Characterization of the SgcF epoxide hydrolase supporting an (R)-vicinal diol intermediate for enediyne antitumor antibiotic C-1027 biosynthesis

Article abstract of DOI:10.1021/ja901242s

C-1027 is a chromoprotein antitumor antibiotic consisting of an apoprotein and the C-1027 chromophore. The C-1027 chromophore possesses four distinct structural moieties - an enediyne core, a deoxy aminosugar, a benzoxazolinate, and an (S)-3-chloro-5-hydroxy-β-tyrosine - the latter two of which are proposed to be appended to the enediyne core via a convergent biosynthetic strategy. Here we report the in vitro characterization of SgcF, an epoxide hydrolase from the C-1027 biosynthetic gene cluster that catalyzes regio- and stereospecific hydrolysis of styrene oxide, serving as an enediyne core epoxide intermediate mimic, to form a vicinal diol. Abolishment of C-1027 production in the ΔsgcF mutant strain Streptomyces globisporus SB1010 unambiguously establishes that sgcF plays an indispensable role in C-1027 biosynthesis. SgcF efficiently hydrolyzes (S)-styrene oxide, displaying an apparent K_m of 0.6 ± 0.1 mM and k_cat of 48 ± 1 min^-1, via attack at the α-position to exclusively generate the (R)-phenyl vicinal diol, consistent with the stereochemistry of the C-1027 chromophore. These findings support the role of SgcF in the proposed convergent pathway for C-1027 biosynthesis, unveiling an (R)-vicinal diol as a key intermediate. Interestingly, SgcF can also hydrolyze (R)-styrene oxide to afford preferentially the (R)-phenyl vicinal diol via attack at the β-position, albeit with significantly reduced efficiency (apparent K_m of 2.0 ± 0.4 mM and k_cat = 4.3 ± 0.3 min^-1). Although the latter activity unlikely contributes to C-1027 biosynthesis in vivo, such enantioconvergence arising from complementary regioselective hydrolysis of a racemic substrate could be exploited to engineer epoxide hydrolases with improved regio- and/or enantiospecificity.

Full text of DOI:10.1021/ja901242s

Published on Web 10/26/2009
Characterization of the SgcF Epoxide Hydrolase Supporting
an (R)-Vicinal Diol Intermediate for Enediyne Antitumor
Antibiotic C-1027 Biosynthesis
Shuangjun Lin,† Geoffrey P. Horsman,† Yihua Chen,† Wenli Li,† and Ben Shen*,†,‡,§
DiVision of Pharmaceutical Sciences, UniVersity of Wisconsin National CooperatiVe Drug
DiscoVery Group, and Department of Chemistry, UniVersity of Wisconsin-Madison,
Madison, Wisconsin 53705-2222
Received February 17, 2009; E-mail: bshen@pharmacy.wisc.edu
Abstract: C-1027 is a chromoprotein antitumor antibiotic consisting of an apoprotein and the C-1027
chromophore. The C-1027 chromophore possesses four distinct structural moietiessan enediyne core, a
deoxy aminosugar, a benzoxazolinate, and an (S)-3-chloro-5-hydroxy-ꢀ-tyrosinesthe latter two of which
are proposed to be appended to the enediyne core via a convergent biosynthetic strategy. Here we report
the in vitro characterization of SgcF, an epoxide hydrolase from the C-1027 biosynthetic gene cluster that
catalyzes regio- and stereospecific hydrolysis of styrene oxide, serving as an enediyne core epoxide
intermediate mimic, to form a vicinal diol. Abolishment of C-1027 production in the ∆sgcF mutant strain
Streptomyces globisporus SB1010 unambiguously establishes that sgcF plays an indispensable role in
C-1027 biosynthesis. SgcF efficiently hydrolyzes (S)-styrene oxide, displaying an apparent Km of 0.6 ( 0.1
mM and kcat of 48 ( 1 min-1, via attack at the R-position to exclusively generate the (R)-phenyl vicinal diol,
consistent with the stereochemistry of the C-1027 chromophore. These findings support the role of SgcF
in the proposed convergent pathway for C-1027 biosynthesis, unveiling an (R)-vicinal diol as a key
intermediate. Interestingly, SgcF can also hydrolyze (R)-styrene oxide to afford preferentially the (R)-phenyl
vicinal diol via attack at the ꢀ-position, albeit with significantly reduced efficiency (apparent Km of 2.0 ( 0.4
mM and kcat ) 4.3 ( 0.3 min-1). Although the latter activity unlikely contributes to C-1027 biosynthesis in
vivo, such enantioconvergence arising from complementary regioselective hydrolysis of a racemic substrate
could be exploited to engineer epoxide hydrolases with improved regio- and/or enantiospecificity.
Introduction
The enediynes are some of the most cytotoxic molecules
known to date, and two members of this family, calicheamicin
and neocarzinostatin (NCS), are currently in clinical use as
anticancer drugs.1,2 The enediyne cores of molecules such as
C-1027 and NCS possess multiple vicinal diols (Figure 1), to
which the various peripheral moieties are attached to impart
the distinct characteristics of each enediyne antitumor antibiotic.
These functional groups may be envisaged to originate from
epoxide precursors that undergo hydrolytic ring-opening cata-
lyzed by epoxide hydrolases (EHs).
The ubiquity of EHs in nature is evidenced by their
widespread occurrence in mammals, plants, insects, and various
microorganisms such as yeast, fungi, and bacteria.3-6 Most EHs
Figure 1. Structures of C-1027 and neocarzinostatin, with vicinal diol
moieties (shaded) predicted to be derived by epoxide hydrolases from
enediyne core epoxide precursors.
belong to the R/ꢀ-hydrolase superfamily,7-9 and crystallographic
studies have confirmed the presence of this structural fold in
several EHs.10-17 Detailed analysis of available sequence and
structural data has revealed that EHs possess a modular
† Division of Pharmaceutical Sciences.
