Repurposing Nonheme Iron Hydroxylases to Enable Catalytic Nitrile Installation through an Azido Group Assistance

Article abstract of DOI:10.1021/jacs.8b13906

Three mononuclear nonheme iron and 2-oxoglutarate dependent enzymes, l-Ile 4-hydroxylase, l-Leu 5-hydroxylase and polyoxin dihydroxylase, are previously reported to catalyze the hydroxylation of l-isoleucine, l-leucine, and l-α-amino-δ-carbamoylhydroxyvaleric acid (ACV). In this study, we showed that these enzymes can accommodate leucine isomers and catalyze regiospecific hydroxylation. On the basis of these results, as a proof-of-concept, we demonstrated that the outcome of the reaction can be redirected by installation of an assisting group within the substrate. Specifically, instead of canonical hydroxylation, these enzymes can catalyze non-native nitrile group installation when an azido group is introduced. The reaction is likely to proceed through C - H bond activation by an Fe(IV)-oxo species, followed by azido-directed C-N bond formation. These results offer a unique opportunity to investigate and expand the reaction repertoire of Fe/2OG enzymes.

Full text of DOI:10.1021/jacs.8b13906

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Communication
Repurposing non-heme iron hydroxylases to enable catalytic
nitrile installation through an azido group assistance
Madison Davidson, Meredith McNamee, Ruixi Fan, Yisong Guo, and Wei-chen Chang
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b13906 • Publication Date (Web): 13 Feb 2019
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Repurposing non-heme iron hydroxylases to enable catalytic
nitrile installation through an azido group assistance
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,b
,a
Madison Davidson, Meredith McNamee, Ruixi Fan, Yisong Guo,* Wei-chen Chang,*
a
b
Department of Chemistry, North Carolina State University, Raleigh, NC 27695 Department of Chemistry, Carnegie
Mellon University, Pittsburgh, PA 15213
Supporting Information Placeholder
ABSTRACT: Three mononuclear non-heme iron dependent
enzymes, L-Ile 4-hydroxylase, L-Leu 5-hydroxylase and polyoxin
dihydroxylase, are reported to catalyze the hydroxylation of L-
isoleucine, L-leucine and L-α-amino--carbamoylhydroxyvaleric
acid (ACV). In this study we showed that these enzymes can
accommodate leucine isomers and catalyze regiospecific
hydroxylation. Based on these results, as a proof-of-concept, we
demonstrated that the outcome of the reaction can be redirected by
installation of an assisting group within the substrate. Specifically,
instead of canonical hydroxylation, these enzymes can catalyze
non-native nitrile group installation when an azido group is
introduced. The reaction is likely to proceed through C-H bond
activation by an Fe(IV)-oxo species, followed by azido-directed
C≡N bond formation. These results offer a unique opportunity to
investigate and expand the reaction repertoire of Fe/2OG enzymes.
Me
Me
Me
CO H
IDO
Me 4
CO H
2
2
NH2
L-isoleucine
OH NH2
1
LdoA
5
Me
CO H
CO H
2
2
HO
Me NH2
Me NH2
L-leucine
2
O
O
OH
PolL
CO H
4
CO H
2
2
H N
2
O
H N
2
O
3
ACV
NH2
OH NH2
Figure 1. Hydroxylation catalyzed by IDO, LdoA and PolL.
For this initial study, we focused on three enzymes, L-Ile 4-
hydroxylase (IDO), L-Leu 5-hydroxylase (LdoA) and polyoxin
Amino acids are an important and widely used class of small
molecules in chemical and biochemical applications. However, the
reactive nature of the carboxylate and amine moieties of amino
acids hampers the development of synthetic approaches to directly
utilize these molecules as reaction substrates. In contrast, amino
acids are common substrates for enzymatic transformations found
in nature. For example, mononuclear non-heme iron and 2-
oxoglutarate dependent (Fe/2OG) enzymes have been reported to
utilize amino acids as substrates to carry out regio- and stereo-
dihydroxylase (PolL), which are known to hydroxylate L-leucine
isomers and L-α-amino--carbamoylhydroxyvaleric acid (ACV)14-
18
(
Figure 1). During Fe/2OG enzyme catalyzed hydroxylation, the
reaction is initiated by a reaction between Fe(II) center with O and
OG to form an Fe(IV)-oxo (Fe(IV)=O) species, succinate and
2
2
2
CO . The reaction is followed by C-H bond activation, or so called
hydrogen atom transfer (HAT), to produce an Fe(III)-OH
intermediate and an substrate radical. Formation of a C-O bond
though the hydroxyl rebound mechanism completes the
1
-4
specific hydroxylation.
