Engineering and analysis of a self-sufficient biosynthetic cytochrome P450 PikC fused to the RhFRED reductase domain

Article abstract of DOI:10.1021/ja075842d

Cytochrome P450 enzymes mediate important oxidative processes in biological systems including regio- and stereospecific hydroxylation and epoxidation reactions. The inherent requirement of these biomolecules for separate redox partner(s) significantly limits their application in biotechnology. To address this challenge, naturally occurring and/or bioengineered self-sufficient P450 systems with covalently fused redox partners have been utilized to harness their catalytic power. In this study, we describe the first in vitro characterization of a bacterial biosynthetic cytochrome P450 PikC fused to a heterologous reductase domain RhFRED that demonstrates single-component self-sufficiency. This novel fusion system not only produces a more active and effective biocatalyst but also suggests a general design for a universal reductase to generate diverse self-sufficient fusions for functional identification or industrial applications of biosynthetic P450s. Copyright

Full text of DOI:10.1021/ja075842d

Published on Web 10/04/2007
Engineering and Analysis of a Self-Sufficient Biosynthetic Cytochrome P450
PikC Fused to the RhFRED Reductase Domain
†
‡
,†,§
Shengying Li, Larissa M. Podust, and David H. Sherman*
Life Sciences Institute, Departments of Medicinal Chemistry, Chemistry, and Microbiology & Immunology,
UniVersity of Michigan, Ann Arbor, Michigan 48109, and Department of Pharmaceutical Chemistry, UniVersity of
California, San Francisco, California 94158
Received August 3, 2007; E-mail: davidhs@umich.edu
Cytochrome P450 enzymes (P450s) are highly attractive bio-
catalysts due to their ability to catalyze a variety of regio- and
stereospecific oxidation reactions of complex organic compounds.
These reactions occur under mild conditions by taking advantage
of the two-electron activated dioxygen that is often challenging in
Scheme 1. Major Physiological Reactions Catalyzed by PikC
1
organic synthesis. To activate molecular oxygen, redox partners
are required to sequentially transfer two reducing equivalents from
2
NAD(P)H to P450. Classically, there are two major redox partner
systems, including an FAD containing reductase with a small iron-
sulfur (Fe
2 2
S ) redoxin for most bacterial and mitochondrial P450s
(
class I) and a single FAD/FMN containing flavoprotein for
3
eukaryotic microsomal P450s (class II). The inherent requirement
of cytochome P450s for separate protein partner(s) significantly
limits their application in biotechnology.
The discovery of the first self-sufficient P450BM3, which is
naturally fused to a eukaryotic-like reductase, represents an effective
solution to this limitation.4 The fusion nature of this enzyme
dramatically improves electron-transfer efficiency and coupling with
the oxidative process, enabling it to be the most efficient P450
that demonstrates high catalytic efficiency. The PikC cytochrome
P450 in this study is involved in the pikromycin biosynthetic
pathway of Streptomyces Venezuelae.10 PikC catalyzes the final
hydroxylation step toward both the 12-membered ring macrolactone
YC-17 (1) and the 14-membered ring macrolactone narbomycin
5
enzyme characterized to date. Based upon the self-sufficiency of
this naturally fused enzyme, a number of engineered proteins of
diverse eukaryotic P450s bearing a reductase domain from P450BM3
have been generated with in Vitro activities. This provides ready
access to the great catalytic versatility of the membrane-bound
eukaryotic P450s. In contrast, the biosynthetic P450s (class I) lack
such a universal reductase that can be used to engineer diverse self-
sufficient P450s for either functional identification or potential
industrial application.