‡ University of Wisconsin National Cooperative Drug Discovery Group.
§ Department of Chemistry.
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10.1021/ja901242s CCC: $40.75  2009 American Chemical Society

SgcF Epoxide Hydrolase for C-1027 Biosynthesis
A R T I C L E S
Figure 2. Proposed mechanism of epoxide hydrolases. The catalytic triad and the oxygen atom from water are shown in red, and the active site residues
reflect those of AnEH from Aspergillus niger.17
architecture consisting of three conserved and three variable
regions (Figure S1, Supporting Information).18,19 The conserved
regions include the N- and C-terminal catalytic regions that
together make up the core catalytic domain of the R/ꢀ-hydrolase
fold, as well as the lid or cap domain. The variable regions
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(ii) an NC-loop linking the lid and the N-terminal catalytic
domain; and (iii) a variable loop in the lid domain (cap-loop).
The active site is located at the interface between the core and
lid domains and contains five residues that are absolutely
conserved among EHs - the nucleophile (Asp or Glu) and His
of the nucleophile-His-acid triad (the acid residue of which is
variable in its position), the 2 residues of the oxyanion hole (X
of the HGXP motif, and the residue following the catalytic
nucleophile), and a conserved Tyr in the fifth helix of the lid
domain (i.e., R8 in Figure S2, Supporting Information). Al-
though most EHs possess a second Tyr in the first helix of the
lid domain (i.e., R4 in Figure S2, Supporting Information), this
residue is not always conserved.19
oxirane oxygen atom,10,20-22 thereby activating the epoxide to
be attacked by the nucleophilic Asp/Glu residue of the catalytic
triad to yield a covalent alkyl-enzyme intermediate.23-25 A water
molecule, activated by a conserved His-Asp charge relay system,
hydrolyzes the monoester intermediate to afford the vicinal diol
as a product. The transient oxyanion tetrahedral intermediate is
stabilized via hydrogen bonds to the backbone amide hydrogens
of the oxyanion hole residues.15-17,26
Although mammalian EHs have been studied for decades for
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attractive for biocatalytic applications due to their regio- and
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their roles in natural product biosynthesis. For an asymmetric
epoxide such as styrene oxide, microbial EHs with various regio-
and enantioselectivity have been reported. Most microbial EHs
preferentially hydrolyze one enantiomer of an epoxide racemate,
and thereby may be utilized to generate enantiopure epoxides
or diols via kinetic resolution. Interestingly, a few EHs have
been reported to catalyze the hydrolysis of both enantiomers to
form enantiomerically enriched diol products.32 This enantio-
convergent process occurs by means of complementary enantio-
The conserved structure and active site residues reflect a
common mechanism for epoxide hydrolysis (Figure 2). The two
tyrosine residues in the lid domain form hydrogen bonds to the
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J. AM. CHEM. SOC. VOL. 131, NO. 45, 2009 16411

A R T I C L E S
Lin et al.
enzyme intermediate at C-1 followed by hydrolysis to afford
the (R)-phenyl vicinal diol exclusively. Interestingly, SgcF also
hydrolyzes (R)-styrene oxide, albeit with significantly reduced
efficiency, and regioselective formation of the monoester
intermediate at the C-2 position also yields the (R)-phenyl vicinal
diol as a preferred product. These data showed that SgcF
catalyzes the net trans-addition of H2O to (S)-styrene oxide in
a Markovnikov manner but followed the opposite stereochemical
course for (R)-styrene oxide in an anti-Markovnikov manner.
Given the critical role EHs play in the biosynthesis of various
natural products, including the enediynes, such enantioconver-
gence arising from complementary regioselective hydrolysis of
a racemic substrate could be exploited to engineer epoxide
hydrolases with improved regio- and/or enantiospecificity to
increase natural product structural diversity.
Figure 3. Proposed biosynthetic pathway for C-1027 involving an SgcF-
catalyzed formation of a (R)-vicinal diol intermediate from a (S)-enediyne
core epoxide precursor. The oxygen atom from water is shown in red.
Experimental Section
Strains, Plasmids and Cosmids. Escherichia coli DH5R and
E. coli BL21(DE3) (Novagen, Madison, WI) were used as hosts
for subcloning and heterologous expression, respectively, E. coli
ET12567/pUZ8002 was used as the donor strain for conjugation,42
and Streptomyces globisporus wild-type strain (C-1027-producing)
has been described previously.43 Plasmid pCDF-2 Ek/LIC was from
Novagen, and pBS1005 was described previously.43 SuperCos1 was
from Stratagene (La Jolla, CA).
DNA Isolation and Manipulation. Plasmid preparation was
carried out using commercial kits (Qiagen, Santa Clarita, CA).
General procedures for DNA restriction digests, ligations and
genetic manipulations in E. coli were performed according to
standard protocols. E. coli-Streptomyces conjugation to introduce
DNA into S. globisporus was performed according to the standard
procedure44 with the following modifications. S. globisporus spores
were suspended in TSB medium and heat-shocked at 50 °C for 10
min, followed by incubation at 30 °C for 6 h. Germinated spores
were mixed with E. coli ET12567/pUZ8002 cells and spread onto
ISP4 plates freshly supplemented with 20 mM MgCl2.
