Mechanistic Investigation of Oxidative Decarboxylation Catalyzed by Two Iron(II)- and 2-Oxoglutarate-Dependent Enzymes

Article abstract of DOI:10.1021/acs.biochem.8b00115

Two non-heme iron enzymes, IsnB and AmbI3, catalyze a novel decarboxylation-assisted olefination to produce indole vinyl isonitrile, an important building block for many natural products. Compared to other reactions catalyzed by this enzyme family, decarboxylation-assisted olefination represents an attractive biosynthetic route and a mechanistically unexplored pathway in constructing a C=C bond. Using mechanistic probes, transient state kinetics, reactive intermediate trapping, spectroscopic characterizations, and product analysis, we propose that both IsnB and AmbI3 initiate stereoselective olefination via a benzylic C-H bond activation by an Fe(IV)-oxo intermediate, and the reaction likely proceeds through a radical- or carbocation-induced decarboxylation to complete C=C bond installation.

Full text of DOI:10.1021/acs.biochem.8b00115

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Communication
Mechanism investigation of oxidative decarboxylation catalyzed
by two iron(II) and 2-oxo-glutarate dependent enzymes
Jhih-Liang Huang, Yijie Tang, Cheng-Ping Yu, Dev Sanyal,
Xinglin Jia, Xinyu Liu, Yisong Guo, and Wei-chen Chang
Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00115 • Publication Date (Web): 27 Feb 2018
Downloaded from http://pubs.acs.org on March 3, 2018
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Biochemistry
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Mechanism investigation of oxidative decarboxylation catalyzed by
two iron(II) and 2-oxo-glutarate dependent enzymes
†
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JhihꢀLiang Huang, Yijie Tang, ChengꢀPing Yu, Dev Sanyal, Xinglin Jia, Xinyu Liu,* Yisong
,
‡
,†
Guo,* Weiꢀchen Chang*
†
Department of Chemistry, North Carolina State University, Raleigh, North Carolina 27695, United States
Department of Chemistry, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, United States
Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, United States.
‡
§
Supporting Information Placeholder
can be formulated for IsnB/AmbI3ꢀtype desaturases. We and othꢀ
ers have previously proposed a pathway involving HAT by a puꢀ
tative Fe(IV)ꢀoxo species followed by subsequent OHꢀrebound to
produce a hydroxylated intermediate, with dehydration assisted by
ABSTRACT: Two nonꢀheme iron enzymes, IsnB and AmbI3,
catalyze a novel decarboxylationꢀassisted olefination to produce
indole vinyl isonitrile, an important building block for many natuꢀ
ral products. Compared to other reactions catalyzed by this enꢀ
zyme family, decarboxylationꢀassisted olefination represents an
attractive biosynthetic route and a mechanistically unexplored
pathway in constructing a C=C bond. Using mechanistic probes,
transient kinetics, reactive intermediate trapping, spectroscopic
characterizations and product analysis, we propose that both IsnB
and AmbI3 initiate stereoselective olefination via a benzylic CꢀH
bond activation by an Fe(IV)ꢀoxo intermediate, and the reaction
likely proceeds through a radical or a carbocation induced decarꢀ
boxylation to complete C=C bond installation.
13, 14
decarboxylation to produce the olefin and CO2
(Scheme 1C,
pathway i). On the other hand, inspired by recent mechanistic
1, 2
studies of Fe/2OG enzymes, other mechanistic possibilities can
be envisioned such as those involving a substrate radical or a catiꢀ
on intermediate. Similar pathways have been suggested for the
15
cytochrome Pꢀ450 enzyme, OleT, the nonꢀheme iron enzyme,
16
17
UndA, and the radical SAM dependent enzyme, MftC
(Scheme 1C, pathway ii). A third possibility, although less likely,
may involve a pathway that utilizes OꢀH bond activation followed
by a CꢀC bond scission and a CꢀH bond cleavage. (Scheme 1C,
pathway iii). To elucidate the catalytic mechanisms of IsnB and
AmbI3, we prepared the substrate 1, its deuterated analog 2, the
product standards 3 and 4, and the amide analog 5. IsnB and Amꢀ
bI3 reactions were characterized using transient state kinetics,
Mössbauer spectroscopy and liquid chromatograph coupled mass
spectrometry (LCꢀMS).
