Conversion of a Dehalogenase into a Nitroreductase by Swapping its Flavin Cofactor with a 5-Deazaflavin Analogue

Article abstract of DOI:10.1002/anie.201703628

Natural and engineered nitroreductases have rarely supported full reduction of nitroaromatics to their amine products, and more typically, transformations are limited to formation of the hydroxylamine intermediates. Efficient use of these enzymes also requires a regenerating system for NAD(P)H to avoid the costs associated with this natural reductant. Iodotyrosine deiodinase is a member of the same structural superfamily as many nitroreductases but does not directly consume reducing equivalents from NAD(P)H, nor demonstrate nitroreductase activity. However, exchange of its flavin cofactor with a 5-deazaflavin analogue dramatically suppresses its native deiodinase activity and leads to significant nitroreductase activity that supports full reduction to an amine product in the presence of the convenient and inexpensive NaBH₄.

Full text of DOI:10.1002/anie.201703628

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Accepted Article
Title: Conversion of a Dehalogenase to a Nitroreductase by Swapping
its Flavin Cofactor with a 5-Deazaflavin Analog
Authors: Qi Su, Petrina A Boucher, and Steve Rokita
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Angewandte Chemie International Edition
10.1002/anie.201703628
COMMUNICATION
Conversion of a Dehalogenase to a Nitroreductase by Swapping
its Flavin Cofactor with a 5-Deazaflavin Analog
Qi Su, Petrina A. Boucher & Steven E. Rokita*
Abstract: Natural and engineered nitroreductases have rarely
supported full reduction of nitroaromatics to their amine products and
more typically transformations are limited to formation of the
hydroxylamine intermediates. Efficient use of these enzymes also
requires a regenerating system for AD(P)H to avoid the costs
associated with this natural reductant. Iodotyrosine deiodinase is a
member of the same structural superfamily as many nitroreductases
but does not directly consume reducing equivalents from NAD(P)H
nor demonstrate a nitroreductase activity. However, exchange of its
flavin cofactor with a 5-deazaflavin analog dramatically suppresses
its native deiodinase activity and gains a significant nitroreductase
activity that supports full reduction to an amine product in the
electron transfer and concurrently activate hydride transfer
[
21]
(Scheme 1).
The resulting mutant IYD also gained an NR
activity as if there is a mechanistic imperative between one- and
two-electron transfer steps and catalysis of dehalogenation and
nitroaromatic reduction, respectively.
4
presence of the convenient and inexpensive NaBH .
Scheme 1. An amino acid switch changes the function of IYD.
Enthusiasm for engineering new enzymes to reduce
nitroaromatic compounds has grown with their increasing
[
1]
contributions to produce fine chemicals, facilitate medical
[
2,3]
[4-6]
[7]
imaging,
toxins and pollutants from the environment.
method for generating the necessary catalysts involves either
activate prodrugs,
silence genes and remove
[
8,9]
The most direct
[
10-12]
mutation of existing nitroreductases (NR)
or mining genomic
Alternatively, oxido-
reductases with little or no propensity for reducing nitroaromatics
Scheme 2. One and two electron chemistry of FMN.
Scheme 3. Structures of the deazaflavin analogs.
[
13,14]
databases for new candidates.
[11]
may still be used as a foundation for developing novel NRs.
Previously, our laboratory converted an iodotyrosine deiodinase
IYD) into a NR by simply mutating a single amino acid that
(
helped to regulate the chemistry of its native FMN (Scheme 1).
This work began from a desire to identify the origins of
mammalian IYD and its relationship to other members of its
Conversion of IYD to NR illustrated a surprisingly facile
switch of activities between distinct enzymes within the nitro-
FMN reductase superfamily. However, this initial demonstration
did not yield a new NR with practical advantages over its
predecessors. Native NRs can be reduced directly by NAD(P)H
whereas IYD and its mutant require an alternative reductant
such as dithionite. NAD(P)H is ineffective as a reducing agent
for IYD in vitro and a purported NAD(P)H reductase for IYD in
vivo has not yet been identified. Use of dithionite precludes
continuous turnover of nitroaromatics since this reductant also
reacts with these substrates. Instead, dithionite has only been
used in stoichiometric amounts to reduce IYD prior to its assay
[15]
nitro-FMN reductase structural superfamily.
