Characterization of the epoxide hydrolase NcsF2 from the neocarzinostatin biosynthetic gene cluster

Article abstract of DOI:10.1021/ol101473t

Neocarzinostatin (1) biosynthesis is proposed to involve a vicinal diol intermediate. It is reported that NcsF2, one of two epoxide hydrolases encoded by the NCS gene cluster, catalyzes regiospecific addition of H₂O to C-2 of both (R)-and (S)-styrene oxides to afford (R)-and (S)-1-phenyl-1,2- ethanediols, respectively, supporting its proposed role in 1 biosynthesis. (R)-1-Phenyl-1,2-ethanediol (87% yield and 99% ee) was obtained from (±)-styrene oxide hydrolysis by cocatalysis using NcsF2 and SgcF, the complementary epoxide hydrolase from the C-1027 biosynthetic pathway.

Full text of DOI:10.1021/ol101473t

ORGANIC
LETTERS
2
010
Vol. 12, No. 17
816-3819
Characterization of the Epoxide Hydrolase
NcsF2 from the Neocarzinostatin
Biosynthetic Gene Cluster
3
†
†
,†,‡,§
Shuangjun Lin, Geoffrey P. Horsman, and Ben Shen*
DiVision of Pharmaceutical Sciences, UniVersity of Wisconsin National CooperatiVe
Drug DiscoVery Group, and Department of Chemistry, UniVersity of
WisconsinsMadison, Madison, Wisconsin 53705
bshen@pharmacy.wisc.edu
Received July 1, 2010
ABSTRACT
Neocarzinostatin (1) biosynthesis is proposed to involve a vicinal diol intermediate. It is reported that NcsF2, one of two epoxide hydrolases encoded
by the NCS gene cluster, catalyzes regiospecific addition of H O to C-2 of both (R)- and (S)-styrene oxides to afford (R)- and (S)-1-phenyl-1,2-
ethanediols, respectively, supporting its proposed role in 1 biosynthesis. (R)-1-Phenyl-1,2-ethanediol (87% yield and 99% ee) was obtained from
()-styrene oxide hydrolysis by cocatalysis using NcsF2 and SgcF, the complementary epoxide hydrolase from the C-1027 biosynthetic pathway.
2
(
Neocarzinostatin (NCS) is an enediyne antitumor antibiotic
produced by Streptomyces carzinostaticus ATCC 15944 as a
chromoprotein complex consisting of an apoprotein and the
diol is thought to arise from hydrolysis of an epoxide intermedi-
ate by one of two putative epoxide hydrolase (EH) enzymes
encoded by the NCS gene cluster, NcsF1 or NcsF2. A similar
2
1
NCS chromophore (1). Characterization of the NCS biosyn-
convergent logic has been proposed for the C-1027 (2)
3
thetic gene cluster from S. carzinostaticus led to a proposed
convergent biosynthetic pathway in which the peripheral
moieties are appended to an enediyne core vicinal diol
biosynthetic pathway in Streptomyces globisporus (Figure 1B),
from which an EH, SgcF, has been characterized using styrene
4
oxide as a substrate mimic. SgcF was shown to catalyze an
2
intermediate to afford 1 (Figure 1A). The presumed (S)-vicinal
(
2) Liu, W.; Nonaka, K.; Nie, L.; Zhang, J.; Christenson, S. D.; Bae, J.;
†
Division of Pharmaceutical Sciences.
University of Wisconsin National Cooperative Drug Discovery Group.
Department of Chemistry.
Van Lanen, S. G.; Zazopoulos, E.; Farnet, C. M.; Yang, C. F.; Shen, B.
Chem. Biol. 2005, 12, 293–302.
‡
§
(3) Liu, W.; Christenson, S. D.; Standage, S.; Shen, B. Science 2002,
297, 1170–1173.
(
1) (a) Ishida, N.; Miyazaki, K.; Kumagai, M.; Rikimaru, M. J. Antibiot.
965, 18, 68–76. (b) Edo, K.; Mizugaki, M.; Koide, Y.; Seto, H.; Furihata,
K.; Otake, N.; Ishida, N. Tetrahedron Lett. 1985, 26, 331–340.
1
(4) Lin, S.; Horsman, G. P.; Chen, Y.; Li, W.; Shen, B. J. Am. Chem.
Soc. 2009, 131, 16410–16417.
1
0.1021/ol101473t  2010 American Chemical Society
Published on Web 08/12/2010