Nonꢀheme mononuclear iron(II) and 2ꢀoxoꢀglutarateꢀdependent
(Fe/2OG) enzymes catalyze a broad range of oxidative transforꢀ
mations involved in the regulation and biosynthesis of cellular
1, 2
metabolites. Among these diverse transformations, the mechaꢀ
nism of hydroxylation has been most extensively investigated.
This process is initiated by Hꢀatom abstraction (HAT) by an
Fe(IV)ꢀoxo species, followed by a rapid CꢀO bond formation
between the substrate radical and Fe(III)ꢀOH species (the OHꢀ
Transient state kinetics via stoppedꢀflow optical absorption
spectroscopy (SFꢀAbs) were employed to reveal intermediates
during the IsnB and AmbI3 catalysis. Reactions were initiated by
rapid mixing of the anaerobic IsnB (or AmbI3)•Fe(II)•2OG•1 (or
2
o
rebound pathway, Scheme 1A). The chemistry of several less
2) complex with equal volume of oxygenated buffer at 5 C with
wellꢀstudied Fe/2OG enzymes, however, does not appear to utiꢀ
lize the “canonical” OHꢀrebound mechanism. Examples of such
final concentrations of ~ 0.5 mM O , 0.3 mM enzyme, 0.27 mM
2
Fe(II), 2.7 mM 2OG, and 0.68 mM substrate. The
IsnB•Fe(II)•2OG•1 complex exhibits a broad absorption band
centered at ~ 540 nm which can be assigned to the Fe(II)ꢀ2OG
metalꢀtoꢀligand charge transfer (MLCT) band commonly obꢀ
3
-5
transformations include the halogenation (e.g., CytC3, SyrB2
and WelO5), the desaturation involving cleavage of two CꢀH
6, 7
bonds (e.g., AsqJ and CarC), and the CꢀO bond formation leadꢀ
6
,
8,
9
18
ing to epoxide and endoperoxide
(e.g., AsqJ, H6H and
served with Fe(II)/2OG enzymes (Fig. S6). After mixing, the
Ftmox1). The oxidative decarboxylation, recently found in vinyl
isonitrile and ethylene production pathways, represent new types
MLCT band depletes over ~ 1 s and two stages of MLCT band
decay were observed (Fig. 1a). Before 0.1 s, the absorption deꢀ
creased near 600 nm with a concomitant increase in absorption
near 320 nm. The isosbestic point observed at ~ 480 nm in the
difference spectra suggests a conversion of the IsnB•Fe(II)•2OG•1
complex to a reactive intermediate. We tentatively assign this
intermediate as an Fe(IV)ꢀoxo species based on similar absorption
10
of Fe/2OG enzyme activity. Compared to ethylene production,
the underlying reaction mechanism of vinyl isonitrile biosynthesis
1
1, 12
is poorly understood.
We have recently shown that the isonitrileꢀcontaining Lꢀ
tryptophan precursor (1) is a substrate for both the transꢀindolyl
vinyl isonitrile (3) synthase IsnB and the cisꢀindolyl vinyl isoniꢀ
changes observed in several wellꢀcharacterized Fe/2OG
19, 20
1
1-13
enzymes.
In the second stage of MLCT decay (> 0.1 s postꢀ
trile (4) synthase AmbI3.
The stereoselective formation of 3
mixing), the features in the 400 – 700 nm region decrease, sugꢀ
gesting the disappearance of the IsnB•Fe(II)•2OG•1 and Fe(IV)ꢀ
oxo complexes. Simultaneously, the absorbance at 320 nm conꢀ
tinues to increase, which can be attributed to the formation of
product 3 (Fig. S16). The negative absorption feature of the
MLCT band after 1 s implies that the IsnB•Fe(II)•2OG•1 complex
and 4 catalyzed by IsnB and AmbI3 is accompanied by the net
loss of a hydride at the benzylic carbon of 1 and the elimination of
11-13
CO2
(Scheme 1B). The IsnB/AmbI3ꢀmediated olefination is
distinct from the reactions catalyzed by other Fe/2OG desaturases
where the C=C bond formation involves two consecutive CꢀH
2
bond cleavages. Toward this end, several mechanistic proposals
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was largely depleted. The partial reformation of Fe(II)ꢀ2OG
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MLCT band is further observed up to 10 s, which could be atꢀ
tributed to the reformation of IsnB•Fe(II)•2OG complex due to
the depletion of O and/or substrate.