IYD is vital for
iodide conservation in organisms that generate thyroid
[
16,17]
hormones
and remarkable for its ability to promote reductive
dehalogenation, a process much more typical of anaerobic
rather than aerobic organisms.
The NR branch of this superfamily has received
considerable attention and has been defined in part by its lack of
[15,18]
sensitivity to molecular oxygen.
This characteristic is
consistent with a suppression of single electron transfer by FMN
[18,19]
(
Scheme 2)
and promotion of an alternative hydride
mechanism for reduction of nitroaromatics. IYD shares many
structural features with NR but instead stimulates the radical
chemistry of FMN. Dehalogenation depends on sequential
transfer of single electrons and participation of an intermediate
[21]
as an NR. Both the mutant IYD and most NRs also share the
limitation of generating hydroxylamine products rather than the
[1,10-13]
more desirable amines.
Many of these issues can now be
[
20,21]
semiquinone radical FMNH.
This pathway is promoted in
addressed through an alternative approach to engineering
catalysis. Substituting FMN in native IYD with its 5-deazaflavin
analog (5dFMN, Scheme 3) effectively generates a new NR that
supports continuous turnover of a nitroaromatic substrate to its
IYD by a substrate-induced alignment of the active site to
establish a hydrogen bond between a Thr side chain and the N5
[
20]
position of FMN for stabilizing the semiquinone FMNH.
Mutation of this Thr to an Ala is sufficient to inhibit single
4
amine product in the presence of NaBH as described below.
This work was inspired by many previous successes in
[
22]
manipulating enzyme activity by cofactor replacement. Flavin
analogs in particular have been very useful in studying reaction
[
a]
Q. Su, P. A. Boucher, Prof. S. E. Rokita
Department of Chemistry, Johns Hopkins University, 3400 N.
Charles St., Baltimore, MD 21218 (USA)
E-mail: rokita@jhu.edu
[23,24]
[25]
mechanisms
and developing new catalysts. The dramatic
change in redox properties offered by a switch from FMN to 5-
dFMN is now shown to support an equally dramatic change in
the catalytic activity of IYD.
Supporting information for this article is given via a link at the end of
the document
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Angewandte Chemie International Edition
10.1002/anie.201703628
COMMUNICATION
Human IYD lacking its N-terminal membrane anchoring
sequence was expressed and purified as described
Monitoring the discharge of electrons from the reduced
form of IYD (IYD•FMNH ) provides a complementary method for
2
[20]
previously. Its apoenzyme was generated with a step gradient
of guanidinium hydrochloride while bound to a nickel affinity
column to prevent precipitation. Reconstitution of IYD was
accomplished on the same column after removal of the
guanidinium hydrochloride and addition of FMN or its analogs,
alternatively. This reconstitution process did not significantly
affect the properties of IYD since binding and deiodination of
assaying enzyme activity. Such a method offered the first
evidence that the parent IYD promoted dechlorination and
debromination in addition to deiodination of the appropriate
[33,34]
halotyrosine.
Each of these substrates rapidly converted
IYD•FMNH to IYD•FMNox as evident by a characteristic gain in
2
absorbance at 450 nm. In contrast, F-Tyr did not promote a
similar response and was not processed by IYD. To test the
diiodotyrosine (I
2
-Tyr) remained similar for the parent IYD (wt)
latent NR activity in the parent IYD, nitrotyrosine (O
previously tested for an ability to oxidize IYD•FMNH
very limited reaction was observed under these conditions.
