New reactions and products resulting from alternative interactions between the P450 enzyme and redox partners

Article abstract of DOI:10.1021/ja4130302

Cytochrome P450 enzymes are capable of catalyzing a great variety of synthetically useful reactions such as selective C-H functionalization. Surrogate redox partners are widely used for reconstitution of P450 activity based on the assumption that the choice of these auxiliary proteins or their mode of action does not affect the type and selectivity of reactions catalyzed by P450s. Herein, we present an exceptional example to challenge this postulate. MycG, a multifunctional biosynthetic P450 monooxygenase responsible for hydroxylation and epoxidation of 16-membered ring macrolide mycinamicins, is shown to catalyze the unnatural N-demethylation(s) of a range of mycinamicin substrates when partnered with the free Rhodococcus reductase domain RhFRED or the engineered Rhodococcus-spinach hybrid reductase RhFRED-Fdx. By contrast, MycG fused with the RhFRED or RhFRED-Fdx reductase domain mediates only physiological oxidations. This finding highlights the larger potential role of variant redox partner protein-protein interactions in modulating the catalytic activity of P450 enzymes.

Full text of DOI:10.1021/ja4130302

Article
pubs.acs.org/JACS
Open Access on 02/12/2015
New Reactions and Products Resulting from Alternative Interactions
between the P450 Enzyme and Redox Partners
Wei Zhang,† Yi Liu,†,‡ Jinyong Yan,† Shaona Cao,† Fali Bai,† Ying Yang,† Shaohua Huang,† Lishan Yao,†
Yojiro Anzai,§ Fumio Kato,§ Larissa M. Podust,∥ David H. Sherman,*,⊥ and Shengying Li*,†
†CAS Key Laboratory of Biofuels, and Shandong Provincial Key Laboratory of Energy Genetics, Qingdao Institute of Bioenergy and
Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong 266101, China
‡University of Chinese Academy of Sciences, Beijing 100049, China
§Faculty of Pharmaceutical Sciences, Toho University, Funabashi, Chiba 274-8510, Japan
∥Center for Discovery and Innovation in Parasitic Diseases, and Department of Pathology, University of California, San Francisco,
California 94158, United States
⊥Life Sciences Institute, Departments of Medicinal Chemistry, Chemistry, and Microbiology and Immunology, University of
Michigan, Ann Arbor, Michigan 48109, United States
S
* Supporting Information
ABSTRACT: Cytochrome P450 enzymes are capable of
catalyzing a great variety of synthetically useful reactions
such as selective C−H functionalization. Surrogate redox
partners are widely used for reconstitution of P450 activity
based on the assumption that the choice of these auxiliary
proteins or their mode of action does not affect the type and
selectivity of reactions catalyzed by P450s. Herein, we present
an exceptional example to challenge this postulate. MycG, a
multifunctional biosynthetic P450 monooxygenase responsible
for hydroxylation and epoxidation of 16-membered ring macrolide mycinamicins, is shown to catalyze the unnatural N-
demethylation(s) of a range of mycinamicin substrates when partnered with the free Rhodococcus reductase domain RhFRED or
the engineered Rhodococcus-spinach hybrid reductase RhFRED-Fdx. By contrast, MycG fused with the RhFRED or RhFRED-Fdx
reductase domain mediates only physiological oxidations. This finding highlights the larger potential role of variant redox partner
protein−protein interactions in modulating the catalytic activity of P450 enzymes.
