 intermediate (3) (Figure 2).8 We have subsequently
confirmed in vitro using purified recombinant enzymes that (i)
SgcC4 catalyzes the conversion of 2 to (S)-â-tyrosine (4) as
* To whom correspondence should be addressed: School of Pharmacy,
University of WisconsinsMadison, 777 Highland Ave., Madison, Wisconsin
53705. Tel.: (608) 263-2673. Fax: (608) 262-5345.
† Division of Pharmaceutical Sciences.
‡ University of Wisconsin National Cooperative Drug Discovery Group.
§ Department of Chemistry.
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10.1021/ja072311g CCC: $37.00 © 2007 American Chemical Society

SgcC3-Catalyzed Chlorination of (S)-
â
-Tyrosyl-S-SgcC2
A R T I C L E S
Figure 1. Bergman cycloaromatization exemplified by the C-1027 chromophore. Upon release from the apoprotein CagA, the enediyne core (shown in red)
undergoes an electronic rearrangement to form a 3,6-diradical species that can abstract hydrogen atoms from DNA. DSB, double-stranded break.
∆sgcC mutant accumulated 22-deshydroxy-C-1027 (9).8 Thus,
the requirement of SgcC3 for chlorination was clearly estab-
lished, although these results provided little insight into the
substrate specificities and mechanistic details of this enzyme.
In general, incorporation of a halogen into natural products
plays an important role in increasing the diversity and biological
activity of natural products. Indeed, 7 was recently shown to
be less proficient at inducing DSB, to be less cytotoxic, and to
have an altered mechanism of action compared to the typical
cellular response induced by treatment with 1.9 Prior to 1997,
halogenation was believed to occur only by the action of
haloperoxidasessenzymes that utilize a heme or vanadium
cofactor and hydrogen peroxide as a cosubstrate.15 Since this
time, several O2-dependent halogenases have been described
and their activities biochemically confirmed. Currently, the O2-
dependent halogenases fall into one of two families: R-keto-
glutarate-dependent halogenases that act on nonactivated ali-
phatic substrates16-21 and flavin-dependent halogenases that
catalyze the regioselective halogenation of primarily aromatic
compounds.16,22-31 SgcC3 has sequence homology to flavin-
dependent halogenases, and combined with the in vivo data,
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Figure 2. Biosynthesis of (S)-3-chloro-4,5-dihydroxy-â-phenylalanine
moiety (3, boxed) of C-1027 (1). (A) A subcluster of genes within the
C-1027 gene cluster that encode biosynthetic proteins for 3 production and
(B) proposed biosynthetic pathway for 3 from L-R-tyrosine (2) and structures
of 1 and engineered analogs 7, 8, and 9.
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T. Nature 2005, 436, 1191-1194.
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the first step10-12 and (ii) SgcC1 activates 4 as an (S)-â-tyrosyl
adenylate (5) and subsequently loads 4 to SgcC2, a peptidyl
carrier protein (PCP), to yield (S)-â-tyrosyl-S-SgcC2 (6) as the
second step.13,14 SgcC3 and SgcC were also identified as the
C-3 halogenase and C-5 hydroxylase, respectively, by gene
inactivation: the ∆sgcC3 mutant produced 20-deschloro-C-1027
(7) and 20-deschloro-22-deshydroxy-C-1027 (8),13 while the
(21) Galonic, D. P.; Vaillancourt, F. H.; Walsh, C. T. J. Am. Chem. Soc. 2006,
128, 3900-3901.
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641.
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A.; Van Pee, K.-H. Chem. Biol. 2005, 12, 445-452.
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H. Angew. Chem., Int. Ed. 2000, 39, 2300-2302.
(25) Dong, C.; Flecks, S.; Unversucht, S.; Haupt, C.; Van Pee, K.-H.; Naismith,
J. H. Nature 2005, 309, 2216-2219.
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102, 3960-3965.
(10) Christenson, S. D.; Liu, W.; Toney, M. D.; Shen, B. J. Am. Chem. Soc.
2003, 125, 6062-6063.
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C. T. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 13843-13848.
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C. T. Biochemistry 2006, 45, 7904-7912.
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Biochemistry 2003, 42, 12708-12718.
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S. D. Biochemistry 2007, 46, 7205-7214.
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Biochemistry 2007, 46, 1284-1292.