Figure 1. Proposed biosynthetic pathways featuring (A) an NcsF2-catalyzed formation of an (S)-vicinal diol intermediate from an (S)-
enediyne core epoxide precursor for NCS (1) and (B) an SgcF-catalyzed formation of an (R)-vicinal diol intermediate from an (S)-enediyne
core epoxide precursor for C-1027 (2). The vicinal diol moieties in 1 and 2 concerned in this study are shaded.
5
enantioconvergent process in which H
2
O attacks primarily at
only a very minor peak, indicating a dramatically less
efficient enzyme (Figure 3). Careful inspection of the selected
EH protein sequences reveals that, in contrast to SgcF and
NcsF2, NcsF1 may lack key amino acid residues conserved
among EHs (Figure S2, Supporting Information). First, the
acid residue of the nucleophile-His-acid catalytic triad has
been replaced by Ala (Asp174-His361-Ala334) at its usual
position after strand ꢀ7, but some EHs are known to harbor
C-2 for (R)-styrene oxide and at the more hindered C-1 for (S)-
4
styrene oxide (Figure 2A). Consequently, the major product
is always the (R)-vicinal diol, but the 37-fold higher specificity
of SgcF for (S)-styrene oxide implicates an (S)-epoxide enediyne
intermediate in C-1027 biosynthesis, consistent with the pro-
3,4
posed pathway (Figure 1B). Here, we report that NcsF2,
but not NcsF1, efficiently hydrolyzes styrene oxide. In
contrast to SgcF, NcsF2 is regiospecific for C-2 such that
hydrolysis of (R)- and (S)-styrene oxides yield (R)- and (S)-
vicinal diol products, respectively (Figure 2B). Although
hydrolysis of an (S)-epoxide enediyne intermediate would
afford the expected (S)-vicinal diol (Figure 1A), the 44-fold
preference of NcsF2 for (R)-styrene oxide is inconsistent with
7
this residue after strand ꢀ6. Second, the two canonical EH
Tyr residues that activate the epoxide oxygen, one of which
7
is absolutely conserved, are missing in NcsF1 (Pro234 and
His303), although we note that the adjacent Tyr304 may
function equivalently. Thus, although NcsF1 is 53% and 59%
identical to SgcF and NcsF2, respectively, its unusual
sequence and relatively inefficient hydrolysis of styrene oxide
suggest an alternative function for this enzyme in 1 biosyn-
thesis, perhaps for hydrolysis of the internal epoxide of the
NCS enediyne core precursor (Figure 1A).
2
our biosynthetic proposal and may be an artifact of using
styrene oxide as a substrate mimic. By exploiting the
complementary activities of NcsF2 and SgcF, we finally
demonstrated the preparation of (R)-1-phenyl-1,2-ethanediol
from racemic styrene oxide with excellent chemical yield
and enantiomeric excess (Figure 2C).
We have previously predicted two genes, ncsF1 and ncsF2,
within the NCS biosynthetic gene cluster, to encode EHs on
the basis of bioinformatics analysis. We first cloned, overpro-
The observed activity of NcsF2 prompted further character-
ization of the enzyme. We then determined the steady-state
kinetic parameters of NcsF2 toward (R)- and (S)-styrene oxides
6,8
using a spectrophotometric assay to continuously monitor
2
theformationof1-phenyl-1,2-ethanediol.TheMichaelis-Menten
duced in E. coli, and purified both NcsF1 and NcsF2 to
homogeneity (Figure S1, Supporting Information). The EH
equation was fitted to plots of initial velocity versus substrate
6
-1
concentration (Figure 4) to yield kcat ) 133 ( 4 min , K
M
-
1
-1
activity of both enzymes toward racemic styrene oxide was then
) 0.5 ( 0.1 mM, and kcat/K
M
) 266 min mM for (R)-
4,6
-1
analyzed using HPLC. Incubation of NcsF2 (50 µM) with
mM styrene oxide produced a single peak corresponding
to 1-phenyl-1,2-ethanediol, and in contrast, NcsF1 produced
styrene oxide and kcat ) 31 ( 2 min , K ) 5.0 ( 0.6
M
-
1
-1
2
mM, and kcat/K
M
) 6 min mM for (S)-styrene oxide,
respectively. Thus, NcsF2 is 44-fold more specific for (R)-
(
5) (a) Faber, K.; Kroutil, W. Tetrahedron: Asymmetry 2002, 13, 377–
(6) See the Supporting Information for full experimental details.
(7) Barth, S.; Fischer, M.; Schmid, R. D.; Pleiss, J. Proteins: Struct.
Funct. Bioinform. 2004, 55, 846–855.
3
1
1
82. (b) Hwang, S.; Choi, C. Y.; Lee, E. Y. Biotechnol. Lett. 2008, 30,
219–1225. (c) Steinreiber, A.; Faber, K. Curr. Opin. Biotechnol. 2001,
2, 552–558. (d) Bellucci, G.; Chiappe, C.; Cordoni, A. Tetrahedron:
Asymmetry 1996, 7, 197–202. (e) Kroutil, W.; Mischitz, M.; Plachota, P.;
Faber, K. Tetrahedron Lett. 1996, 37, 8379–8382.
(8) (a) Doderer, K.; Lutz-Wahl, S.; Hauer, B.; Schmid, R. D. Anal.
Biochem. 2003, 321, 131–134. (b) Mateo, C.; Archelas, A.; Furstoss, R.
Anal. Biochem. 2003, 314, 135–141
.
Org. Lett., Vol. 12, No. 17, 2010
3817