systems have been discovered continuously,10,12 among which
the FMN/Fe2S2-containing reductase domain termed
INTRODUCTION
■
The superfamily of cytochrome P450 enzymes (CYPs) is one
“RhFRED” that is naturally fused with the P450 enzyme
of the most versatile biocatalyst systems in nature.1−3 CYPs are
from Rhodococcus sp. NCIMB 978413 has been extensively
capable of catalyzing more than 20 distinct types of reactions
including regio- and stereoselective oxidation of unactivated
C−H bonds (hydroxylation and epoxidation), dealkylation, C−
studied in our laboratories.14−16
For practical purposes due to the difficulty of obtaining
C bond cleavage, aromatic coupling, and others.4,5 Recently,
native partners, one or more surrogate redox partners, acting
more novel activities such as decarboxylation,6 nitration,7
either in isolation or as artificially fused protein complexes, are
farnesene synthase activity,8 and carbene transfer9 have been
often employed in functional characterization or synthetic
applications of CYPs. The choice of surrogate partners or their
mode of action is not necessarily expected to affect the type and
reported, reflecting the ability to further diversify the function
of these versatile enzymes.
selectivity of reactions catalyzed by P450s. Although alternative
Catalytic activities of the vast majority of CYPs require one
redox partners may influence catalytic efficiency and/or product
or more redox partner proteins to sequentially deliver two
electrons from NAD(P)H to the heme iron reactive center for
distribution,17−22 the chemical identity of products themselves
dioxygen activation.1,2 Classically, there are two major redox
is apparently determined by the P450 enzyme. Our previous
partner systems,10,11 including (1) a two-component system for
experience constructing and characterizing various self-
sufficient biosynthetic P450 enzymes (whose activity is
most bacterial and mitochondrial CYPs comprising a small
iron−sulfur redoxin and an FAD-containing redoxin reductase
and (2) a single FAD/FMN-containing CYP reductase that
independent of isolated redox partners) fused with the
RhFRED reductase domain14−16 also supports this “postulate”.
serves in eukaryotic microsomal P450s as a separate partner
protein or in a small number of bacterial CYPs (e.g., P450BM3
as a fused functional domain. Interestingly, nonclassical redox
)
Received: December 22, 2013
Published: February 12, 2014
© 2014 American Chemical Society
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Figure 1. Oxidation and demethylation of M-IV catalyzed by MycG using different redox systems (2 h reactions). (A) Scheme for MycG catalyzed
reactions. The novel demethylation product dMe-M-IV is shown in box. The introduced hydroxyl and epoxy groups are labeled in red. The
demethylated group is highlighted in blue. (B) Left panel: LC traces at 280 nm of different reaction extracts. The redox systems used are shown at
the left side of the corresponding traces. Mycinamicin derivatives are colored differently for clarity. Due to close polarity, M-V and M-II were
coeluted, and dMe-M-IV and M-I were coeluted. Since M-I and M-II lacking of the diene moiety only have weak absorbance at 280 nm, the
formation of products appears not to be proportional to substrate consumption. Right panel: MS/MS analysis of each mycinamicin compound (see
Figure S1 for explanations of the secondary mass spectra). (C) Comparison of the 1H NMR spectra (see Figures S2 and S3 for full spectra) of M-IV
and dMe-M-IV. The chemical shift and integrated peak area of the N-monomethyl group in dMe-M-IV are apparently different from those of the N-
dimethyl group in M-IV.
Notably, previous work revealed that cytochrome b5 is able
to modulate the bifunctional role of human CYP17A1 as a 17α-
hydroxylase or a 17,20-lyase in steroid biosynthesis.23 This well-
studied example demonstrates that the catalytic type of a P450
enzyme can be altered by a third-party protein effector via
alternative interactions among the P450 enzyme, P450
reductase, effector protein, and distinct substrates.24−27
However, there have been no reports for a redoxin protein or
a P450 reductase acting as the specific effector by itself to
endow the serving P450 enzyme with new catalytic activity.
In this work, novel demethylated mycinamicin products were
generated by P450 MycG supported by a stand-alone form of
the RhFRED reductase domain, as opposed to RhFRED fused
to MycG, thus challenging the generally accepted postulate.