(4) to produce methymycin/neomethymycin (2/3) and pikromycin
(5) as major products (Scheme 1).11 Recently, we elucidated the
structural basis for the remarkable substrate flexibility by analyzing
ligand-free and substrate-bound structures of PikC.12 However, since
the native redox partner of PikC remains unknown, its in Vitro
activity has depended on expensive spinach ferredoxin reductase
6
(
Fdr) and ferredoxin (Fdx) (Scheme 2A), as are many other
13
biosynthetic P450s. To investigate an alternative electron-transfer
pathway mimicking the fusion organization in P450RhF (Scheme
Recently, a new class of self-sufficient cytochrome P450s
exemplified by P450RhF from Rhodococcus sp. NCIMB 9784 was
2B), pikC was linked to the RhFRED gene including the native 16
2 2
discovered to be naturally fused to a novel FMN/Fe S containing
amino acid linker sequence. The hybrid sequence was cloned into
pET28b(+), and overexpressed in E. coli BL21 (DE3) to generate
N-terminal His -tagged PikC-RhFRED. After Ni-NTA chromatog-
6
raphy, the purified red-colored recombinant P450 displayed (upon
reduction) the signature peak at 450 nm in the CO-difference
spectrum. Interestingly, gel filtration chromatography indicated that
PikC-RhFRED predominantly dimerizes in storage buffer solution
containing 0.2 mM dithioerythritol (DTE). In contrast, wild type
7
reductase partner. Although the physiological function of P450RhF
remains unknown, its reductase domain (RhFRED), which is similar
to the phthalate family of dioxygenase reductases, is capable of
transferring electrons from NADPH to the heme domain of the
monooxygenase, supporting 7-ethoxycoumarin dealkylation activ-
8
ity. Moreover, recent reports from Misawa et al. demonstrated that
this reductase domain could be used to reconstitute the catalytic
activities of various class I P450s in ViVo through expression of
corresponding genes fused to RhFRED in Escherichia coli cells.9
This suggests that RhFRED might be developed into a generally
effective redox partner for biosynthetic bacterial P450s. However,
the lack of corresponding in Vitro data could not unambiguously
exclude in trans involvement of additional cellular redox partners.
Herein, we describe the first in Vitro characterization of a single
component bacterial biosynthetic cytochrome P450 fused to RhFRED
(wt) PikC was shown to be monomeric under the same conditions.
It was thus unclear whether the intermonomer electron transfer could
14
occur in the dimeric PikC-RhFRED as in P450BM3
.
We next tested the ability of PikC-RhFRED to hydroxylate 1
and 4 in Vitro when provided electron donor NADPH. We were
gratified to observe that this chimeric protein showed significantly
improved catalytic activity compared to wt PikC in the presence
of exogenous redox partners (spinach Fdr and Fdx), producing
higher yields of 2/3 and 5 under identical reaction conditions (Figure
1). This result unambiguously confirms that PikC-RhFRED is a
self-sufficient P450 enzyme. Interestingly, we also constructed the
pET21b(+)-pikC-RhFRED and obtained the purified C-terminal
†
Life Sciences Institute and Department of Medicinal Chemistry, University
of Michigan.
‡
University of California, San Francisco.
Departments of Chemistry and Microbiology & Immunology, University of
§
Michigan.
12940
9
J. AM. CHEM. SOC. 2007, 129, 12940-12941
10.1021/ja075842d CCC: $37.00 © 2007 American Chemical Society

C O M M U N I C A T I O N S
NADPH depletion assay,11,16 suggesting that the stoichiometric ratio
between NADPH and substrate hydroxylation could not be 1:1.
The presumed decoupling between electron transfer and hydroxy-
lation might account for this difference.
Scheme 2. Two Redox Partner Systems (Electron-Transfer
Pathways) Used in This Study for PikC
a
Finally, when RhFRED was fused to another prototype biosyn-
thetic P450 EryF,13 a more active self-sufficient biocatalyst was
obtained once again (see Supporting Information). Together with
previous in ViVo work,9 our studies demonstrate that further
development of RhFRED as the basis for an efficient cost-effective
redox partner for bacterial biosynthetic P450s is warranted. Further
efforts to understand this unique reductase, especially the electron-
transfer process involving heterologous fusion systems, are now
in progress.
a
A, three component system; B, one component RhFRED system.
Acknowledgment. We thank Dr. Norihiko Misawa for gener-
ously providing the construct pRED containing reductase domain
gene from P450RhF, and Dr. Gary W. Ashley for the gift of
6-deoxyerythronolide. The authors thank Dr. Jeffrey D. Kittendorf
and Liangcai Gu for helpful discussions. We are grateful for support
from NIH RO1 Grant GM078553 (to D.H.S. and L.M.P.).