Figure 4
and (R)-styrene oxide showing single substrate kinetic plots for (A)
R)-styrene oxide with 2 µM NcsF2 and (B) (S)-styrene oxide with
µM NcsF2.
. Kinetic analysis of NcsF2-catalyzed hydrolysis of (S)-
(
8
of NCS and C-1027 (Figure 1), but only if NcsF2 was
regioselective for C-2 and catalyzed stereochemical inversion
of the preferred (R)-styrene oxide substrate to yield the (S)-
vicinal diol.
Figure 2. SgcF- and NcsF2-catalyzed hydrolysis of styrene oxide to
-phenyl-1,2-ethanediol (vicinal diol): (A) SgcF-catalyzed regiospecific
hydrolysis of (S)-styrene oxide to (R)-vicinal diol and regioselective
hydrolysis of (R)-styrene oxide to both (R)- and (S)-vicinal diol with
enantioselectivity (E ) 37) for the (R)-styrene oxide; (B) NcsF2-
catalyzed regiospecific hydrolysis of (S)- and (R)-styrene oxides to
1
We therefore examined the stereochemistry of the NcsF2
4,6
reaction products using chiral HPLC (Figure 5) and surpris-
ingly found no inversion of stereochemistry. Specifically,
NcsF2 transformed (R)-styrene oxide to (R)-1-phenyl-1,2-
ethanediol (Figure 5A, panel II, 98% ee) and (S)-styrene
oxide to (S)-1-phenyl-1,2-ethanediol (Figure 5A, panel V,
(
4
S)- and (R)-vicinal diols, respectively, with enantioselectivity (E )
4) for the (R)-styrene oxide; (C) SgcF/NcsF2-catalyzed preparation
of (R)-vicinal diol from racemic styrene oxide.
90% ee). Similarly, NcsF2 hydrolyzed the racemic substrate
to afford racemic product (Figure 5A, panel III). This finding
strongly suggested that, unlike SgcF (Figure 2A), NcsF2
catalyzes addition of water exclusively at C-2 for both
enantiomers (Figure 2B).
We next verified the C-2 regiospecificity of NcsF2 using an
18
4,6
O-labeling experiment. Hydrolysis of (R)- or (S)-styrene
1
8
oxide by NcsF2 was performed in the presence of [ O]-
1
3
2
H O, and the products were analyzed by C NMR. As
expected, a 2.4 Hz chemical shift was observed exclusively
at the C-2 position for the 1-phenyl-1,2-ethanediol product
resulting from either the (R)- or (S)-styrene oxide substrate
(
Figure 6). In contrast to SgcF, which adds H
2
O to either
Figure 3. HPLC profiles for NcsF1- or NcsF2-catalyzed hydrolysis
of racemic styrene oxide: (I) 1-phenyl-1,2-ethanediol standard (O); (II)
with 50 µM NcsF2; (III) with 50 µM NcsF1; and (IV) with 50 µM
NcsF2 (boiled) as a negative control.
C-1 or C-2 depending on the stereochemistry of the substrate
4
(
(
Figure 2A), NcsF2 is regiospecific for C-2 of styrene oxide
Figure 2B).
Finally, the complementary activities of NcsF2 and SgcF
were exploited to prepare enantiopure (R)-1-phenyl-1,2-
ethanediol from racemic styrene oxide (Figure 2C). For
example, SgcF has 37-fold higher specificity for (S)-styrene
oxide, which is transformed almost exclusively to (R)-1-
phenyl-1,2-ethanediol, while the slower reacting (R)-styrene
styrene oxide (Figure 2B), in contrast to the 37-fold
preference of SgcF for (S)-styrene oxide (Figure 2A). The
opposite enantioselectivities of NcsF2 and SgcF were
consistent with the respective vicinal diol stereochemistries
4
3818
Org. Lett., Vol. 12, No. 17, 2010