Further engineering of RhFRED led to didemethylated
mycinamicin macrolides, demonstrating a novel route for
structural diversification of natural products. Mechanistically,
our results suggest that variant protein−protein interactions
with the redox partner may serve as a modulator of P450
function by dramatically affecting the substrate binding mode in
the P450 active site.
RESULTS
■
MycG is the multifunctional P450 monooxygenase involved in
the biosynthetic pathway of 16-membered ring macrolide
mycinamicins in the rare actinomycete Micromonospora
griseorubida.19,28,29 Physiologically, it catalyzes the C14
hydroxylation, and the C12C13 epoxidation of mycinamicin
IV (M-IV) leading to mycinamicin V (M-V) and mycinamicin I
(M-I), respectively. M-V is subsequently epoxidized by MycG,
giving rise to the major final product mycinamicin II (M-II),
while M-I cannot be further modified by MycG, thus
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Table 1. Quantitative Measurements of M-IV Conversions Mediated by Different MycG Catalytic Systems (2 h reactions)
a
relative AUC280 nm of oxidized
products
relative AUC280 nm of deMe- isolated yield of deMe-
rate constant (k,
min−1
coupling
b
IV
IV
)
efficiency
MycG + RhFRED
MycG + RhFRED-Fdx
MycG-RhFRED
22.1 1.1%
5.8 0.8%
54.1 2.0%
5.0 0.4%
27.9 1.4%
18.5%
24.0%
−
0.036 0.008
0.036 0.007
0.052 0.012
0.017 0.004
29.4 0.8%
24.2 0.9%
18.7 0.5%
34.5 1.5%
−
−
MycG-RhFRED-Fdx
−
21.3 0.7%
a
b
Numbers of relative AUC280 nm (areas under curve at 280 nm) were calculated from the integrated area of certain peaks on HPLC traces Coupling
efficiencies were calculated as the percentage of NADPH used for product formation over the total NADPH consumption
representing the other terminal product in the pathway (Figure
1A). All diglycosylated mycinamicins display strong antibiotic
activity against Gram-positive bacteria and mycoplasma,30 and
M-II has been developed into a veterinary anti-infective agent.31
The multifunctional activities of MycG were previously
reconstituted in vitro by using the commercial surrogate redox
partners spinach ferredoxin (Fdx) and spinach ferredoxin-
NADP+ reductase (FdR), since the native redox system driving
catalytic activity of MycG remains unknown.19 Motivated by
our recent work14−16 and that of others32−35 in enhancing
catalytic activity by fusing the RhFRED reductase domain to
the C-terminus of various biosynthetic P450s, we constructed
the MycG-RhFRED fusion protein. Similar to MycG supported
by separate spinach Fdx/FdR, this single-component self-
sufficient P450 system driven by NADPH achieved all
physiological reactions known for MycG (Figure 1B).
To investigate if the free form of the RhFRED domain can
support the catalytic function of MycG, the ability of this two-
component system to convert mycinamicin IV (M-IV) was
tested. Surprisingly, in addition to the expected oxidative
products, M-I, M-II, and M-V (coeluted with M-II), a new
product formed with 18.5% isolated yield (Table 1) with the
same retention time as M-I was observed in LC-MS analysis of
the reaction mixture (Figure 1B). Because of its strong
absorbance at 280 nm, resembling that of M-IV and M-V,
but distinct from M-I, we assumed that the chemical structure
of the new compound likely retained an intact diene moiety.
Indeed, its m/z value of 682.3 is 14 amu less than that of
protonated M-IV, suggesting a demethylated product, which
was also supported by high-resolution mass spectrometry ([M
+ H]+: obsd, 682.4161; calcd, 682.4160). Furthermore, the MS/
MS spectrum of the demethylated M-IV N-demethylated
mycinamicin IV (dMe-M-IV) showed two fragmentation ions
of 144.2 and 102.2, consistent with a demethylated desosamine
moiety, while the corresponding fragmentation ions for intact
desosamine are 158.2 and 116.115,36 (Figures 1B and S1).