1-4
hydroxylation and reforms the Fe(II) species (Scheme 1a). We
In Fe/2OG enzymes, non-native reactivity is generally achieved
by changing the coordinating ligand or amino acid residues around
the iron center of the enzyme. For example, in the halogenase,
SyrB2, replacement of a halide that is coordinated to the iron center
with an azide altered the reaction outcome from halogenation to
reasoned that if the minimum substrate structural requirements to
maintain efficient substrate binding were achieved, a functional
group, such as N , could be appended onto the substrate to interact
3
with the intermediate, and redirect the reaction outcome. (Scheme
1b).
5
azidation. A second example is the structure-guided mutation
We first established substrate flexibility and reactivities of these
enzymes. IDO, LdoA and PolL were overexpressed and purified
6
study that converted SadA, a hydroxylase, into a halogenase. In
contrast, for cytochrome P450 enzymes, non-native chemical
(see SI). To test the activity and the substrate promiscuity of these
transformations, such as C-C, C-N, or C-Si bond formations, have
enzymes, an anaerobic reaction mixture containing enzyme (IDO,
LdoA or PolL), Fe(II), 2OG and substrate (1, 2, or 3) with final
concentrations of 110 µM enzyme, 100 µM Fe(II), 1.0 mM 2OG,
and 0.5 mM of substrate was prepared. The reaction mixture was
7-10
been achieved by using substrate analogs.
Even though
redirecting reaction outcomes using substrate analogs has been
reported in Fe/2OG enzymes, e.g. replacing L-Thr with L-norvaline
1
1
changes reactivity of SyrB2 from chlorination to hydroxylation,
2
then exposed to air for 20 minutes to react with O . Analysis by
expanding Fe/2OG enzyme reaction repertoire and investigating
plausible reaction pathways using a substrate-oriented approach
has not been carefully elucidated. Inspired by the mechanistic
liquid chromatography coupled mass spectrometry (LC-MS)
revealed that IDO and LdoA have a strong preference for catalyzing
C4- or C5-hydroxylation (m/z: 132.1  148.1) of L-leucine
isomers, respectively; with the exception that L-isoleucine (1)
cannot be converted by LdoA. These results are consistent with
3
, 12-13
understanding of Fe/2OG enzyme catalyzed hydroxylation,
herein, as a proof-of-concept, we used an azido group as the
assisting group to divert the reaction outcome from hydroxylation
to nitrile formation on an amino acid substrate in three Fe/2OG
hydroxylases.
16-18
previously reported IDO and LdoA reactivity.
In addition, PolL
shows high fidelity for C4-hydroxylation towards all tested leucine
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isomers with equal or better efficiency than those of IDO or LdoA
under the current conditions (Figure 2 and S21a).
hydroxylases.13, 19 A reaction mixture containing 0.6 mM PolL,
0.54 mM Fe(II), 12 mM 2OG, and 3 mM 3 was prepared
anaerobically. The reaction was initiated by mixing the solution
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6
H
R
a
R
H
CO2-
containing the PoIL•Fe(II)•2OG•3 complex (the enzyme
O2
CO2-
R
H
R
OH
D/N
O
FeIV
O
o
succ
succ
quaternary complex) with O saturated buffer at 5 C. A rapid
FeII
O
(succ)
2
O
FeIII
FeII
H
O
depletion of an absorption feature centered at ~ 520 nm was
H
(2OG)
O
O
detected up to ~ 0.1 s, which was followed by an almost complete
recovery of this spectral feature after ~ 2.0 s. Meanwhile, a spectral
feature centered ~ 320 nm developed to a maximum within ~ 0.03
s and subsequently decayed (Figure 3). Compared with the pre-
steady state kinetics reported on several characterized Fe/2OG
enzymes, the former absorption feature (~520 nm) decay can be
CO2
b
H
H
H
R
H
R
N3
R
N3
HO
O
R
FeIV
N
H
i-a
OH
N
FeIV
FeII
FeII
H2O
C-H
OH
activation
-
OH
N2
0
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H
R
II
rebound
HO
R
H
attributed to the kinetics of Fe -2OG metal-to-ligand charge
N3
i-b
transfer band, which reflects the rapid reaction of the enzyme
OH
FeIII
R
N
R
N
13,
N
N
N
FeII
H2O
quaternary complex with O
2
(the depletion of the 520 nm band).