Chemicals and Instruments. Substrates including racemic
styrene oxide, (R)-styrene oxide, and (S)-styrene oxide, products
including racemic 1-phenyl-1,2-ethanediol, (R)-1-phenyl-1,2-
ethanediol, and (S)-1-phenyl-1,2-ethanediol, and [18O]-H2O were
purchased from Sigma-Aldrich (St. Louis, MO). Dithiothreitol
(DTT) was purchased from Research Products International Corp
(Mt. Prospect, IL). Complete protease inhibitor tablet, EDTA-free,
was from Roche Applied Science (Indianapolis, IN). Medium
components and buffers were from Fisher Scientific (Pittsburgh,
PA). Synthetic DNA oligonucleotides were purchased from the
University of Wisconsin-Madison Biotechnology Center (Madison,
WI). PCR was performed with a PerkinElmer GeneAmp 2400.
Electrospray ionization-mass spectroscopy (ESI-MS) was performed
with an Agilent 1100 MSD SL ion trap mass spectrometer (Agilent
Technologies, Inc. Santa Clara, CA). NMR spectra were recorded
using a Varian UI-500 spectrometer (Varian, Inc., Palo Alto, CA).
High performance liquid chromatography (HPLC) analyses were
carried out on a Varian HPLC system equipped with Prostar 210
pumps, a photodiode array (PDA) detector, and an Alltech Appolo
C18 reverse phase column (5 µm, 4.6 × 250 mm, Grace Davison
Discovery Sciences, Deerfield, IL), using a 20 min linear gradient
from 0 to 60% acetonitrile in H2O. The enantiomeric separation
was performed on a Waters HPLC system equipped with 600
pumps, a 996 PDA detector, and a Chiralcel OD-H chiral column
and regioselective activities, whereby nucleophilic attack may
occur at either epoxide carbon.
We have been studying the biosynthesis of the 9-membered
enediynes C-1027, NCS, and maduropeptin as model systems
to gain fundamental insight into the biochemical logic of
enediyne biosynthesis. The biosynthetic gene clusters of each
have been cloned, the involvement of which in enediyne
biosynthesis has been confirmed by in vivo inactivation of
selected genes within each cluster,33-35 and in vivo and in vitro
characterizations have unveiled numerous examples of unprec-
edented biochemistry and enzymology,36-41 including a con-
vergent biosynthetic strategy for this family of metabolites
featuring vicinal diol intermediates (Figure 3). Sequence analysis
indeed revealed one gene, sgcF, from the C-1027 biosynthetic
gene cluster and two genes, ncsF1 and ncsF2, from the NCS
biosynthetic gene clusters, respectively, whose deduced gene
products show high amino acid homology to known microbial
EHs, including the Asp-His-Asp catalytic triad (Figure S2,
Supporting Information). These findings are consistent with the
proposed biosynthetic pathways, presenting outstanding op-
portunities to study the regio- and enantioselectivity of EHs
involved in natural product biosynthesis.
Here we report the in vitro characterization of SgcF as an
EH, supporting an (R)-vicinal diol as a key intermediate for
C-1027 biosynthesis. SgcF efficiently catalyzes regio- and
enantiospecific hydrolysis of (S)-styrene oxide, which serves
as an enediyne core epoxide mimic, forming a covalent alkyl-
(32) Lee, E. Y. Biotechnol. Lett. 2008.
(33) Liu, W.; Christenson, S. D.; Standage, S.; Shen, B. Science 2002, 297,
1170–1173.
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J.; Van Lanen, S. G.; Zazopoulos, E.; Farnet, C. M.; Yang, C. F.;
Shen, B. Chem. Biol. 2005, 12, 293–302.
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B. J. Am. Chem. Soc. 2007, 129, 13082–13094.
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Soc. 2003, 125, 6062–6063.
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12432–12438.
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6616–6623.
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2009, 106, 4183–4188.
(42) MacNeil, D. J.; Gewain, K. M.; Ruby, C. L.; Dezeny, G.; Gibbons,
P. H.; MacNeil, T. Gene 1992, 111, 61–68.
(40) Van Lanen, S. G.; Lin, S. J.; Shen, B. Proc. Natl. Acad. Sci. U.S.A.
2008, 105, 494–499.
(43) Liu, W.; Shen, B. Antimicrob. Agents Chemother. 2000, 44, 382–
392.
(41) Zhang, J.; Van Lanen, S. G.; Ju, J. H.; Liu, W.; Dorrestein, P. C; Li,
W. L.; Kelleher, N. L.; Shen, B. Proc. Natl. Acad. Sci. U.S.A. 2008,
105, 1460–1465.
(44) Kieser, T.; Bibb, M. J.; Buttner, M. M.; Chater, K. F.; Hopwood, D. A.
Practical Streptomyces Genetics; The John Innes Foundation: Norwich,
UK, 2000.
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SgcF Epoxide Hydrolase for C-1027 Biosynthesis
A R T I C L E S
(5 µm, 4.6 × 250 mm, Grace Davison Discovery Sciences) using
a 60 min isocratic elution with 2.5% isopropanol in n-hexane.
Inactivation of sgcF. The sgcF gene was inactivated by the λ-red
mediated PCR-targeting method according to the literature proto-
col.45 Since the backbone of pBS1005 is pOJ446,43 which carries
the replication origin of plasmid SCP2*, a 19-kb XbaI fragment
from pBS1005 was subcloned into the same site of SuperCos1 to
afford pBS1094. The apramycin (Apr) resistance gene aac(3)IV/
oriT cassette was amplified by using the following pair of primers:
using a PD-10 column (GE Healthcare, Piscataway, NJ) twice to
completely remove glycerol, concentrated with a 10 K MWCO
Vivaspin ultrafiltration device (Sartorius, Edgewood, NY), and
stored at -80 °C in 100 µL aliquots. The purity of SgcF was
assessed by SDS-PAGE on a 12% gel, and its concentration was
determined from the absorbance at 280 nm using a molar absorp-
tivity (ε ) 96.9 mM-1 cm-1) calculated using the program
Protparam (http://www.expasy.ch/tools/protparam.html).