Detection of formaldehyde in the reaction by Purpald reagent37
provides additional evidence for oxidative demethylation (see
Supporting Information). To confirm the site of demethylation,
the 1H NMR of enzymatically prepared dMe-M-IV was
analyzed using M-IV as reference (Figures 1C, S2 and S3).
As expected, the 2.35 ppm singlet peak of M-IV that
corresponds to the N-dimethyl group disappeared in the
spectrum of dMe-M-IV. Instead, a new singlet peak was
observed with half the integrated area at 2.72 ppm, indicating
the −NHMe group resulted from the MycG-catalyzed
demethylation.
between MycG and RhFRED. In all cases, both oxidative and
demethylated products were detected (Figure S4), indicating
the ability of MycG to catalyze demethylation is not the
consequence of a stoichiometric effect of RhFRED. A time
course of the reaction (Figure S5) showed proportional
accumulation of oxidative and demethylated mycinamicin
products.
Sequence analysis shows that RhFRED consists of two
functional domains, the FMN domain and the Fe2S2
domain.38,39 To analyze the impact of each domain on
demethylation by MycG, the Fe2S2 domain of RhFRED,
which is presumably involved in direct interaction with the
P450 enzyme, was exchanged for the spinach Fdx, yielding a
hybrid Rhodococcus-spinach reductase, RhFRED-Fdx. When
partnered with this hybrid reductase, the MycG-catalyzed
reaction produced even greater amounts of dMe-M-IV (24.0%
isolated yield, see Table 1), while the oxidized products M-I, M-
V, and M-II were also detectable (Figure 1B). This is in
contrast to the MycG-RhFRED-Fdx fusion system which,
similarly to MycG-RhFRED, generated only oxidized products,
albeit in lower quantities. These results suggest that MycG-
mediated demethylation is mainly determined by the mode of
interaction between MycG and its redox partner rather than by
the redox partner itself.
Quantitatively, by fitting the substrate consumption data into
the one phase exponential decay curve, the rate constants (k) of
the four studied MycG catalytic systems were determined
(Table 1 and Figure 2A). In comparison, isolated RhFRED and
RhFRED-Fdx served MycG with close efficiency despite
distinct product profiles. The M-IV oxidation mediated by
MycG-RhFRED and MycG-RhFRED-Fdx demonstrated the
apparent reaction velocity at 500 μM of M-IV being 26.0 6.0
μM min−1 and 8.5 2.0 μM min−1 (k × [S]), respectively. The
steady-state Michaelis−Menten kinetics of MycG could not be
ascertained due to multiple reactions occurring simultaneously.
It is well-known that the use of an unnatural redox partner in
a CYP catalyzed reaction likely uncouples the electron
generation upon NAD(P)H consumption and P450 product
formation.40−42 Separation of the fused P450 and reductase
domains could have a similar effect.43 The uncoupling process
generates reactive oxygen species (ROS) such as superoxide
−
anions (O2 ) and H2O2, which could direct the P450 catalytic
route into the “peroxide shunt pathway”.1,2 To examine
whether the demethylation of mycinamicins is a consequence
of the peroxygenase activity by undergoing this shunt pathway,
we first determined the NADPH coupling efficiency of the four
catalytic systems using M-IV as substrate. As shown in Table 1,
the coupling efficiencies for P450-reductase fusions (18.7% for
MycG-RhFRED and 21.3% for MycG-RhFRED-Fdx) were
lower than those of corresponding reactions using separated
reductases (29.4% for MycG + RhFRED and 24.2% for MycG
+ RhFRED-Fdx). These data indicate that separation of
This observation is intriguing because a heretofore unknown
dMe-M-IV product was obtained following separation of the
previously fused protein domains, MycG and RhFRED. Next,
we measured the oxidation and demethylation activities of
MycG under a variety of molar ratios (1:1, 1:10, and 1:100)
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Next, we comparatively tested the activity of MycG, driven
by each of the four different redox systems, against M-IV
analogues M-I, II, III, and V, isolated from fermentation
cultures of wild-type or mutant Micromonospora griseorubi-