E.T.
1
9-21
H
It is then followed by reformation of the PoIL•Fe(II)•2OG
HO FeII
R
ii-a
N
N
N
complex (the reformation of the 520 nm band). The latter
absorption feature (~320 nm) change can be attributed to the
H
+
H2O
H ,N2
H2O
H
R
1
9, 22
kinetics of the Fe(IV)=O intermediate.
using a three step kinetic model (PoIL•Fe(II)•2OG•3 + O
Fe(IV)=O  PoIL•Fe(II)•product  PoIL•Fe(II)•2OG•3) derived
A kinetic simulation
ii-b N2
N
2

Me
OH
c
Me
CO2H
CO H
Me
CO2H
2
Me
5
-1 -1
-1
the following rate constants: k
=
1
~ 60 mM s ; k
61 s . The same model has been used to delineate the pre-steady
state kinetic behaviors of several Fe/2OG enzymes, such as TauD
2
= 172 s ; and k
3
1
Me NH2
CO H
2
NH2
NH2
3
-1
N3
4
CO2H
2
CO2H
1
3, 19, 23
NC
N3
and SyrB2.
Thus, PoIL catalyzed hydroxylation follows a
NH2
NH2
NH2
similar mechanism as that of other Fe/2OG enzymes where an
Fe(IV)=O species is used as the key intermediate to carry out HAT.
Combined with the LC-MS results, these observations suggest that
all three enzymes recognize the L-leucine isomers and can trigger
HAT to afford the corresponding hydroxylated products. In
addition, these enzymes are promiscuous toward substrates and
may serve as good candidates to test the azide-assisted reaction
outcome hypothesis.
4
5
6
N
CO2H
NH2
NC
CO2H
HO
7
NH2
8
Scheme 1. a. A generally accepted mechanism of the Fe/2OG
enzyme catalyzed hydroxylation. b. The possible pathways
for azido-directed nitrile group formation. c. Substrates and
products used in this study.
Figure 3. SF-Abs of PoIL reaction with 3. Left: Change in
absorbance at various times after mixing the PoIL•Fe(II)•2OG•3
with O
2
. The spectra were obtained by subtracting the spectrum at
0
.002 s. Right: Kinetic traces used to follow the kinetics of the
Fe(IV)=O intermediate (320 nm) and the quaternary complex (520
nm). Simulations are shown in black.
Based on the specific HAT site preference of IDO, LdoA, and
PolL, mechanistic probes with site-specifically incorporated azide
group were designed. Namely, the probes (4 and 6) having an azido
4 5
group appended next to C and C were prepared. Comparing to the
established hydroxylation profile, we anticipate that IDO, LdoA
and PolL can employ 4 and 6 as the substrates. During the IDO and
PolL reactions with 4, we envision that the reaction will likely
Figure 2. LC-MS chromatograms of IDO, LdoA and PolL
catalyzed regiospecific hydroxylation when 1-3 were used. IDO
and PolL catalyze C -hydroxylation and LdoA catalyzes C -
4 5
hydroxylation. All peaks are detected at the corresponding
hydroxylation m/z values.
4
proceed via C -HAT, and followed by hydroxylation. The Fe(II)
center would then engage with the azido group to form the
corresponding Fe(IV)-nitrene intermediate. A Fe(IV)-nitrene
intermediate has been recently reported in P450 enzyme catalyzed
Next, we carried out kinetics studies using stopped-flow
absorption spectroscopy to elucidate the reaction intermediates and
to illustrate that the hydroxylation carried out by PolL follows a
similar reaction mechanism as that of other Fe/2OG
2
4
C-N formation. Due to its high reactivity, the nitrene species
would rearrange to an oxime or an iminol and then be converted to
the corresponding nitrile 5 (pathway i, scheme 1b). Alternatively,
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the substrate radical can be stabilized by the azido group and further
1
2
3
4
5
6
7
8
9
converted to a carbocation species via an electron transfer step. In
this pathway, the cationic species can be used as the key
intermediate to trigger nitrile formation (pathway ii, scheme 1b).