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Kelleher, N. L.; Shen, B. J. Am. Chem. Soc. 2005, 127, 11594-11595.
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A R T I C L E S
Lin et al.
was washed with 5-column volumes of Buffer B followed by 5-column
volumes of Buffer B containing 20 mM imidazole. The His6-tagged
proteins were eluted with 6-column volumes of Buffer B containing
250 mM imidazole. SgcC2 was dialyzed in 50 mM Tris-HCl, pH 7.5,
50 mM NaCl, and 1 mM dithiothreitol. SgcC1, SgcC3, SgcE6, and
Fre were desalted using a PD-10 column (GE Healthcare, Piscataway,
NJ). After concentration with an Amicon Ultra-4 (5K or 10K), the
purified proteins were stored at -25 °C in 40% glycerol. Protein purity
was assessed as >90% by 12-15% SDS-PAGE. Protein concentrations
were determined using the Bradford assay (Bio-Rad, Hercules, CA).
SgcC3 was predicted to incorporate chloride using flavin as a
redox cofactor and O2 as an oxidizing agent.
Given the importance of halogen atoms in natural products
and the intriguing variety and complexity of halogenases,16 we
set out to characterize SgcC3 in vitro as part of our continuous
efforts to investigate the biosynthetic pathway of 1. In this study,
we report the functional characterization of recombinant SgcC3
as a flavin adenine dinucleotide (FAD)-dependent halogenase
that catalyzes the regiospecific chlorination of 6, requiring O2
and reduced FAD (FADH2), to afford (S)-3-chloro-â-tyrosyl-
S-SgcC2 (10) as the third step for the biosynthesis of 3 from 2
(Figure 2). The general catalytic properties of SgcC3 are
reported, and SgcC3 now is the second biochemically character-
ized flavin-dependent halogenase that acts on a carrier protein-
tethered substrate, which likely represents a general strategy
for oxidative halogenation of secondary metabolites that are
assembled via carrier protein-dependent biosynthetic machinery.
The results established herein, along with the evidence that
SgcC5 has relaxed specificity as implied by the isolation of 7,
8, and 9, affords the opportunity to generate new C-1027 analogs
by combinatorial biosynthesis methods.
Determination of the SgcC3 Flavin Cofactor. SgcC3 was denatured
by heat or with 50% methanol (final concentration) to release any
noncovalently bound cofactor. After centrifugation the supernatant was
analyzed on a C18 reverse phase column (250 × 4.6 mm, Alltech
Associates Inc. Deerfield, IL) using a linear gradient from 0 to 60%
acetonitrile (in water) at a flow rate of 1 mL min-1 with detection at
266 nm.
Enzymatic Synthesis of â-Aminoacyl-S-SgcC2. Post-translational
modification of apo-SgcC2 into holo-SgcC2 was achieved in a reaction
mixture containing 100 mM Tris-HCl, pH 7.5, 160 µM apo-SgcC2,
0.8 mM coenzyme A (CoA), 12.5 mM MgCl2, 2.0 mM tris(2-
carboxyethyl)phosphine hydrochloride (TCEP), and 5 µM Svp.34 After
incubation at room temperature for 45 min, an equal volume of loading
solution consisting of 2 mM 4 [4 mM for (R)-â-tyrosine (11) or
3-hydroxy-â-tyrosine], 4 mM adenosine triphosphate (ATP), 2.0 mM
TCEP, and 12.5 mM MgCl2 (final concentration in the loading reaction
mixture) was added. Amino acid loading was initiated by the addition
of SgcC1 to a final concentration of 2 µM (6 µM for 11 or 3-hydroxy-
â-tyrosine), and the resulting solution was incubated at room temper-
ature for an additional 60 min as described previously.13 The resulting
â-aminoacyl-S-SgcC2 substrates [i.e., 6, (R)-â-tyrosyl-S-SgcC2 (12),
or 3-hydroxy-â-tyrosyinl-S-SgcC2] were purified from loading mixture
with a 5-mL HiTrap Q anion-exchange column (GE Healthcare,
Piscataway, NJ). The column was pre-equilibrated with 20 mM sodium
phosphate buffer, pH 7.0, and the crude SgcC2-tethered substrate
preparations were loaded and eluted using a linear gradient of 0 to
100% of 1 M NaCl for 25-column volumes and flow rate of 3 mL
min-1. The purified â-aminoacyl-S-SgcC2 substrates, which were eluted
between 0.35 and 0.4 M NaCl, were desalted using size-exclusion
chromatography (Superose 12, GE Healthcare, Piscataway, NJ) and
concentrated prior to use in SgcC3 assays.