together should generate (R)-1-phenyl-1,2-ethanediol in high
enantiomeric excess (Figure 2C). Indeed, we carried out the
reaction with a 3.5:1 ratio of SgcF/NcsF2 with 14 mM
styrene oxide and observed (R)-1-phenyl-1,2-ethanediol in
6
8
7% yield with 99% ee (Figure 5B). Such systems employ-
ing complementary enzymes have previously been used to
9
generate enantiomerically enriched products. For example,
a potato EH and an engineered EH from Agrobacterium
radiobacter AD1 together transformed racemic styrene oxide
to (R)-1-phenyl-1,2-ethanediol with 98% ee after complete
9a
conversion. Interestingly, these cases resulted from deliber-
ate efforts involving screening many enzymes to obtain a
suitable biocatalytic process. In contrast, our results arose
fortuitously from studies of enediyne biosynthesis and
highlight the biocatalytic potential available to be harnessed
from our ever-expanding knowledge of natural product
biosynthetic machineries.
In conclusion, we have demonstrated that the NcsF2
enzyme is active as an epoxide hydrolase toward styrene
oxide, an activity consistent with its proposed function in
hydrolyzing a terminal epoxide enediyne intermediate. We
Figure 5
. Chiral HPLC analysis for determination of stereochem-
istry of 1-phenyl-1,2-ethanediol resulting from NcsF2- or NcsF2/
SgcF-catalyzed hydrolysis of styrene oxide.
6,10
A: (I) (R)-1-phenyl-
1
,2-ethanediol standard; (II) NcsF2 with (R)-styrene oxide; (III)
2
further showed that NcsF2 regiospecifically adds H O to the
NcsF2 with (()-styrene oxide; (IV) (S)-1-phenyl-1,2-ethanediol
standard; (V) NcsF2 with (S)-styrene oxide. B: (I) (()-1-phenyl-
less hindered C-2 position of styrene oxide such that the
stereochemistry is retained in the vicinal diol product.
Surprisingly, the enantioselectivity of NcsF2 for (R)-
styrene oxide to (S)-styrene oxide (E ) 44) is inconsistent
with preferential production of an (S)-vicinal diol bio-
synthetic intermediate, as would be expected on the basis
of the structure of 1. However, NcsF2 remains our best
candidate for this function, and its unexpected selectivity
may reflect the inadequacy of styrene oxide as a substrate
mimic. Moreover, the natural enediyne core epoxide
precursor is likely a single enantiomer, in which case
regioselective hydrolysis of an (S)-epoxide enediyne
intermediate would produce the expected (S)-vicinal diol
stereochemistry (Figure 1A).
1
,2-ethanediol standard; (II) NscF2/SgcF with (R)-styrene oxide;
(
III) (R)-1-phenyl-1,2-ethanediol standard. ([), (R)-1-phenyl-1,2-
ethanediol; (b), (S)-1-phenyl-1,2-ethanediol.
Acknowledgment. We thank the Analytical Instrumentation
Center of the School of Pharmacy, Dr. Qiu Cui of the National
Magnetic Resonance Facility at the University of Wisconsins
Madison for support in obtaining MS and NMR data, and Prof.
W. Tang, School of Pharmacy, UWsMadison, for providing
access to chiral HPLC. This work is supported in part by NIH
Grant Nos. CA78747 and CA113297. G.P.H. is the recipient
of an NSERC (Canada) postdoctoral fellowship.
Figure 6 1
3C NMR spectra for C-1 and C-2 of 1-phenyl-1,2-ethanediol
.
produced by NcsF2-catalyzed hydrolysis of (A) racemic styrene oxide
18
2
in H O, (B) (R)-styrene oxide in [ O]-H
2
O (∼80% final concentration),
18
and (C) (S)-styrene oxide in [ O]-H
2
O (∼60% final concentration).
Supporting Information Available: Detailed experimen-
tal procedures, SDS-PAGE (Figure S1), and sequence
alignment (Figure S2). This material is available free of
charge via the Internet at http://pubs.acs.org.
oxide produces a minor but significant amount of unwanted
S)-vicinal diol (Figure 2A). Conversely, NcsF2 preferentially
(
hydrolyzes (R)-styrene oxide to afford the (R)-vicinal diol,
but slower reacting (S)-styrene oxide yields unwanted (S)-
vicinal diol product (Figure 2B). Thus, for a given enzyme,
the “slow” enantiomer that yields unwanted product is the
substrate for the other enzyme, which rapidly generates the
correct product. These complementary specificities and
product stereochemistries predict that the two enzymes
OL101473T
(
9) (a) Cao, L.; Lee, J.; Chen, J. W.; Wood, T. K. Biotechnol. Bioeng.
2
006, 94, 522–529. (b) Pedragosa-Moreau, S.; Archelas, A.; Furstoss, R.
J. Org. Chem. 1993, 58, 5533–5536. (c) Manoj, K. M.; Archelas, A.; Baratti,
J.; Furstoss, R. Tetrahedron 2001, 57, 695–701.
(
10) The discrepency in retention times between A and B results from
the use of a new OD-H column in the latter.
Org. Lett., Vol. 12, No. 17, 2010
3819