da.30,45 The two fused systems displayed the typical
physiological set of the hydroxylation and/or epoxidation
products (Figure 3). However, the reactions driven by free
redox partners gave rise to more diversity of demethylated
products. Specifically, both RhFRED and RhFRED-Fdx
efficiently supported the demethylation of M-I and M-III by
MycG, generating the new products dMe-M-I and dMe-M-III,
respectively. M-II and M-V were demethylated by MycG at a
low level driven by RhFRED-Fdx, but not by RhFRED. Of
particular interest, MycG partnered by the stand-alone form of
RhFRED-Fdx was able to further remove the second N-methyl
group from monodemethylated dMe-M-I and dMe-M-III,
leading to the unique double-demethylated product d2Me-M-
I and d2Me-M-III (Figure 3), respectively. Previously, in both
the in vivo30,45 and in vitro reactions that employed the
surrogate spinach Fdx/FdR system,19 M-I and M-II have been
shown to be the two end products of the mycinamicin
biosynthetic pathway, and M-III bearing the monomethoxy
sugar javose is oxidized by MycG at a very low level. Here, the
change of the redox partners and their interaction mode has led
to seven novel demethylated products (Figures 1, 3, and S8),
demonstrating a new route for structural diversification of
natural products.
DISCUSSION
Figure 2. (A) Enzymatic consumption of M-IV (calculated from
HPLC areas under curve, AUC) fit into the one phase exponential
decay curve. The rate constants (k) are shown in Table 1. (B) The
effects of catalase (20 U), superoxide dismutase (2 U), ascorbate (10
mM), and the combination of these three ROS scavengers on the
activity of MycG/RhFRED against M-IV. Solid bar is the ratio of
demethylated product/oxidized products relative to that of the control
reaction without addition of any scavengers, and open bar is the overall
conversion percentage calculated based on the substrate M-IV
consumption. The percentage numbers from the control reaction are
arbitrarily assigned to be 100%.
■
In this study we show for the first time the ability of the
multifunctional P450 monooxygenase MycG to catalyze N-
demethylation of various mycinamicin natural product
molecules. This novel functionality of MycG occurs when the
monooxygenase is partnered with either the free Rhodococcus
reductase domain, RhFRED, or the Rhodococcus-spinach hybrid,
RhFRED-Fdx. In contrast, no similar demethylation activity
was observed with the corresponding fused redox partner
systems. The minor differences in the NADPH coupling
efficiency upon P450-reductase domain separation (Table 1)
and dose-independent effects of catalase, SOD, and the
chemical radical scavenger ascorbate on both MycG activity
and the demethylation/oxidation product distribution (Figure
2B) strongly suggest that the unnatural demethylation activity
is primarily due to heme-iron-oxo catalysis, as opposed to the
peroxide shunt pathway. Thus, this finding highlights a broader
role of redox partner protein−protein interactions in
modulating catalytic activity of the P450 enzyme. It is well-
known that alternative or surrogate redox partner protein(s)
might not provide the optimal supporting activity toward
mismatched P450 enzymes17,19,22 and could influence product
distribution.14,16 The modulating effect of a third party protein,
cytochrome b5, on the hydroxylase/lyase bifunction of
CYP17A1 supported by cytochrome P450 reductase23−26 has
also been demonstrated. However, to the best of our
knowledge, the entirely new catalytic activity of a P450 enzyme
associated with an alternative redox partner is unprecedented.
The novel catalytic activity demonstrated by MycG likely
stems from the poor accessibility of the substrate binding site,
as evidenced by the X-ray structure analysis of the MycG-
substrate complexes.46 Thus, the bulky and conformationally
restrained mycinamicin substrates were largely unable to
penetrate the L-shaped binding site to adopt a catalytically
productive binding mode. NMR modeling using paramagnetic
reductase and P450 domains did not result in greater
uncoupling in these P450 catalytic systems.