Experimental Procedures
Synthesis of the â-Tyrosine Analogues. Syntheses of the â-tyrosine
analogues [i.e., 3-chloro-â-tyrosine, 3-bromo-â-tyrosine, 3-hydroxy-
â-tyrosine, and 3-chloro-5-hydroxy-â-tyrosine] were achieved by
following literature procedure32 (see Supporting Information for details).
Cloning of sgcC2, sgcC3, sgcE6, and E. coli fre Genes for
Heterologous Expression. The genes encoding SgcC2, SgcC3, SgcE6
and E. coli flavin reductase (Fre) were amplified by PCR using Platinum
Pfx DNA polymerase following the program and conditions provided
by Invitrogen (see Table S1 for primers used in Supporting Information).
Templates utilized for PCR were pBS103413 (for sgcC2), pBS10058
(for sgcC3), pBS10068 (for sgcE6), and E. coli DH5R genomic DNA
(for fre), respectively. Purified PCR product of sgcC2 was cloned into
the pCDF-2Ek/LIC vector using ligation-independent cloning procedure
as described by Novagen (Madison, WI) to give pCDF-2Ek/LIC-SgcC2
(pBS1040). The PCR products of sgcC3, sgcE6, and fre were similarly
cloned into the pET-30Xa/LIC vector (Novagen, Madison, WI) to yield
pET-30Xa/LIC-SgcC3 (pBS1041), pET-30Xa/LIC-SgcE6 (pBS1042),
and pET-30XaLIC-Fre (pBS1043), respectively. All cloned PCR
products were confirmed by DNA sequencing. The sgcC1 expression
construct pBS1033 has been described previously.13
Overproduction and Purification of SgcC1, SgcC2, SgcC3,
SgcE6, and Fre. E. coli BL21 (DE3) introduced with pBS1033 (for
SgcC1), pBS1040 (for SgcC2), pBS1041 (for SgcC3), pBS1042
(SgcE6), or pBS1043 (for Fre) was cultured in a LB medium33
supplemented with streptomycin (50 µg mL-1 for pBS1040) or
kanamycin (50 µg mL-1 for pBS1033, pBS1041, pBS1042, and
pBS1043), respectively. All cells were grown at 18 °C and induced
with IPTG (final concentration of 0.1 mM) when OD600 reached ∼0.5.
They were further cultured at 18°C for an additional 15 h. Cells were
harvested by centrifugation (4°C, 8000 rpm for 15 min) and resuspended
in Buffer A (100 mM sodium phosphate, pH 7.5, containing 300 mM
NaCl) supplemented with a complete protease inhibitor tablet, EDTA-
free (Roche Applied Science, Indianapolis, IN). The cells were lysed
by sonication (4 × 30 s pulsed cycle), and the debris was removed by
centrifugation (4°C, 15000 rpm for 50 min). The clarified supernatant
was loaded onto a pre-equilibrated Ni-NTA agarose column (Qiagen,
Valencia, CA) with Buffer B (Buffer A plus 10% glycerol). The column
In Vitro Activity Assay for SgcC3. The SgcC3 assay solution
contained 50 µM 6 (or 12 or 250 µM 3-hydroxy-â-tyrosyl-S-SgcC2),
5 mM â-nicotinamide adenine dinuceotide, reduced (NADH), 0.10 mM
FAD, 100 mM NaCl, 1 mM TCEP, 20 µM SgcC3, and 5 µM SgcE6
in 50 mM sodium phosphate buffer, pH 6.0. Reactions were incubated
at 37°C for 1 h. The reaction was terminated by the addition of 70%
trichloroacetic acid (TCA) to a final concentration of 10% to precipitate
all proteins. After incubation on ice for 15 min, the precipitate was
separated by centrifugation (4°C, 14000 rpm for 15 min). The resulting
pellet was washed twice with 200 µL of 5% TCA and once with 200
µL of ethanol. After drying by speed-vac, the protein pellet was
redissolved in 150 µL of 0.1 M KOH and incubated at 70 °C for 15
min to hydrolyze all SgcC2-tethered â-amino acids. After removal of
the proteins by centrifugation, the solution was concentrated by speed-
vac and analyzed for 4 (or 11 or 3-hydroxy-â-tyrosine) and the expected
product (S)-3-chloro-â-tyrosine (13) [or (R)-3-chloro-â-tyrosine (14)
or 3-chloro-5-hydroxy-â-tyrosine] by a Varian ProStar 210 HPLC
System equipped with a C18 reverse-phase column (250 × 4.6 mm,
Alltech Associates Inc., Deerfield, IL), using a 24-min linear gradient
from 0 to 40% (25% for 3-hydroxy-â-tyrosine and 3-chloro-5-hydroxy-
â-tyrosine) acetonitrile (0.1% TFA) at a flow rate of 1 mL min-1 and
(32) Tan, C. Y. K.; Weaver, D. F. Tetrahedron 2002, 58, 7449-7461.