Supporting Information Available: Plasmid maps for constructs,
SDS-PAGE analysis, UV-visible absorption spectrum for PikC-
RhFRED, gel filtration chromatography results, substrate binding
affinity measurements, steady-state kinetics data, mass spectrometry
data for EryF-RhFRED reaction analysis, and experimental procedures.
This material is available free of charge via the Internet at http://
pubs.acs.org.
Figure 1. HPLC analysis of reactions (1 h) catalyzed by wt PikC and fusion
enzyme PikC-RhFRED. (a) Negative control of 1 in absence of P450. (b)
1
with wt PikC in presence of Fdr, Fdx, and NADPH. (c) 1 with PikC-
RhFRED in presence of only NADPH. (d) Negative control of 4 in absence
of P450. (e) 4 with wt PikC in presence of Fdr, Fdx, and NADPH. (f) 4
with PikC-RhFRED in presence of only NADPH.
References
His
difference spectrum as its N-terminal His
not shown). However, it lacks catalytic activity, which is consistent
with a similar C-terminal His -tagged form of original P450RhF.
6
This provides additional evidence for the importance of the C
terminus of RhFRED for electron transfer.
6
-tagged PikC-RhFRED. This protein showed a similar CO-
(
1) (a) Ortiz de Montellano, P. R. Cytochrome P450: Structure, Mechanism
and Biochemistry, 2nd ed.; Ortiz de Montellano, P. R., Ed.; Plenum
Press: New York, 1995; p 473. (b) Guengerich, F. P. Chem. Res. Toxicol.
2001, 14, 611.
6
-tagged counterpart (data
8
(
2) Hannemann, F. B. A.; Ewen, K. M.; Bernhardt, R. Biochim. Biophys.
Acta 2007, 1770, 330.
(
3) (a) Lewis, D. F. V.; Hlavica, P. Biochim. Biophys. Acta 2000, 1460, 353.
(
b) Munro, A. W.; Girvan, H. M.; McLean, K. J. Nat. Prod. Rep. 2007,
As mentioned above, one benefit of the fusion arrangement is
that the covalent linkage presumably stabilizes the interaction
between the P450 and redox partner, thus enhancing electron-
transfer efficiency. As such, one would expect this to improve the
catalytic activity in terms of kcat, whereas the substrate specificity
2
4, 585.
(
4) (a) Ruettinger, R. T.; Fulco, A. J. J. Biol. Chem. 1981, 256, 5728. (b)
Otey, C. R.; Bandara, G.; Lalonde, J.; Takahashi, K.; Arnold, F. H.
Biotechnol. Bioeng. 2005, 93, 494.
(5) Munro, A. W.; Leys, D. G.; McLean, K. J.; Marshall, K. R.; Ost, T. W.
B.; Daff, S.; Miles, C. S.; Chapman, S. K.; Lysek, D. A.; Moser, C. C.;
Page, C. C.; Dutton, P. L. Trends Biochem. Sci. 2002, 27, 250.
(6) (a) Fairhead, M.; Giannini, S.; Gillam, E. M. J.; Gilardi, G. J. Biol. Inorg.
Chem. 2005, 10, 842. (b) Dodhia, V. R.; Fantuzzi, A.; Gilardi, G. J. Biol.
Inorg. Chem. 2006, 11, 903.
15
would not be changed significantly. To test whether this also
applies to PikC-RhFRED, we first determined the substrate binding
affinity of 1 and 4 toward both PikC and PikC-RhFRED. As
(7) (a) De Mot, R.; Parret, A. H. A. Trends Microbiol. 2002, 502. (b) Roberts,
G. A.; Grogan, G.; Greter, A.; Flitsch, S. L.; Turner, N. J. J. Bacteriol.
expected, 1 and 4 bind to PikC-RhFRED with K
0
1
d
values of 92.6 (
.5 µM and 215.0 ( 4.2 µM, respectively, which are similar to
12.9 ( 1.9 µM (1) and 288.3 ( 7.1 (4) toward wt PikC. This
2002, 184, 3898.