2
N-Tyr) was
and its FMN reconstituted derivative (Table 1). In contrast,
reconstitution of IYD with 5dFMN (IYD•5dFMN) suppressed the
deiodination activity by at least four orders of magnitude. This
2
. Only a
Oxidation of bacterial IYD•FMNH
2
stalled at its semiquinone
The same response was
[21]
result is not a consequence of poor binding since I
2
-Tyr exhibits
form (IYD•FMNH, Scheme 2).
a similar affinity for IYD•5dFMN and the FMN reconstituted IYD.
Instead, this result most likely reflects the dependence of
reductive dehalogenation involving single electron transfer, a
evident for human IYD used in the current investigation (Figure
S3 in the Supporting Information). A partial oxidation was also
observed for IYD•1dFMNH
2 2
after addition of O N-Tyr (Figure S4
[26]
process not supported by 5dFMN. The low reduction potential
in the Supporting Information).
[27]
[28]
of 5dFMN (-311 mV)
versus FMN (-205 mV)
might also
have been implicated in the loss of deiodinase activity. However,
this explanation seems less likely since reconstitution of IYD
with 1-deazaflavin (1dFMN) retained an ability to catalyze
2 m
deiodination of I -Tyr with a kcat/K value equivalent to almost
50% of that expressed by IYD reconstituted with FMN (Table 1).
The reduction potential of 1dFMN (-280 mV) is also lower than
that of FMN but this analog retains the ability to promote both
[26,29]
one- and two-electron transfers.
efficiency of deiodination by IYD containing 1dFMN
IYD•1dFMN) was anticipated since substitution of the polar N1
Some decrease in the
(
group of flavin with a CH group should disrupt interaction with an
Arg side chain that is highly conserved in both IYD and
[
15,30,31]
NR.
Loss of a flavin N1-Arg interaction in another
[
32]
flavoprotein had a profound effect on catalysis.
Table 1. Binding and Kinetic Constants for IYD Derivatives.
IYD (wt)
FMN
IYD reconstituted with
FMN
5-dFMN
75-90%
1-dFMN
cofactor
bound
9
6%
80-85%
75-90%
[
a]
K
d
I
2
-Tyr
[b]
[c]
1
.5 ± 0.2
1.4 ± 0.2
0.28 ± 0.01
0.33 ± 0.05
23 ± 3
0.11± 0.01
-
(
M)
[
d
a]
K
O
M)
2
N-Tyr
[c]
-
0.40 ± 0.002
-
(
k
cat
I
2
-Tyr
-5
-
1
0.48 ± 0.06
~ 3 x 10
0.097±0.008
16 ± 2
(s )
K
m
I
2
-Tyr
28 ± 8
--
--
(
M)
Figure 1. Stability and reactivity of IYD reconstituted with 5-dFMN as
monitored by UV-Vis spectroscopy. (A) The oxidized form of IYD
(IYD•5dFMNox, 55 μM) in 100 mM MES pH 6.0, 500 mM NaCl and 10%
glycerol was reduced to IYD•5dFMNH2 under aerobic conditions by a 5% aq.
k
cat/K
x10 M s )
m
I
2
-Tyr
4
-1 -1
1.7
1.4
0.6
(
NaHCO
conditions in the presence (IYD•5dFMNH
55 mM, IYD•5dFMNH /O ). (B) Alternatively, O
indicated concentrations to the reduced enzyme (IYD•5dFMNH
anaerobic conditions in 100 mM MES pH 6.0, 500 mM NaCl and 10% glycerol
at room temperature.
3
solution of NaBH
4
and then incubated for 60 min under ambient
/O + I -Tyr) and absence of I -Tyr
N-Tyr was added in the
,18 μM) under
a]
[
Values represent the average of three independent determinations of
2
2
2
2
ligand-dependent quenching of cofactor fluorescence and the error represents
the standard deviation. From Hu et al.
not fluorescent. See Figures S1 and S2 in Supporting Information for more
details.