(33) Sambrook, J.; Fritsch, E. F.; Maniatis, T. Molecular cloning: A Laboratory
Manual 2000, 3rd ed.; Cold Spring Harbor Laboratory Press: Cold Spring
Harbor, NY, 2000.
(34) Sanchez, C.; Du, L.-C.; Edwards, D. J.; Toney, M. D.; Shen, B. Chem.
Biol. 2001, 8, 725-738.
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SgcC3-Catalyzed Chlorination of (S)-
â
-Tyrosyl-S-SgcC2
A R T I C L E S
detection at 280 nm and authentic 4, 11, 13, 14, 3-hydroxy-â-tyrosine,
or 3-chloro-5-hydroxy-â-tyrosine as references. Control experiments
were carried out under identical conditions but with boiled SgcC3.
Formation of 3-Bromo-â-Tyrosyl-S-SgcC2. Both SgcC3 and
SgcE6 were prepared by purification using Ni-NTA columns in buffers
without chloride. After 4 was loaded onto holo-SgcC2 by SgcC1, and
the resultant product 6 was purified from the loading reaction mixture
through a 5-mL HiTrap Q column. Chloride was then completely
removed by passing through a size-exclusion column twice (Superose
12). The SgcC3-catalyzed bromination of 6 was performed in a solution
identical to above except that 0.1 M NaBr replaced NaCl in the assay
mixture and TCEP was excluded. The reaction was quenched with 10%
TCA to precipitate proteins at 10, 30, and 60 min as described above.
The precipitate was treated with 150 µL of 0.1 M KOH to release all
SgcC2-tethered â-amino acids. The resultant free â-amino acids [i.e.,
4 and the expected product 3-bromo-â-tyrosine (15)] were subjected
to HPLC and MS analysis. The standard curve was calibrated with
synthetic 15 to correlate the peak area with product amount formed in
each reaction. The product formation was fitted into a linear equation
to obtain the initial velocity.
Figure 3. Purification and characterization of proteins used in this study
as judged by SDS-PAGE: lane 2, SgcC2; lane 3, SgcC3; lane 4, SgcE6;
lane 5, Fre; lane 7, SgcC1; lanes 1 and 6, low-range molecular weight
standards.
Initial Velocity of SgcC3 with (S)-â-Tyrosyl-S-SgcC2 and (R)-
â-Tyrosyl-S-SgcC2. Conversion of apo-SgcC2 to holo-SgcC2 was
carried out in a 1.8 mL reaction containing 400 µM SgcC2, 1.6 mM
CoA, and 10 µM Svp at room temperature for 60 min. For preparation
of 6 from 4, the loading condition was identical to the standard assay
condition described above. For preparation of 12 from 11, the reaction
proceeded with 4 mM 11 and 6 µM SgcC1 and was incubated at room
temperature for 90 min. Chlorination assays were performed in 200
µL of reaction mixture containing 200 µM 6 or 12, 0.1 M NaCl, 5
mM NADH, 0.1 mM FAD, 25 µM SgcC3, and 3 µM SgcE6. The
reaction was quenched by the addition of 30 µL of 70% TCA at 10,
20, 40, and 60 min and analyzed as described above to determine the
rate for (S)-3-chloro-â-tyrosyl-S-SgcC2 (16) or (R)-3-chloro-â-tyrosyl-
S-SgcC2 (17) formation. A standard calibration curve was created with
synthetic 13, and product formation was fitted to a linear equation to
obtain the initial velocity. The specific activity was calculated from
the initial velocity divided by the concentration of SgcC3 as determined
using the Bradford dye-binding procedure.