For LdoA, similar pathways can be envisioned where 6 is converted
5
to 7 through C -HAT, respectively.
To test the hypothesis that a substrate analog can enable a
designed reaction outcome, enzymatic assays using 4 or 6 as the
substrate were carried out under analogous conditions as described
above. LC-MS analysis of the PolL catalyzed reaction with 4
showed a peak with the corresponding m/z value of the predicted
nitrile product (m/z: 145.1  115.1, Figure 4a). Meanwhile, no
1
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2
2
2
2
2
2
2
3
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3
3
3
3
3
3
3
4
4
4
4
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4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
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1
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hydroxylation could be detected. When 2OG or O was absent, this
putative nitrile product was not observed. To validate this result,
the nitrile product (5) was prepared synthetically and showed the
same retention time and isotope distribution as the product obtained
from the PolL reaction. Compound 4 was also tested with IDO and
LdoA. No obvious product formation, i.e. hydroxylation or nitrile
formation, was detected. Combined with the C
when 1-3 were used, PolL catalyzed nitirile formation likely
initiates from C -HAT. When LdoA was used, no reactivity was
due to the replacement of C , the preferred C-H activation site, by
the N . Following the same hypothesis, one would expect that
LdoA can convert 6 to the corresponding 7 via C -HAT, and PolL
and IDO will produce the corresponding C -hydroxylated product.
4
-hydroxylation
4
5
3
5
4
Figure 4. LC-MS chromatograms of IDO, LdoA and PolL reaction
with 4 and 6. (a) and (b) PolL and LdoA can catalyze nitrile 5 and
Indeed, when 6 was tested with LdoA, the major product detected
has an identical retention time and m/z value (m/z = 129.1) as the
synthetic nitrile 7 (Figure 4b). In contrast, the PolL reaction with 6
revealed a possible hydroxylated product (m/z: 159.1  171.1,
Figure 4c). No obvious hydroxylation or nitrile formation was
detected when IDO was employed. Combining with the
regiospecific hydroxylation results, it is likely that nitrile (5 and 7)
7
formation. (c) PolL is able to catalyze hydroxylation using 6. All
analytes are detected using selected ion monitoring.
To probe the possible reaction pathway, the oxime analog (8)
was prepared. We reasoned that if an oxime is an intermediate, one
should expect to observe a formation of the nitrile (5) when 8 is
incubated with PolL. A similar observation has been demonstrated
in a heme-containing enzyme, aldoxime dehydratase, and a Reiske-
cluster containing enzyme, toluene dioxygenase, where incubating
4 5
formation catalyzed by PoIL and LdoA is initiated by C -H and C -
H bond activation, respectively. The reaction is then followed by
the azido group directed nitrile formation. In contrast, IDO is
unable to employ either 4 or 6 as a substrate. Compared to the
the proposed oxime intermediates with enzymes produced the
steady state kinetics of LdoA catalyzed nitrile formation (K
M
= 1.30
25-28
corresponding nitriles.
In our case, no nitrile product (5)
-1
±
0.32 mM, kcat = 1.90 ± 0.18 min ), the observed kcat for nitrile
formation can be detected, even after 60 minute incubation with 8
Figure S20). The competition experiment using 8 and 2 suggests
that 8 binds to the active site of PolL and prevents hydroxylation of
(Figure S21b). Even though it is still possible that 8 binds to PoIL
M
formation is ~ 2 fold higher for PolL (K = 1.49 ± 0.65 mM, kcat =
(
-1
4.08 ± 0.74 min , Figure S22). Under the current conditions, PolL
and LdoA can catalyze nitrile formation of 4 and 6 with total
turnover numbers of ~ 150 and 180, respectively.
2
at different orientation than L-leucine isomers or 4, these results
imply that the pathway involving an oxime as the intermediate is
unlikely to operate in the PolL catalyzed nitrile formation and is
different from the proposed mechanism where the nitrile is
2
5-26
generated through an oxime intermediate.
In PolL and LdoA
reaction, the pathway involving an iminol or a cationic specie is
likely to be utilized.