(
2
2
2
[
b]
[20] [c]
Not determined since 1-dFMN is
2
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Angewandte Chemie International Edition
10.1002/anie.201703628
COMMUNICATION
Exchange of FMN with 5dFMN yielded a unique catalyst
that exhibits behavior quite distinct from its IYD parent. First,
Supporting Information). In general, nitroaromatics also do not
react with NaBH
such as Ag/TiO
of the corresponding amines.
4
spontaneously and instead require a catalyst
NaBH
4
is able to reduce IYD•5dFMNox but not the parent IYD
FMNox,its T173A mutant with FMNox (from
or IYD reconstituted with
dFMNox (Figures 1A and S5 in the Supporting Information).
Second, the reduced enzyme IYD•5dFMNH is stable to
2
or transition metal sulfides to promote formation
[40,41]
with
4
The inability of NaBH to
[
21]
Haliscomenobacter hydrossis)
reduce nitro- and hydroxylamine-containing aromatics provided
the first opportunity to test the continuous turnover of an IYD
1
2
derivative designed to act as an NR. Ten-fold excess O
and 100-fold excess NaBH were added to IYD•5dFMN (1 M).
Consumption of O N-Tyr rather than generation of H N-Tyr was
monitored by reverse-phase HPLC since the reduced product
2
N-Tyr
molecular oxygen unlike standard IYD (Figure 1A). This was
anticipated since 5dFMN is known to behave more like
4
2
2
[
35,36]
nicotinamide than flavin.
detectably oxidized over 60 min by I
vanishingly low rate of deiodination (Table 1, Figure 1A). Third
and most importantly, IYD•5dFMNH is efficiently and completely
oxidized by O N-Tyr in contrast to the results for the parent IYD
IYD•5dFMNH
2
was also not
[21]
2
-Tyr, a substrate with a
elutes with the void volume. Multiple turnovers were confirmed
by consumption of 3 M O N-Tyr after a 4 h incubation (Figure
2). In the absence of enzyme, consumption of less than 0.5 M
N-Tyr was observed over the same 4 h. Since NaBH
4
2
2
2
O
2
and its 1dFMN derivative (Figure 1B versus Figures S3A and
S4A in the Supporting Information). No spectral intermediates
decomposes under aqueous conditions, this reductant was
alternatively added hourly in four aliquots of 25 mM in an
otherwise equivalent incubation (Figure 2). Under these
were observed during oxidation of IYD•5dFMNH
IYD•5dFMNox Unlike initial reduction of IYD•5dFMNox
2
to
its
.
,
2
conditions, consumption of O N-Tyr increased to 6 M. This
subsequent oxidation was performed under anaerobic conditions
represents a minimum of 6 turnovers over 4 h if only the nitroso
intermediate had formed but more likely up to 18 turnovers if the
amine was produced. Obviously, this system does not yet offer
the efficiency required for most applications but it does represent
a very large gain of function over the immeasurable ability of
[37,38]
to limit potential side reactions of the intermediate products.
Titration with O N-Tyr suggests an ability to oxidize three
equivalents of IYD•5dFMNH . This stoichiometry offered the first
evidence that IYD•5dFMNH could produce the fully reduced
aminotyrosine (H N-Tyr). Prior mutation of IYD generated an NR
2
2
2
2
2
native IYD to reduce O N-Tyr (Figure S3 in the Supporting
equivalent containing FMN that was limited, like most all other
NRs, to the formation of a hydroxylamine product. Accordingly,
only approximately 2 equivalents of this mutant were consumed
Information). Significant increases in catalysis can be expected
in the future after the rate determining features of this process
are identified and addressed by engineering both the
polypeptide and cofactor.
[
21]
per substrate.