Activity Assay of SgcF. HPLC-based assays were carried out
in 200-µL reaction mixture containing 2 mM racemic styrene oxide
and 50 mM phosphate buffer (pH 7.5). The reaction was initiated
by the addition of 100 µM SgcF and incubated at 28 °C for 90
min. The reaction was quenched by extracting with ethyl acetate
(3 × 200 µL). After the solvent was removed using a speed-vac,
the resulting residue was dissolved in 50 µL of acetonitrile, 25 µL
of which was subjected to HPLC analysis. Control reactions were
carried out under the identical conditions except with SgcF that
had been boiled for 5 min.
To determine the kinetic parameters of SgcF-catalyzed hydrolysis
of styrene oxide, a spectrophotometric assay method was adopted
to achieve continuous and accurate determination of epoxide
hydrolase activity.47,48 Prior to kinetic analysis, the optimal pH
for SgcF activity was tested in three different bufferss50 mM
sodium acetate (pH 5.0, 5.5, and 6.0), 50 mM sodium phosphate
buffer (pH 5.5 to pH 8.5), and 50 mM glycine-NaOH (pH 8.5, 9.0,
and 10.0). The reactions were carried out in 1-mL reaction mixture
containing 10 µL of 300 mM sodium periodate in DMF, 20 µL of
5 M styrene oxide in DMSO, and 50 mM buffer. The reactions
were initiated by the addition of 10 µM SgcF and carried out in
triplicate. The absorbance at 290 nm was monitored in a 1-mL
quartz cell, and the velocity was calculated based on the rate of
change of absorbance over several minutes. Steady-state kinetic
parameters were obtained from reactions carried out in 50 mM
phosphate buffer (pH 8.0) with varying styrene oxide concentrations
[0.10 to 6.4 mM for (S)-styrene oxide and 1.0 to 24 mM for (R)-
styrene oxide]. The assays were initiated by addition of SgcF [3.2
µM for (S)-styrene oxide and 9.6 µM for (R)-styrene oxide] and
carried out in triplicate. The velocity was determined based on the
change of the absorbance at 290 nm over several minutes. The
Michaelis-Menten equation was fitted to plots of velocity of
1-phenyl-1,2-ethanediol formation versus substrate concentration
to extract values for Km and kcat.
sgcFF1:
GAGGGTGGATTCACTATTCCGGGGATCCGTCGACC-3′/ sgc-
FR1: 5′-TCCGGCCGACGGGCGCTCCACTTCGTAT-
5′-CGCGCCGCCCCGCTCCCACAATCAC-
GTGCCCTACTGGTTGTAGGCTGGAGCTGCTTC-3′ (underlined
letters represent the oligonucleotide homologous to the flanking
DNA regions of sgcF). The resulting PCR product was introduced
into E. coli BW25113/pIJ790 harboring pBS1094 by electropora-
tion, yielding the ∆sgcF mutated cosmid pBS1095 through λ-red
mediated homologous recombination. Cosmid pBS1095 was then
introduced into S. globisporus by E. coli-Streptomyces conjugation.
Exconjugants that were apramycin resistant and kanamycin sensitive
were selected as double crossover mutants and named SB1010, the
genotype of which was confirmed by PCR using the following pair
of primers: sgcFF1: 5′-CCGGGGACGGTCAATCTAG-3′/ sgcFR1:
5′-GCTCCCACAATCACGAGG-3′.
C-1027 Production, Isolation, and Analysis. Fermentation of
S. globisporus wild-type and the ∆sgcF mutant strain SB1010 for
C-1027 production was carried out using A9 medium as described
previously.46 Briefly, a spore suspension of the S. globisporus wild-
type or recombinant strain was inoculated into 50 mL A9 medium
in a 250 mL baffled flask and incubated at 28 °C, 250 rpm for 2
days. Five milliliters of the resultant seed culture was then used to
inoculate 50 mL of A9 medium in 250 mL baffled flasks, and
incubation continued at 28 °C, 250 rpm for 5 days. The culture
was centrifuged (4000 rpm, 4 °C, 10 min) to collect the supernatant.
This supernatant was first adjusted to pH 4.0 with 1 N HCl followed
by centrifugation (4000 rpm, 4 °C, 8 min). The resultant supernatant
was then added (NH4)2SO4 to 70% saturation and readjusted to pH
4.0 to precipitate the C-1027 chromoprotein complex. The precipi-
tated C-1027 chromoprotein complex was finally collected by
centrifugation (4150 rpm, 4 °C, 20 min) and was extracted with
acetone to isolate the C-1027 chromophore, which was subjected
to HPLC analysis immediately.