Moreover, catalase, superoxide dismutase (SOD), ascorbic
acid, and the combination of these three ROS scavengers were
added into the MycG/RhFRED/M-IV reaction to remove ROS
possibly generated from the uncoupling process. Although
these ROS scavengers lowered the M-IV conversion (except for
ascorbate) as well as the ratio of demethylated product to
oxidized products (except for SOD) in the majority of reactions
(Figure 2B), these effects were not dose dependent, and the N-
demethylation activity remained even at the highest concen-
tration of catalase, SOD, and ascorbate (Figure S6).
These results, together with the small change of coupling
efficiency upon reductase domain separation, suggest that dMe-
M-IV is unlikely to be a product of the peroxide shunt pathway.
However, the lowered demethylated product/oxidized product
ratio implies superoxide species might still contribute to some
of the observed demethylation. This could be supported by
detection of a low level of H2O2 in MycG reactions (Figure S7)
and the fact that addition of ascorbic acid improved the overall
conversion of M-IV (Figure 2B) since this chemical scavenger
is capable of protecting the enzyme and substrate/products
from radical damage by superoxides.44
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Figure 3. LC-MS analysis of the MycG reactions using M-I, M-II, M-III, and M-V as substrates. The structures of substrates and products are shown
on top. The reactions catalyzed by the same enzyme(s) are aligned in each row. The reactions using the common substrate are arranged in the same
column. The LC traces of M-I and M-II were recorded at 240 nm. The LC traces of M-III and M-V were recorded at 280 nm. In the M-V reactions
catalyzed by two fusion MycG enzymes, the accumulation of M-II (seen at 240 nm) that is coeluted with the unreacted M-V is shown in insets. The
selected MS/MS results are displayed in insets, whose colors are consistent with those of the corresponding LC peaks. The asterisked numbers
indicate the mass for the secondary ion fragment of double-demethylated mycinamicins (Figure S1). The structure of M-IX (the hydroxylated M-III
labeled by the diamond symbol) is shown in Figure S8.
restraints suggests that displacement of a number of the protein
secondary structure elements takes place in order to
accommodate mycinamicin substrates in an orientation
conducive to the observed oxidation pattern.46 By analogy to
P450cam47,48 and the human P450 3A4,49 reorganization of the
MycG active site may be driven by binding of the redox partner.
As has been recently demonstrated by elegant work from the
Poulos laboratory,48 binding of the endogenous redox partner
putidaredoxin stabilizes the “open” conformation of P450cam,
facilitating substrate access to the active site.
Consistent with the structurally resolved MycG-substrate
complexes,46 mycinamicins exhibiting pseudo two-fold symme-
try enter the active site in one of the two possible orientations:
“mycinose-in-desosamine-out” (Figure 4A) or “desosamine-in-
mycinose-out” (Figure 4B). The desosamine-in-mycinose-out
entry results in the catalytically nonproductive binding mode
represented by the M-III costructure (PDB ID code 2YCA).
Remarkably, the mycinose-in-desosamine-out entry in the
absence of the redox partner resulted in both the M-IV and
M-V substrates being stalled in the intermediate nonproductive
binding pose in all the structurally resolved complexes.46 If
substrate relocation to the catalytically competent mode is
indeed initiated by specific interactions with the endogenous
redox partner, the alternative surrogates may to some extent
mimic this allosteric effect, either facilitating or hampering
substrate progression to the catalytic site. Tethered to MycG,
both RhFRED and RhFRED-Fdx afforded physiological
product profiles, with the qualification that the product yield
was low for MycG-RhFRED-Fdx. Alternatively, when used as
stand-alone redox partners, both the RhFRED and RhFRED-
Fdx domains produced demethylated (RhFRED) or dideme-
thylated (RhFRED-Fdx) products of not only the native MycG
substrates M-IV and M-V but also the M-III precursor, an
otherwise poor substrate for MycG oxidation, which was
efficiently demethylated (Figure 3).