2
A
R
R
OH
R
R
H
O
(succ)
-
2
CO
H
H
FeII
O2
O
Asp/Glu
His
O
O
-
succ
succ
CO
2
FeIV
O
FeIII
FeII
His
O
(2OG)
CO2
O
C-H
activation
C-O bond
formation
NC
B
O
IsnB
N
H
R
O
, H-
CO
2
H
3
4
O
NC
0
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0
O
R
NC
N
NC
AmbI3
H
1
NC
N
H
Figure 1. Kinetic evidence for the C ꢀH activation in the IsnB
catalysis. (A) Changes in absorbance at the indicated reaction
β
C
OH
O
+H
+
OH
2
O
-CO , -OH
2
2
H
O
O
O
i
Cβ-H
activation
R
O
R
O
times after mixing IsnB•Fe(II)•2OG•1 with O . The spectra at the
R
2
NC
O
NC
R
O
R
FeII
indicated time points were obtained by subtracting the spectrum at
0.002 s. (B) Kinetic traces at 400 nm is used to indicate the forꢀ
mation and the decay of the Fe(IV)ꢀoxo intermediate in the reacꢀ
tions using 1 (red) or 2 (blue), with simulations shown in black.
The SFꢀAbs results of AmbI3 reaction are shown in Figure S9.
NC
NC
HO
HO
O
FeIII
CN
FeIV
R
O
FeIII
OR
O
NC
ii
O
R
O
-CO2
HO
FeII
NC
R
O
NC
H
O
H
O
O
O-H
pathway
H
R
O
To corroborate the results obtained from the SFꢀAbs studies,
freezeꢀquench coupled Mössbauer experiments were performed
(see the SI for more discussion). The IsnB•Fe(II)•2OG•1 (or 2)
complex exhibited a quadrupole doublet in the Mössbauer specꢀ
trum measured at 4.2 K with isomer shift (δ) of 1.24 mm/s and a
R
O
R
O
NC
NC
NC
O
iii
HO
2
H O
FeIV
FeIII
FeII
D
O
O
D
D
D
O
O
D
O
NH
2
D
NC
NC
NC
quadrupole splitting (|ꢁE |) of 3.09 mm/s, which is typical for
N
N
H
D
N
H
Q
H
1
D
2
5
highꢀspin ferrous species (Fig. 2). In addition to this species
which represents ~ 75% of the total iron in the sample, the reꢀ
maining 25% of the iron in the sample belonged to a species exꢀ
Scheme 1. (A) Schematic representation of hydroxylation
catalyzed by Fe/2OG enzyme; (B) Left: decarboxylation-
assisted desaturation catalyzed by IsnB, AmbI3 and other
Fe/2OG enzymes. Right: outcomes of IsnB and AmbI3
catalyzed stereoselective vinyl isonitrile production; (C)
Possible pathways for decarboxylation-assisted olefination;
hibiting a quadrupole doublet with δ = 0.24 mm/s, and |ꢁE | =
Q
0.54 mm/s. The high field measurement revealed that this species
has a diamagnetic ground state (S = 0) with parameters resemꢀ
bling those of isonitrileꢀiron(II) complexes (Fig. S13, Table S3).
Therefore, we reasoned that this minor species originates from the
coordination of the isonitrile group of 1 to the Fe(II) center. Folꢀ
(D) Substrates and analog used in this study.
The timeꢀdependent changes of the absorption at 400 nm (Fig.