(8) (a) Roberts, G. A.; C¸ elik, A.; Hunter, D. J. B.; Ost, T. W. B.; White, J.
H.; Chapman, S. K.; Turner, N. J.; Flitsch, S. L. J. Biol. Chem. 2003,
4
8914. (b) Hunter, D. J. B.; Roberts, G. A.; Ost, T. W. B.; White, J. H.;
indicates that attachment of the heterologous reductase domain has
no significant impact on substrate binding to PikC. Subsequently,
we compared the kinetic parameters of PikC-RhFRED with those
M u¨ ller, S.; Turner, N. J.; Flitsch, S. L.; Chapman, S. K. FEBS Lett. 2005,
579, 2215.
(
9) (a) Kubota, M.; Nodate, M.; Yasumoto-Hirose, M.; Uchiyama, T.; Kagami,
O.; Shizuri, Y.; Misawa, N. Biosci. Biotechnol. Biochem. 2005, 69, 2421.
(
b) Nodate, M.; Kubota, M.; Misawa, N. Appl. Microbiol. Biotechnol.
of the PikC-Fdr-Fdx three component system. As previously
2
006, 71, 455.
reported,1
1,16
substrate inhibition was observed in all cases when
(
10) Xue, Y.; Zhao, L.; Liu, H.-w.; Sherman, D. H. Proc. Natl. Acad. Sci.
U.S.A. 1998, 95, 12111.
substrate concentration was greater than 250 µM. Moreover, the
solubility limitation (less than 500 µM) of macrolides in aqueous
(
11) (a) Xue, Y.; Wilson, D.; Zhao, L.; Liu, H.-w.; Sherman, D. H. Chem.
Biol. 1998, 5, 661. (b) Lee, S. K.; Park, J. W.; Kim, J. W.; Jung, W. S.;
Park, S. R.; Choi, C. Y.; Kim, E. S.; Ahn, J. S.; Sherman, D. H.; Yoon,
Y. J. J. Nat. Prod. 2006, 69, 847.
solution prevented us from deducing the K
i
value. Therefore, we
determined the apparent specificity constants (kcat/K
m
) by fitting
(
12) Sherman, D. H.; Li, S.; Yermalitskaya, L. V.; Kim, Y.; Smith, J. A.;
Waterman, M. R.; Podust, L. M. J. Biol. Chem. 2006, 281, 26289.
13) (a) Lambalot, R. H.; Cane, D. E. Biochemistry 1995, 34, 1858. (b)
Andersen, J. F.; Hutchinson, R. C. J. Bacteriol. 1992, 174, 725. (c) Ogura,
H.; Nishida, C. R.; Hoch, U. R.; Perera, R.; Dawson, J. H.; Ortiz de
Montellano, P. R. Biochemistry 2004, 43, 14712.
the low-concentration data to the linear region of the Michaelis-
Menten curve. By directly monitoring the substrate consumption
(
m
by HPLC, the kcat/K values of PikC-RhFRED were determined to
-1
-1
be 0.96 and 1.20 µM ‚min for 1 and 4, respectively. In contrast,
(14) Neeli, R.; Girvan, H. M.; Lawrence, A.; Warren, M. J.; Leys, D.; Scrutton,
the specificity constants of wt PikC partnered by Fdr and Fdx were
N. S.; Munro, A. W. FEBS Lett. 2005, 579, 5582.
-
1
-1
-1
-1
(15) (a) Munro, A. W.; Girvan, H. M.; McLean, K. J. Biochim. Biophys. Acta.
0
.24 µM ‚min for 1 and 0.31 µM ‚min for 4. It is evident
2007, 1770, 345. (b) Yabusaki, Y. Biochimie 1995, 77, 594.
that the fusion enhanced catalytic activity approximately 4-fold for
both 1 and 4. Notably, the kinetic parameters of wt PikC differ
significantly from those previously determined indirectly, using a
(16) Graziani, E. I.; Cane, D. E.; Betlach, M. C.; Kealey, J. T.; McDaniel, R.
Bioorg. Med. Chem. Lett. 1998, 3117.
JA075842D
J. AM. CHEM. SOC.
9
VOL. 129, NO. 43, 2007 12941