Figure 4. HPLC chromatograms of SgcC3 assays showing authentic (S)-
â-tyrosine (4) standard (I), incubation of (S)-â-tyrosyl-S-SgcC2 (6) with
SgcC3 at 37 °C for 15 min (II), 30 min (III), 60 min (IV), and synthetic
3-chloro-â-tyrosine (V). (b) 4; ([) 13 or 3-chloro-â-tyrosine.
Results
SgcC3 completely released the flavin group, indicating that the
cofactor is noncovalently bound to SgcC3. HPLC analysis of
the released flavin prosthetic group with authentic FMN (flavin
mononucleotide) and FAD as standards established its identity
as FAD with a 1:0.18 molar ratio of SgcC3: FAD (see Figure
S1 in Supporting Information).
Overproduction and Purification of SgcC1, SgcC2, SgcC3,
SgcE6, and Fre. The sgcC2 gene was subcloned from pBS103413
into pCDF-2Ek/LIC vector to eliminate most of the extra
N-terminal residues engineered into the recombinant protein
used in previous studies.13,14 After overproduction in E. coli,
SgcC2 was purified to homogeneity as an N-terminal His6-
tagged fusion protein containing an additional 13 amino acids.
Purification of SgcC3 to homogeneity afforded a yellow solution
whose UV-vis spectrum showed the characteristic absorption
maxima of 375 and 450 nm, indicative of the presence of a
flavin prosthetic group. Since it has been previously reported
that FADH2-dependent halogenases require a separate flavin
reductase to supply flavin cofactor in FADH2 form,22-31 the
putative flavin reductase SgcE6 within the C-1027 biosynthetic
gene cluster (Figure 2A) and E. coli Fre were individually
overproduced and purified to homogeneity. Similar to SgcC3,
the purified SgcE6 and Fre also had UV-vis profiles charac-
teristic of flavoproteins. SgcC1 was overproduced and purified,
and the activity was accessed as previously described.13,14 The
homogeneity of the purified proteins was confirmed by SDS-
PAGE analysis (Figure 3).
Substrate Preparation and Activity Assay of SgcC3 with
(S)-â-Tyrosyl-S-SgcC2. Holo-SgcC2 was generated enzymati-
cally using the Svp phosphopantetheinyl transferase (PPTase).34
Subsequently, the 4-specific adenylation enzyme SgcC1 was
used to load holo-SgcC2 with 4 to generate 6. After purification
using anion exchange, 6 was incubated with NaCl in the
presence of SgcC3, SgcE6, FAD, and NADH under aerobic
conditions at 37°C for 1 h, and the reaction was monitored by
subjecting aminoacyl-S-SgcC2 substrate 6 and product 10 to
alkaline hydrolysis followed by HPLC analysis. A new peak
appeared eluting after 4 (obtained from hydrolysis of substrate
6), and this new peak had an identical retention time to authentic
13 (Figure 4). The identity of the hydrolyzed product as 13 was
confirmed by ESI-MS (electrospray ionization-mass spectrom-
etry), yielding a pair of [M + H]+ ions at m/z ) 216.2 and
218.1 with a 3:1 ratio, characteristic for the mono-chlorinated
13 (molecular formula C9H10O3NCl, calcd 215.0 and 217.0) (see
Figure S2 in Supporting Information). Time course analysis
showed that SgcC3 catalyzed the conversion of 6 to 10 in a
Identity of the SgcC3 Flavin Cofactor. As predicted from
bioinformatics analysis, the purified SgcC3 had a UV-vis
spectrum reminiscent of a flavin cofactor. Heat denaturation of
9
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A R T I C L E S
Lin et al.
Figure 5. SgcC3-catalyzed halogenation of (S)-â-tyrosyl-S-SgcC2 (6) or (R)-â-tyrosyl-S-SgcC2 (11). (A) The reaction scheme for SgcC3 depicting a catalytic
cycle involving the FAD reductase SgcE6. (B) HPLC chromatograms examining substrate and cofactor requirement for SgcC3-catalyzed chlorination of 6.