In summary, these results represent the first example of using an
assisting group approach to alter the reaction outcome of Fe/2OG
enzyme catalysis from hydroxylation to a designed non-native
reaction outcome. Through initial studies, we propose that nitrile
formation likely initiates from regioselective C-H bond activation
by an Fe(IV)-oxo species. Next, the reaction may proceed through
2
hydroxylation. Followed by N departure, the nitrene species
rearranges to an iminol intermediate, which further converts to the
nitrile (pathway i-b, scheme 1b). Alternatively, subsequent to HAT,
2
9
the cation intermediate can be produced. The reaction is followed
by deprotonation and N
2
elimination to complete nitrile formation
(
pathway ii-a, scheme 1b). The second pathway is analogous to the
3
0-31
Schmidt reaction.
intermediate
The pathway involving an oxime
2
5-26
is unlikely to operate in PolL catalyzed nitrile
formation. This work reveals the plausibility to use the assisting
group approach to redirect reaction outcome. It also underscores
the importance of employing mechanistic understanding to
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Page 4 of 5
redesign substrates in order to enhance the generality of Fe/2OG
enzymes as potential biocatalysts.
positioning controls the partition between halogenation and hydroxylation
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2. Bollinger, J. M., Jr.; Chang, W.-c.; Matthews, M. L.; Martinie,
R. J.; Boal, A. K.; Krebs, C., Mechanisms of 2-Oxoglutarate-Dependent
Oxygenases: The Hydroxylation Paradigm and Beyond. In 2-Oxoglutarate-
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(
1
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS
Publications website.
Detailed experimental methods and Figures S1-22.
13.
Price, J. C.; Barr, E. W.; Tirupati, B.; Bollinger, J. M., Jr.; Krebs,
C., The first direct characterization of a high-valent iron intermediate in the
reaction of an alpha-ketoglutarate-dependent dioxygenase: a high-spin
FeIV complex in taurine/alpha-ketoglutarate dioxygenase (TauD) from
Escherichia coli. Biochemistry 2003, 42 (24), 7497-508.
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AUTHOR INFORMATION
Corresponding Author
14.
Qi, J.; Wan, D.; Ma, H.; Liu, Y.; Gong, R.; Qu, X.; Sun, Y.;
Deng, Z.; Chen, W., Deciphering Carbamoylpolyoxamic Acid Biosynthesis
Reveals Unusual Acetylation Cycle Associated with Tandem Reduction
and Sequential Hydroxylation. Cell. Chem. Biol. 2016, 23 (8), 935-44.
*
*
ysguo@andrew.cmu.edu
wchang6@ncsu.edu
15.
Shi, F.; Niu, T.; Fang, H., 4-Hydroxyisoleucine production of
recombinant Corynebacterium glutamicum ssp. lactofermentum under
optimal corn steep liquor limitation. Appl. Microbiol. Biotechnol. 2015, 99
(9), 3851-63.
Author Contributions
1
6. Hibi, M.; Kawashima, T.; Sokolov, P. M.; Smirnov, S. V.;
The manuscript was written through contributions of all authors.
All authors have given approval to the final version of the
manuscript.
Kodera, T.; Sugiyama, M.; Shimizu, S.; Yokozeki, K.; Ogawa, J., L-leucine
5-hydroxylase of Nostoc punctiforme is a novel type of Fe(II)/alpha-
ketoglutarate-dependent dioxygenase that is useful as a biocatalyst. Appl.
Microbiol. Biotechnol. 2013, 97 (6), 2467-72.
17.
Ogawa, J.; Kodera, T.; Smirnov, S. V.; Hibi, M.; Samsonova, N.
Notes
N.; Koyama, R.; Yamanaka, H.; Mano, J.; Kawashima, T.; Yokozeki, K.;
Shimizu, S., A novel L-isoleucine metabolism in Bacillus thuringiensis
generating (2S,3R,4S)-4-hydroxyisoleucine, a potential insulinotropic and
anti-obesity amino acid. Appl. Microbiol. Biotechnol. 2011, 89 (6), 1929-
The authors declare no competing financial interests.
ACKNOWLEDGMENT
3
1
8.
8.
This work was supported by North Carolina State University and
Carnegie Mellon University. M.M. acknowledges Undergraduate
Research Summer Grant at North Carolina State University.
Hibi, M.; Kawashima, T.; Kodera, T.; Smirnov, S. V.; Sokolov,
P. M.; Sugiyama, M.; Shimizu, S.; Yokozeki, K.; Ogawa, J.,
Characterization of Bacillus thuringiensis L-isoleucine dioxygenase for
production of useful amino acids. Appl. Environ. Microbiol. 2011, 77 (19),
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hydroxylation
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