The HPLC analysis was carried out on a C18 column (5 µm,
250 mm × 4.6 mm, Alltech, Lexington, KY). The column was
equilibrated with 25% solvent A-75% solvent B and eluted with
25% solvent A-75% solvent B for 10 min followed by a linear
gradient to 10% solvent A-90% solvent B in 15 min at a flow
rate of 1 mL/min and with UV detection at 350 nm (solvent A, 10
mM phosphate buffer, pH 7.2 and solvent B, acetonitrile). Bioassay
against Micrococcus luteus ATCC 9431 was performed as de-
scribed.43
Cloning, Overproduction, and Purification of SgcF. The sgcF
gene was amplified by PCR from cosmid pBS100533,43 using
Platinum Pfx polymerase (Invitrogen, Carlsbad, CA) with the
following primers: forward 5′-GAC GAC GAC AAG ATG CGT
CCC TTC CGT ATC GA-3′ and reverse 5′-GAG GAG AAG CCC
GG TCA GCG GAG CGG AGG GT-3′ (starting and stop codons
underlined). The gel-purified PCR products were cloned into the
pCDF-2 Ek/LIC vector using ligation-independent cloning as
described by Novagen (Madison, WI) to give pBS1096 for E. coli
expression. Overproduction in E. coli BL21 (DE3) and purification
of SgcF by affinity chromatography using an NTA-Ni agarose
column (Qiagen, Valencia, CA) were performed following the
previously described procedure.37,38 The purified SgcF was desalted
Large-Scale Reaction. To prepare sufficient quantities of product
for NMR characterization, the reaction was carried out in 1-mL
reaction mixture containing 10 mM styrene oxide and 200 µM SgcF
in 50 mM phosphate buffer (pH 8.0). During the course of the
reaction, 10 mM styrene oxide and 100 µM SgcF were added every
hour. The reaction was allowed to proceed at 25 °C for 8 h, after
which the products were extracted from the reaction mixture with
ethyl acetate (5 × 0.5 mL). The solvent was removed by
evaporation under reduced pressure. Purification by a flash column
chromatography finally yielded the phenyl-1, 2-ethanediol product
for NMR analysis and chiral analyses. [18O]-labeled products were
obtained following a similar procedure. The reactions were carried
out in 0.5-mL reaction mixtures containing 80% [18O]-H2O. For
(S)-styrene oxide, 5 mM substrate and 20 µM SgcF were added
every 30 min. For (R)-styrene oxide, 20 mM substrate and 75 µM
SgcF were added every hour.
Results
Bioinformatics Analysis. Close examination of the genes
within the C-1027 biosynthetic gene cluster revealed a single
gene, sgcF, whose deduced gene product showed high amino
acid sequence homology to known microbial EHs.33 Most EHs
(45) Gust, B.; Challis, G. L.; Fowler, K.; Kieser, T.; Chater, K. F. Proc.
Natl. Acad. Sci. U.S.A. 2003, 100, 1541–1546.
(46) Van Lanen, S. G.; Dorrestein, P. C.; Christenson, S. D.; Liu, W.; Ju,
J. H.; Kelleher, N. L.; Shen, B. J. Am. Chem. Soc. 2005, 127, 11594–
11595.
(47) Doderer, K.; Lutz-Wahl, S.; Hauer, B.; Schmid, R. D. Anal. Biochem.
2003, 321, 131–134.
(48) Mateo, C.; Archelas, A.; Furstoss, R. Anal. Biochem. 2003, 314, 135–
141.
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A R T I C L E S
Lin et al.
belong to the Rꢀ-hydrolase superfamily of proteins that share
this common structural fold. SgcF contains several motifs that
are conserved among this family of EHs: (i) the “nucleophilic
elbow” motif Sm-X-Nu-X-Sm-Sm containing the Asp175
nucleophile (GGD175WGK) that functions as a member of the
nucleophile-His-acid catalytic triad (D175, D336, H363); (ii) the
oxyanion hole HGXP motif (HGW99P); (iii) and the G-X-Sm-
X-S/T motif18,19,49 (G137YGFS) that has been proposed to
stabilize the interface between the lid and core domains,18 and
(iv) the conserved Tyr304 in helix 5 of the lid domain.
Interestingly, the second Tyr in helix 1 of the lid domain (helix
R4 in Figure S2, Supporting Information) has been changed to
Trp236. Taken together, the bioinformatic analysis predicts that
SgcF is an EH responsible for the hydrolysis of an enediyne
intermediate bearing an oxirane ring.
Inactivation of sgcF. The ∆sgcF mutant strain SB1010
completely lost its ability to produce C-1027. Inactivation of
sgcF in S. globisporus was accomplished by following the λ-red-
mediated PCR-targeting method to replace the entire sgcF gene
with the aac(3)IV/oriT cassette, affording the ∆sgcF mutant
strain SB1010 (Figure S3A and B, Supporting Information).
Bioassays using M. luteus as a reference strain showed that the
production of C-1027 was totally abolished in SB1010 either
cultured on solid (ISP4) (Figure S3C, Supporting Information)
or in liquid (A9) media (Figure S3D, Supporting Information).
It was further confirmed by HPLC analysis that no C-1027 was
produced by SB1010 when fermented in A9 media for 5 days
(Figure S3E, Supporting Information), indicating that sgcF is
indispensable for C-1027 biosynthesis.
Figure 4. HPLC profiles for SgcF-catalyzed hydrolysis of racemic styrene
oxide: (I) 1-phenyl-1,2-ethanediol standard; (II) with 100 µM SgcF for 90
min; and (III) with boiled SgcF for 90 min.
Overproduction and Purification of SgcF. The sgcF gene was
amplified from the cosmid pBS100533,43 and cloned into
pCDF-2 Ek/LIC. After overproduction in E. coli BL21 (DE3),
SgcF (∼70 mg/L) was purified to homogeneity as an N-terminal
His6-tagged fusion protein using NTA-Ni affinity column
chromatography. SDS-PAGE showed a single protein band
consistent with the predicted molecular weight of SgcF (44.5
KDa) (Figure S4, Supporting Information).