(C) HPLC chromatograms examining alternative substrates for SgcC3. (b) 4 or 11; ([) 13, 14, or 3-chloro-â-tyrosine; (]) 15 or 3-bromo-â-tyrosine.
time-dependent reaction with a specific activity of 0.93 ( 0.14
oxygenase.35,36 However, optimal SgcC3 activity requires the
exogenous supply of a flavin reductase (Figure 5B, IV vs II
and III). Finally, there appears to be no specific interaction
between SgcC3 and the flavin reductase since SgcE6 and Fre
can equally support the halogenase activity of SgcC3 (Figure
5B, II and III). This finding is consistent with the observation
that SgcE6 is the only flavin reductase within the C-1027
cluster,8 which likely serves all of the flavin-dependent enzymes
involved in C-1027 biosynthesis.
pH Optimum for SgcC3 Activity. To estimate the pH
optimum for SgcC3, chlorination of 6 was carried out in 50
mM sodium acetate, sodium phosphate, and Tris-HCl buffer,
ranging from pH 5.0 to pH 9.0, under the standard assay
conditions.
h-1 (Figure 4).
Cofactor and Cosubstrate Requirements for SgcC3-
Catalyzed Chlorination. The substrate and cofactor requirement
for the SgcC3-catalyzed chlorination of 6 to 10 as depicted in
Figure 5A was next examined (results summarized in Table 1).
Both O2 and NADH are required for SgcC3 catalysissthe
removal of either completely abolished 10 formation (Figure
5B, VI and VII). When FAD was excluded from the assay
solution, a minute amount of 10 was detected by HPLC,
consistent with the observation that SgcC3 is co-purified with
sub-stoichiometric amounts of FAD that can be utilized for
SgcC3 catalysis (Figure 5B, V). In the absence of a flavin
reductase, a small amount of 10 was produced, and this activity
was likely due to co-purification of SgcC3 with E. coli Fre as
has been reported for other halogenases27 and flavin-dependent
(35) Eichhorn, E.; van de Ploeg, J. R.; Leisinger, T. J. Biol. Chem. 1999, 274,
26639-26646.
(36) Fieschi, F.; Niviere, V.; Frier, C.; Decout, J. L.; Fontecave, M. J. Biol.
Chem. 1995, 270, 30392-30400.
9
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SgcC3-Catalyzed Chlorination of (S)-
â
-Tyrosyl-S-SgcC2
A R T I C L E S
Table 1. Cofactor and Cosubstrate Requirement for SgcC3-
Catalyzed Chlorination of (S)-â-Tyrosyl-S-SgcC2 (6) in Vitro
azolinate moiety, and a â-amino acid moiety (Figure 1). Indeed,
as we have recently reported, small structural permutations to
the benzoxazolinate and â-amino acid moieties have profound
effects on the biological activity of C-1027, including the ability
to induce DSB, the in vivo cytotoxicity, and the mechanism of
the cellular response upon treatment with C-1027.9
entrya
condition
% conversion
relative rate
II
complete assay
38
39
6
11
0
100
100
16
30
0
III
IV
V
VI
VII
complete assay - SgcE6 + E. Coli Fre
complete assay - SgcE6
complete assay - FAD
complete assay - NADH
complete assay - O2
The 3-chloro-4,5-dihydroxy-â-phenylalanine moiety of 1
contributes critical interactions with the apoprotein CagA and,
as suggested from the studies regarding the bioactivity of 7 and
9,9 modulates the stability and reactivity of the enediyne core
via π-π stacking interaction.38 This moiety is activated and
incorporated into C-1027 by first generating a (S)-â-tyrosyl-S-
SgcC2 intermediate 6, which subsequently serves as a substrate
for chlorination and likely hydroxylation prior to attachment to
the enediyne core.8,13 The process of activation and incorporation
is achieved by enzymes with homology to protein domains
found in nonribosomal peptide synthetases (NRPS), and these
enzymessSgcC1, SgcC2, and SgcC5sare located in a sub-
clustered region within the ∼80 kb C-1027 biosynthetic gene
cluster (Figure 2A). In contrast, a single candidate for halogena-
tion, SgcC3, was located upstream and distant from the NRPS
locus. It only became apparent that SgcC3 was involved in
chlorination of the â-amino acid moiety upon inactivation of
sgcC3 and isolation of the expected deschloro analog 7.13 To
unravel the molecular details of this transformation, we cloned
sgcC3 and overproduced and purified the recombinant protein
for in vitro characterization.