The stoichiometry of IYD•5dFMNH
confirmed by a complementary assay based on consumption of
N-Tyr as detected by reverse-phase HPLC (Figure S6 in the
2
oxidation was also
O
2
[21]
Supporting Information).
the chromatograms when added in excess of 0.33 equivalents
relative to IYD•5dFMNH . Comparable analysis with the reduced
form of either the parent IYD or its reconstituted 1dFMN
derivative suggested that N-Tyr was not consumed
irreversibly during incubation under anaerobic conditions.
Although the UV-vis spectra revealed a O N-Tyr-dependent
This substrate was only evident in
2
O
2
2
formation of the flavin semiquinone, no change in the
concentration of the substrate was evident after quenching and
HPLC separation under aerobic conditions (Figures S3B and
S4B in the Supporting Information). This behavior is reminiscent
of the oxygen-sensitive nitroreductases for which electrons pass
2
Figure 2. Catalytic reduction of O N-Tyr by IYD•5dFMN. Solutions in the
absence and presence of IYD•5dFMNox (1 M) in 100 mM MES (pH 6), 500
mM NaCl and 10% glycerol were reduced under anaerobic conditions (Ar) by
[39]
from enzyme to nitro organic compound to molecular oxygen.
In contrast, formation of N-Tyr by IYD•5dFMNH was
NaBH
sequential additions of 25 M each at 1 h intervals as indicated. Reaction
was initiated by O N-Tyr (10M) and terminated, alternatively, after 1.5 and 4
4 3
(final 100 M) from a stock in 5% aq NaHCO using a single addition or
H
2
2
4
confirmed after dansylation, HPLC separation and mass spectral
characterization (Figure S7 in the Supporting Information). Again,
2
h by addition of TFA to a final concentration of 1.5 % under aerobic conditions.
Precipitated protein was removed by centrifugation and the supernatant was
analyzed with an internal standard of m-cresol by reverse-phase HPLC (0.1 %
aq TFA, 10 min, with 0 - 45 % acetonitrile over a subsequent 10 min and 45 -
2
reaction of 3 equivalents of IYD•5dFMNH and one equivalent of
O
2
N-Tyr yielded only the fully reduced amino product.This
represents a very rare example of an NR equivalent that is
capable of generating an aromatic amine rather than its
100 % acetonitrile over a final 15 min, 1 ml/min).
[
13]
corresponding hydroxylamine intermediate.
Equivalent
reduction was also not supported by prior mutation of IYD that
suppressed the one-electron chemistry of its native FMN and
produced an NR-like catalyst.
Previously, a simple mutation of the parent IYD was able
to inhibit its native dehalogenase activity and gain an NR
activity reminiscent of other enzymes in the same structural
[
21]
[21]
superfamily. Now, a much more dramatic shift in activity has
been possible by the alternative approach of switching
cofactors from FMN to 5dFMN. The native deiodinase activity
The final reduction step converting the hydroxylamine to
amine is unlikely the result of excess NaBH since
phenylhydroxylamine is inert to this reductant (Figure S8 in the
4
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Angewandte Chemie International Edition
10.1002/anie.201703628
COMMUNICATION
is suppressed by many orders of magnitude after this
substitution and the NR activity becomes easily detectable.
Most significantly, a new NR capable of generating the fully
reduced aromatic amine has been created. The basis for the
strict correlation between the catalytic specificity within this
structural superfamily and the nature of its flavin chemistry is
not yet apparent. Certainly, other types of enzyme are capable
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two additional advantages beyond conferring the NR activity.
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could be used to replace the natural and, as of
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[
We thank David Ballou and Bruce Palfey for initial samples of
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Entry for the Table of Contents
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Qi Su, Petrina A. Boucher & Steven E.
Rokita*
Page No. – Page No.
Swapping Cofactors as an Alternative
to Engineering Proteins
A new catalyst from old: A dehalogenase has been reconstituted into a
nitroreductase by exchange of its native flavin cofactor for a 5-deazaflavin analog.
The resulting enzyme catalyzes full reduction of nitrotyrosine to aminotyrosine in
4
the presence of NaBH .
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