Activity Assay for SgcF. Due to the unavailability of the
oxirane-containing enediyne core that is predicted to be the
substrate for SgcF, styrene oxide was chosen as a substrate
mimic with which to test SgcF activity (Figure 3). The HPLC-
based assay was carried out as described in the Experimental
Section, and a single peak was eluted with a retention time
identical to that of authentic 1-phenyl-1,2-ethanediol (Figure
4). The new peak was collected and analyzed by ESI-MS,
yielding a [M + Na]+ ion at m/z 161.2 (calcd [M + Na]+ ion
for molecular formula C8H10O2 is 161.1). The absence of a
styrene oxide peak in the HPLC chromatogram was attributed
to its evaporation under reduced pressure, as indicated by the
control experiments performed without enzyme.
Optimization of SgcF Activity. Prior to kinetic analysis, the
pH dependence of SgcF-catalyzed hydrolysis was examined.
The pH profile exhibited a bell-shaped curve between pH 6.0
to 10.0 with an optimal activity at pH 8.0 (Figure S5A,
Supporting Information). As a result, all subsequent assays were
performed in 50 mM phosphate buffer at pH 8.0. The reactions
with different concentrations of SgcF showed that the SgcF-
catalyzed hydrolysis of styrene oxide is enzyme-dependent
(Figure S5B, Supporting Information).
Figure 5. Kinetic analysis of SgcF-catalyzed hydrolysis of (S)- and (R)-
styrene oxide showing single substrate kinetic plots for (A) (S)-styrene oxide
and (B) (R)-styrene oxide.
tioselectivity of SgcF was investigated using enantiomerically
pure (R)- and (S)-styrene oxide substrates. A spectrophoto-
metric assay was adopted to continuously monitor the
formation of 1-phenyl-1,2-ethanediol,47,48 thereby determin-
ing the steady-state kinetic constants for each enantiomer
(Figure 5A and B). A plot of initial velocity versus the
concentration of (S)-styrene oxide displayed Michaelis-Menten
kinetics yielding a Km of 0.6 ( 0.1 mM and kcat of 48 ( 1
min-1, while assays with variable (R)-styrene oxide gave a
Km of 2.0 ( 0.4 mM and kcat of 4.3 ( 0.3 min-1, respectively.
Enantioselectivity of SgcF. Because most EH-catalyzed
reactions exhibit stereo- and/or regio-selectivity, the enan-
(49) van Loo, B.; Kingma, J.; Arand, M.; Wubbolts, M. G.; Janssen, D. B.
Appl. EnViron. Microb. 2006, 72, 2905–2917.
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A R T I C L E S
Figure 7. Regioselectivity of SgcF-catalyzed hydrolysis of (S)- and (R)-
styrene oxide in [18O]-H2O: regioselective attack of the [18O]-labeled (red)
nucleophile Asp175 at (A) C-1 of (S)-styrene oxide and (B) C-1 and C-2
of (R)-styrene oxide, and (C) 13C NMR spectra of C-1 and C-2 of 1-phenyl-
1,2-ethanediol obtained from SgcF-catalyzed hydrolysis of (I) racemic
styrene oxide in H2O, (II) (S)-styrene oxide in [18O]-H2O, and (III) (R)-
styrene oxide in [18O]-H2O.
Figure 6. HPLC profiles for determination of the stereochemistry of
1-phenyl-1,2-ethanediol resulted from SgcF catalysis: (I) racemic 1-phenyl-
1,2-ethanediol standard; (II) (R)-1-phenyl-1,2-ethanediol standard ([); (III)
SgcF-catalyzed hydrolysis of racemic styrene oxide; (IV) SgcF-catalyzed
hydrolysis of (S)-styrene oxide; (V) (S)-1-phenyl-1,2-ethanediol standard
(b); and (VI) SgcF-catalyzed hydrolysis of (R)-styrene oxide.
epoxide enantiomers were hydrolyzed via the same mechanism
(e.g., regioselective attack at the R-carbon). Consequently, the
predominance of the (R)-vicinal diol product must arise from
complementary stereo- and regioselective SgcF-catalyzed hy-
drolysis of each styrene oxide enantiomer. This intriguing
finding prompted further investigation of this apparent enan-
tioconvergent activity of SgcF.
Thus, SgcF preferentially hydrolyzes (S)-styene oxide with
a 37-fold greater specificity constant (E ) 37), implicating
an (S)-epoxide enediyne intermediate in C-1027 biosynthesis
(Figure 3).
Investigation of Enantioconvergent Mechanisms of
SgcF-Catalyzed Hydrolysis. A few EHs have been reported
to produce enantiomerically enriched diol products from
racemic epoxides via complementary enantio- and regio-
selectivity.30 The regioselectivity of the SgcF-catalyzed
hydrolysis of (S)- and (R)-styrene oxide was investigated by
performing the reaction for each individual enantiomer
substrate in the presence of [18O]-H2O. 13C NMR analysis of
the resultant product from SgcF-catalyzed hydrolysis of (S)-
styrene oxide showed a 2.8-Hz upfield shift that was
exclusively associated with the C-1 position (Figure 7C), and
chiral HPLC analysis confirmed the identity of the product
as pure (R)-1-phenyl-1,2-ethanediol (Panel IV in Figure 6).