0
0
a Entry number corresponds to the chromatogram shown in Figure 5B.
SgcC3 showed the highest activity at pH 6.0 (see Figure S3 in
Supporting Information), and therefore all subsequent assays
were performed in 50 mM phosphate buffer at pH 6.0.
Substrate Specificity of SgcC3. The substrate specificity of
SgcC3 was initially examined with free amino acids as
substrates, including 4, 11, and 3-hydroxy-â-tyrosine. HPLC
analysis showed no activity under all conditions tested, con-
sistent with the previous proposal that chlorination occurs after
4 is tethered to SgcC2 as 6 (Figure 2B).8,10-14
The substrate specificity of SgcC3 was next investigated by
using a series of â-aminoacyl-S-SgcC2 as substrates (Figures
5A and 5C). Taking advantage of SgcC1’s ability to activate
and load other â-amino acids to SgcC2,13,14 12 and 3-hydroxy-
â-tyrosyl-SgcC2 were prepared and tested, in comparison with
6, for activity with SgcC3. Unexpectedly, SgcC3 catalyzes the
chlorination of 12 to generate 16 with a specific activity of 1.1
( 0.20 h-1, nearly identical to that found with 6 (Figure 5C, II
and III). In contrast, no chlorination was detected with 3-hy-
droxy-â-tyrosyl-SgcC2 in spite of the inclusion of a 5-fold
amount of SgcC3 and prolonged incubation time. The latter
finding is consistent with 6 as the bona fide substrate of SgcC3
and supports the timing of the individual steps proposed for 3
biosynthesis (Figure 2B).8,10-14
Finally, halogen specificity of SgcC3 was examined. After
purification of all proteins in the absence of chloride, SgcC3
efficiently catalyzed the bromination of 6 to afford the corre-
sponding brominated product 3-bromo-â-tyrosyl-SgcC2 (17)
with a specific activity of 0.36 ( 0.10 h-1, which is ap-
proximately 2-fold less than chlorination under similar reaction
conditions (Figure 5C, V and VI). However, neither fluorination
nor iodination was observed under the identical condition, a
finding that is consistent with all flavin-dependent halogenases
known to date.16,22 The identity of 17 was confirmed by HPLC
analysis of the free acid 15 using synthetic 15 as a standard.
ESI-MS analysis of 15 yielded a pair of [M + H]+ ions at m/z
) 260.0 and 262.0 with 1:1 ratio, in agreement with the mono-
bromonated 15 (molecular formula C9H10O3NBr, calcd 259.0
and 261.0 around 1:1 ratio).
Initial activity tests revealed that SgcC3 does not catalyze
the chlorination of free amino acid substrates, including 4. As
a result, 6 was enzymatically prepared from 4 in vitro using
the Svp PPTase,34 the SgcC1 adenylation enzyme, and the
SgcC2 PCP, all of which have been previously characterized.13,14
After purification of the resulting aminoacyl-S-SgcC2 and prior
to activity tests, hydrolysis of â-amino acids from SgcC2 was
optimized to afford a stoichiometric release for detection by
HPLC. The activity of SgcC3 was examined with NaCl as a
halide donor, and a single, new peak was observed only when
(i) exogenous FAD was supplied to the reaction mixture, (ii)
an NADH-dependent flavin reductase was included to provide
diffusible FADH2, and (iii) the reaction was performed in an
aerobic environment. This new peak was confirmed to be 13
based on comparisons to synthetic standard and ESI-MS
analysis. These results provided unambiguous evidence that
SgcC3 is a FADH2, O2-dependent halogenase that requires a
carrier protein-tethered substrate for activity. The cofactor and
cosubstrate requirements are similar to those observed for the
halogenase PltA involved in pyoluteorin biosynthesis,27 and
these two enzymes now represent a growing family of FADH2,
O2, and carrier protein-dependent halogenases for which several
have been predicted from bioinformatics analysis.16
Discussion
Like all enediyne natural products, C-1027 shows a potent
antitumor activity. Despite sharing a common enediyne core,
however, C-1027 is unique within the family in that it is
estimated to be 1000 times more potent than other 9-membered
enediynes such as neocarzinostatin or 10-membered enediynes
such as calicheamicin.37 This difference in cytotoxicity can be
attributed in part to the moieties decorating the enediyne core,
for which C-1027 has threesa deoxy aminosugar, a benzox-
We previously reported that SgcC1 specifically activates 4,
but also activates other â-tyrosine analogues including 11,
3-chloro-â-tyrosine, 3-hydroxy-â-tyrosine, and 3-chloro-5-hy-
droxy-â-tyrosine, albeit with minimally 25-fold less effi-
ciency.13,14 This indicates that C-3 chlorination and C-5
(38) Okuno, Y.; Otsuka, M.; Sugiura, Y. J. Med. Chem. 1994, 37, 2266-2273.