To explain this phenomenon we speculate that while the
electron transfer between MycG and the surrogate redox
partners is retained, the allosteric effect facilitating substrate
progression to the normal catalytically competent binding
mode (“mycinose-in-desosamine-out”) is likely diminished, if
not entirely lost. This might be due to intrinsically low affinity
between the surrogate partners and MycG, particularly if
concentration dependence in binary complex formation
becomes a rate-limiting issue in the bimolecular reaction.
Also, loss of the positioning effect of the linker following
domain separation may be a factor in changing the interaction
mode of free reductase and P450 domains. We conclude that in
the absence of the allosteric influence of the redox partner,
mycinamicins are more inclined to be stalled in the “desos-
amine-in-mycinose-out” noncatalytic entry pose, increasing the
probability of N-demethylation occurring in an otherwise
atypical reaction site.
Despite discovery of a new N-demethylation activity for
monooxygenase MycG, caution should be exercised in
interpreting reconstituted P450 activity generated in a surrogate
redox system, particularly until the generality of this approach is
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Figure 4. Dual binding modes of the mycinamicin substrates in the active site of MycG. (A) A putative binding pose of M-IV leading to physiological
C14 hydroxylation or/and C12−C13 epoxidation, which might be derived from the noncatalytic “mycinose-in-desosamine-out” entry pose as
observed in the 2Y98 (PDB ID) structure. (B) A putative “desosamine-in-mycinose/javose-out” binding pose yielding N-demethylation is likely to
be induced or stabilized by protein−protein interactions between MycG and separate redox partners. The noncatalytic conformation observed in the
2YCA structure may be derived from this pose. The mycinamicin chemical structures are simplified for clarity. Oxygen atoms are shown in red and
nitrogen in blue. Heme is shown in dark green. The reactive sites are asterisked. Slice through the MycG binding site shows M-IV or M-III in
virtually orthogonal orientations experimentally observed in the crystal structures.46 Protein surface is colored by hydrophobicity, hydrophobic areas
are in orange, and hydrophilic areas in blue.
further investigated. However, an interesting hypothesis is
suggested: P450 enzymes could be even more versatile in vivo
than previously considered, because these biocatalysts might
interact with a variety of redox partners to gain alternative
activities. This may impart evolutionary advantages to the host
organisms through detoxifying a more diverse range of
xenobiotics or synthesizing more secondary metabolites to
adapt to ever-changing environments. Clear indications that the
P450-omes of microbes (e.g., Streptomyces coelicolor)17,22 are
capable of interacting with a limited set of redox partners are
consistent with a broader role for redoxin-mediated selectivity
in these fascinating enzyme systems.
ACKNOWLEDGMENTS
■
This work was supported by funding from “Recruitment
Program of Global Experts, 2012” (S.L.) and NIH grant
GM078553 (D.H.S. and L.M.P.). We are also grateful for the
supports from National Natural Science Foundation of China
(NSFC 31270855 to S.L. and NSFC 31300075 to W.Z.). We
thank Dr. Norihiko Misawa for generously providing the
construct pRED containing the DNA sequence encoding
RhFRED, and Mr. Potter Wickware for his attentive reading
of the manuscript.
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ASSOCIATED CONTENT
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* Supporting Information
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Experimental methods, DNA sequences for the synthesized fdx
gene and the gene encoding the RhFRED-Fdx hybrid protein,
NMR spectra, the data for effects of catalase, superoxide
dismutase, and ascorbate on MycG activity. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Authors
davidhs@umich.edu
lishengying@qibebt.ac.cn
Notes
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The authors declare no competing financial interest.
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