This indicates a mechanism involving regiospecific nucleo-
philic attack at C-1 of (S)-styrene oxide (Figure 7A). By
contrast, the product generated from (R)-styrene oxide
revealed upfield shifts at both C-1 (2.8-Hz) and C-2 (2.4-
Hz) (Figure 7C), and chiral HPLC analysis showed that (R)-
Stereochemistry of the Vicinal Diol Product. To determine
the stereochemistry of the product, SgcF-catalyzed hydrolysis
of racemic styrene oxide was performed on a large scale (1 mL)
and the reaction was quenched by extraction of ethyl acetate
when the reaction was complete as judged by thin layer
chromotography. After ethyl acetate extraction and complete
1
removal of solvent, H NMR analysis in CDCl3 confirmed the
product as 1-phenyl-1,2-ethanediol by comparison to the
authentic standard. Specifically, five protons at δ 7.3-7.4 ppm
correspond to the hydrogens of the monosubstituted benzene
ring, a doublet-doublet signal (δ 4.90 ppm, J ) 8.1, 3.4 Hz)
is associated with the proton at C-1, and two doublet-doublet
signals (δ 3.83 ppm, J ) 11.5, 3.4 Hz and δ 3.73 ppm, J )
11.0, 8.1 Hz) represent the two protons at C-2. Chiral HPLC
analysis, however, revealed that (R)-1-phenyl-1,2-ethanediol was
the dominant product (Panel III in Figure 6), a surprising finding
given that no enantioselectivity would be expected if both
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A R T I C L E S
Lin et al.
1-phenyl-1,2-ethanediol was produced with 20% ee (Panel
VI in Figure 6). This labeling pattern is consistent with a
lack of regiospecificity of the enzyme in attacking both C-1
and C-2 of (R)-styrene oxide (Figure 7B). These data establish
that the enantiomerically enriched (R)-vicinal diol product
from SgcF-catalyzed hydrolysis of racemic styrene oxide
originates from complementary enantio- and regioselectivities
of SgcF.
vivo data showed that no C-1027 production was observed
in the ∆sgcF mutant strain SB1010, unambiguously estab-
lishing that sgcF is indispensable for C-1027 biosynthesis.
In vitro characterization of SgcF unveiled a canonical EH
activity as demonstrated by the efficient hydrolysis of styrene
oxide, a substrate mimic of the putative enediyne core
epoxide (Figure 3). Specifically, SgcF efficiently hydrolyzes
(S)-styrene oxide, displaying an apparent Km of 0.6 ( 0.1
mM and kcat of 48 ( 1 min-1, via attack at the R-position to
exclusively generate the (R)-phenyl vicinal diol, consistent
with the stereochemistry of the C-1027 chromophore. These
findings support the role of SgcF in the proposed convergent
pathway for C-1027 biosynthesis, unveiling an (R)-vicinal
diol as a key intermediate (Figures 1 and 3). Interestingly,
SgcF can also hydrolyze (R)-styrene oxide to afford prefer-
entially the (R)-phenyl vicinal diol via attack at the ꢀ-position,
albeit with significantly reduced efficiency (apparent Km of
2.0 ( 0.4 mM and kcat ) 4.3 ( 0.3 min-1). However, the
latter activity unlikely contributes to C-1027 biosynthesis in
vivo since the oxygenase that catalyzes the formation of the
enediyne core epoxide intermediate most probably would
yield a single enediyne core epoxide, that is, the (S)-
enantiomer, as a substrate for SgcF.
EHs are believed to be involved in the biosynthesis of a
variety of natural products, including polyether antibiotics like
nanchangmycin60 and monensin,61 plant cutin,29 and insect
pheromones.62 The large diversity of putative EHs identified
from genome sequences49 implies that comparative studies may
potentially reveal a great deal about the determinants of regio-
and enantiospecificity of these enzymes. Indeed, comparative
analyses of enediyne biosynthetic gene clusters33-35 has pre-
dicted two EHs involved in the production of NCS - NcsF1
(53% identity to SgcF) and NcsF2 (62% identity to SgcF).34 In
a biosynthetic strategy that parallels SgcF, one would predict
NcsF1 or NcsF2 to catalyze the hydrolysis of an (R)-enediyne
core epoxide intermediate to afford an (S)-vicinal diol product
for NCS biosynthesis (Figure 1). Comparative studies of SgcF
and NcsF1 or F2 therefore would present a great opportunity
to investigate how EHs evolve and acquire their exquisite regio-
and enantioselectivity.
Discussion
C-1027, a chromoprotein antitumor antibiotic produced by
Steptomyces globisporus, is composed of an apoprotein (CagA)
and the C-1027 chromophore. The C-1027 chromophore consists
of four distinct moieties - an enediyne core, a deoxy aminosugar,
a benzoxazolinate, and an (S)-3-chloro-5-hydroxy-ꢀ-tyrosine
moiety (Figure 1).50-52 The 9-membered enediyne core readily
undergoes Bergman cycloaromatization to generate a highly
reactive diradical intermediate that abstracts hydrogen atoms
from DNA, leading to both double-stranded breaks (DSBs) and
interstrand cross-links (ICLs) that ultimately result in cell
death.53-55 Although the enediyne core directly confers cyto-
toxicity, the three peripheral moieties appended to the core are
required for activity and stability. For instance, the benzoxazo-
linate moiety aids in binding to CagA that protects and carries
the enediyne chromophore,56,57 while the (S)-3-chloro-5-hy-
droxy-ꢀ-tyrosine also contributes critical binding interactions
with CagA58,59 and modulates the reactivity of the enediyne
core via π-π interactions.59 A comparison of the biosynthetic
gene clusters of C-1027,33 NCS34 and maduropeptin35 suggested
that these two moieties might be appended to a (R)-vicinal diol-
containing C-1027 enediyne core, which in turn may originate
from a (S)-enediyne core epoxide intermediate via EH-catalyzed
ring-opening (Figure 3).