(39) van Berkel, W. J. H.; Kamerbeek, N. M.; Fraaije, M. W. J. Biotechnol.
2006, 124, 670-689.
(40) Orser, C. S.; Lange, C. C.; Xun, L.; Zahrt, T. C.; Schneider, B. J. J.
Bacteriol. 1993, 175, 411-416.
(37) Wang, X.-W.; Hong, X. Drugs Future 1999, 24, 847-852.
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A R T I C L E S
Lin et al.
hydroxylation of 4 most likely occurs after 4 is tethered to
SgcC2, a prediction that is now confirmed by the finding that
4 is not a substrate for SgcC3. However, these results fell short
of revealing any insight into the timing of halogenation after 4
is tethered to SgcC2. Therefore, both 12 and 3-hydroxy-â-
tyrosyl-S-SgcC2 were enzymatically prepared by taking advan-
tage of the promiscuous nature of SgcC1. Remarkably, SgcC3
catalyzes the chlorination of 12 into 16 with a specific activity
that is almost identical to that for 6. While this may appear to
be unexpected, it in fact is consistent with the early finding
that SgcC1 has a 25-fold selectivity for 4 over 11, serving as
the “gate-keeper” that controls the â-amino acid to be incor-
porated into 1 in vivo.13,14 In contrast, no chlorination was
detected with 3-hydroxy-â-tyrosyl-S-SgcC2, the alternative
substrate for SgcC3 if C-5 hydroxylation occurred prior to C-3
halogenation. This is consistent with 6 as the preferred substrate
of SgcC3 and further supports the timing of the individual steps
proposed for 3 biosynthesis (Figure 2B). Finally, similar to other
halogenases, chloride can be substituted with bromide but not
fluoride or iodide, and structural analysis of SgcC3 should
provide insights into this selectivity.
analogs 7, 8, and 9 suggests enzymes downstream of SgcC3
have relaxed substrate specificity. Along with the results for
SgcC3 provided here, application of precursor-directed biosyn-
thesis and combinatorial biosynthesis methods to the C-1027
biosynthetic machinery presents a unique opportunity to generate
novel C-1027 analogs, some of which could have improved
biological activity.
Acknowledgment. We thank Dr. Y. Li, Institute of Medicinal
Biotechnology, Chinese Academy of Medical Sciences, Beijing,
China, for the wild-type S. globisporus strain and the Analytical
Instrumentation Center of the School of Pharmacy, University
of WisconsinsMadison, for support in obtaining MS and NMR
data. This work is supported in part by NIH grants CA78747
and CA113297. S.V.L. is the recipient of an NIH postdoctoral
fellowship (CA1059845).
Supporting Information Available: Full experimental details
for the synthesis of â-tyrosine analogs as substrates and authentic
standards of products, PCR primers used for amplification of
the sgcC2, sgcC3, sgcE6, and E. coli fre genes for heterologous
expression (Table S1), HPLC chromatogram for determination
of the SgcC3 cofactor (Figure S1), ESI-MS spectra of (S)-3-
chloro-â-tyrosine (Figure S2), and pH profile for SgcC3 (Figure
S3). This material is available free of charge via the Internet at
http://pubs.acs.org.
In conclusion, SgcC3 has been shown to catalyze the
regioselective chlorination of 6, and this activity is dependent
on O2 and FADH2. The requirements for SgcC3 catalysis are
consistent with the catalytic cycle proposed for FADH2, O2-
dependent halogenases as depicted in Figure 5A. The fact that
recombinant strains of S. globisporus can produce C-1027
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12438 J. AM. CHEM. SOC. VOL. 129, NO. 41, 2007