To test this hypothesis, we first carried out extensive
bioinformatics analysis of the 56 genes within C-1027
biosynthetic gene cluster, and identified two genes, sgcF and
sgcI, encoding R/ꢀ- hydrolases.33 While bioinformatics
analysis alone fell short of assigning SgcI as an EH, SgcF
possesses such EH characteristics as the nucleophilic elbow
(Sm-X-Nu-X-Sm-Sm), the G-X-Sm-X-S/T motif, the H-G-
X-P oxyanion hole, and the Asp-His-Asp catalytic triad, and
hence was annotated as an EH. Interestingly, SgcF contains
only one of the two Tyr residues usually conserved in the
lid domain (i.e., Y304) that serve to activate the oxirane ring
to nucleophilic attack, and the other is changed to Trp (i.e.,
W236) (Figure S2, Supporting Information).10,15-17,20-22,26 In
Most known EHs catalyze the hydrolysis of each enantiomer
of a racemic mixture at a different rate to yield enantiomerically
enriched vicinal diol products. However, this method of
separation, or kinetic resolution, is limited by a maximum
possible yield of 50%. In contrast, SgcF can not only catalyze
the hydrolysis of (S)-styrene oxide to afford (R)-1-phenyl-1,
2-ethanediol exclusively but also hydrolyze (R)-styrene oxide
to yield predominately (R)-1-phenyl-1, 2-ethanediol with 20%
ee. Taken together, SgcF can generate (R)-1-phenyl-1, 2-ethanediol
from racemic styrene epoxide with a minimal enantiomeric
excess of 60% ee. SgcF therefore represents an excellent starting
point for protein engineering efforts directed toward developing
a biocatalyst for preparing enantiomerically pure diols from
racemic epoxides.
(50) Hu, J.; Xue, Y. C.; Xie, M. Y.; Zhang, R.; Otani, T.; Minami, Y.;
Yamada, Y.; Marunaka, T. J. Antibiot. 1988, 41, 1575–1579.
(51) Otani, T.; Minami, Y.; Marunaka, T.; Zhang, R.; Xie, M. Y. J. Antibiot.
1988, 41, 1580–1585.
(52) Otani, T.; Yasuhara, T.; Minami, Y.; Shimazu, T.; Zhang, R.; Xie,
M. Y. Agric. Biol. Chem. 1991, 55, 407–417.
(53) Kennedy, D. R.; Gawron, L. S.; Ju, J. H.; Liu, W.; Shen, B.; Beerman,
T. A. Cancer Res. 2007, 67, 773–781.
(54) Kennedy, D. R.; Ju, J.; Shen, B.; Beerman, T. A. Proc. Natl. Acad.
Sci. U.S.A. 2007, 104, 17632–17637.
In conclusion, the in vitro characterization of SgcF as an EH
significantly advances our understanding of C-1027 biosynthesis,
(55) Xu, Y. J.; Zhen, Y. S.; Goldberg, I. H. Biochemistry 1994, 33, 5947–
5954.
(56) Matsumoto, T.; Okuno, Y.; Sugiura, Y. Biochem. Biophys. Res.
Commun. 1993, 195, 659–666.
(60) Sun, Y.; Zhou, X.; Dong, H.; Tu, G.; Wang, M.; Wang, B.; ZX, D.
Chem. Biol. 2003, 10, 431–441.
(57) Okuno, Y.; Otsuka, M.; Sugiura, Y. J. Med. Chem. 1994, 37, 2266–
2273.
(61) Gallimore, A.; Stark, C.; Bhatt, A.; Harvey, B.; Demydchuk, Y.;
Bolanos-Garcia, V.; Fowler, D.; Staunton, J.; Leadlay, P.; Spencer, J.
Chem. Biol. 2006, 13, 453–460.
(58) Okuno, Y.; Iwashita, T.; Sugiura, Y. J. Am. Chem. Soc. 2000, 122,
6848–6854.
(59) Tanaka, T.; Fukuda-Ishisaka, S.; Hirama, M.; Otani, T. J. Mol. Biol.
2001, 309, 267–283.
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SgcF Epoxide Hydrolase for C-1027 Biosynthesis
A R T I C L E S
Instrumentation Center of the School of Pharmacy and Dr. Q. Cui,
the National Magnetic Resonance Facility at UW-Madison for
support in obtaining MS and NMR data, and Prof. W. Tang, School
of Pharmacy, UW-Madison for the use of chiral HPLC. This work
is supported in part by NIH grants CA78747 and CA113297. G.P.H.
is the recipient of an NSERC postdoctoral fellowship.
supporting a (R)-vicinal diol as a key intermediate. The
preference of SgcF for hydrolyzing (S)-styrene oxide to generate
(R)-phenyl vicinal diol is consistent with the proposed conver-
gent logic of C-1027 biosynthesis in which the benzoxazolinate
and ꢀ-amino acid moieties are attached to an (R)-vicinal diol
functionality of an (S)-enediyne core epoxide intermediate
(Figure 3).39,41 Furthermore, the complementary regioselectivity
of SgcF, which leads to the enantioconvergent production of
(R)-vicinal diol as the dominant product from a racemic epoxide,
sets the stage to investigate the stereochemistry of EH catalysis
and to engineer EHs with improved regio- and/or enantiospeci-
ficity.
Supporting Information Available: The structural model of
AnEH (Figure S1), sequence alignment of SgcF (Figure S2),
sgcF inactivation results (Figure S3), SDS-PAGE of purified
recombinant SgcF (Figure S4), and pH (Figure S5A) and
concentration (Figure S5B) dependence of SgcF. This material
is available free of charge via the Internet at http://pubs.acs.org.
Acknowledgment. We thank Dr. Y. Li, Institute of Medicinal
Biotechnology, Chinese Academy of Medical Sciences, Beijing,
China for the wild-type S. globisporus strain, the Analytical
JA901242S
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