1b) were analyzed using a twoꢀstep kinetic model (enzyme subꢀ
lowing addition of O , it remains unchanged (Table S2). Thus it is
2
not involved in IsnB catalysis. The Mössbauer spectra of samples
quenched at various time points after mixing with oxygenated
buffer revealed the accumulation of an Fe(IV)ꢀoxo intermediate,
which is evidenced by the observation of a quadrupole doublet
strate complex + O → Fe(IV)ꢀoxo → enzyme product complex,
2
see the SI for more discussion) that has been used in other
19
Fe/2OG enzymes. The 320 nm trace normally selected to anaꢀ
19,21
lyze Fe(IV)ꢀoxo kinetics
is not used here due to the interferꢀ
with δ = 0.31 mm/s, and |ꢁE | = 0.92 mm/s. The Fe(IV)ꢀoxo
Q
ence of product absorption at ~ 320 nm (Fig. S16). The rate conꢀ
stants for the formation and decay of the Fe(IV)ꢀoxo intermediate
species accumulated to a maximum of ~ 28% of the total iron in
the sample quenched at 0.13 s and decayed to less than 4% at 1 s.
In addtion, a quadrupole doublet representing another highꢀspin
ꢀ
1
ꢀ1
ꢀ1
were simulated to be ~ 30 mM s and 4.0±0.2 s and are similar
to those of reported Fe/2OG enzymes, such as TauD, SyrB2 and
ferrous species with δ = 1.15 mm/s, and |ꢁE | = 3.24 mm/s, was
Q
4, 19, 21
CarC.
When 2 was used, the rate of Fe(IV)ꢀoxo formation
formed in concomitance with the decay of the Fe(IV)ꢀoxo species.
This quadrupole doublet is different from the substrate bound
complex and likely represents the IsnBꢀFe(II)ꢀproduct complex.
When 2 was used, the amount of Fe(IV)ꢀoxo species observed at
0.03 s was similar (~25%) with that in the case of 1; however, it
accumulated to a higher level (~ 45%) at 0.13 s, and hardly deꢀ
cayed at 1 s. In a sample quenched at 10 s, the Fe(IV)ꢀoxo species
still accounted for ~ 20% of the total iron. These observations are
consistent with the SFꢀAbs experiments results that a large H/D
KIE is observed on the decay of the Fe(IV)ꢀoxo species (Fig.
S10).
was not perturbed (Fig. 1, S7 and S8); however, the lifetime of the
Fe(IV)ꢀoxo intermediate was greatly extended (Fig. 1b, blue
trace) and did not fully decay up to ~ 30 s (Fig. S8). Thus, the
observed rate constant for the Fe(IV)ꢀoxo decay is reduced to
ꢀ
1
0.05±0.01 s . When AmbI3 was examined, analogous results
were obtained where the rate constants for Fe(IV)ꢀoxo formation
ꢀ
1
ꢀ1
ꢀ1
and decay were found to be ~ 55 mM s and 13±1 s or ~ 55
ꢀ
1
ꢀ1
ꢀ1
mM s and 0.25±0.05 s when 1 or 2 were used (Fig. S9). The
greater accumulation and slow decay of the Fe(IV)ꢀoxo species
when 2 was used is likely due to the H/D kinetic isotope effect
(H/D KIE of ~80 for IsnB and ~50 for AmbI3) on the CꢀH activaꢀ
tion step, which has been documented in several Fe/2OG enꢀ
2, 20, 21
zymes.
Together, these results support the hypothesis that an
Fe(IV)ꢀoxo species is responsible for initiating the benzylic CꢀH
activation in IsnB/AmbI3.
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Biochemistry
was used indicates that it is likely an offꢀpathway product during
1
2
3
4
5
6
7
8
9
IsnB reaction.
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1
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1
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0
1
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0
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0
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0
1
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3
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9
0
1
2
3
4
5
6
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8
9
0
Figure 2. 4.2 K zero field Mössbauer spectra of IsnB reaction. The
black hashed lines represent spectra of samples freeze quenched at
various time points by rapid mixing of IsnB•Fe(II)•2OG•substrate
(
1: left panel and 2: right panel) with O . The red lines, the green
2
Figure 3: LCꢀMS analysis of IsnB and AmbI3 catalysis. (a) Forꢀ
mation of 3 and 4 when reacting 1 with IsnB and AmbI3. (b) 1ꢀ
OH formation when 1 was reacted with IsnB or AmbI3 (m/z 213.1
lines, the blue lines and the purple lines represent the spectral
simulations of the Fe(IV)ꢀoxo species, the isonitrileꢀFe(II) comꢀ
plex, the enzymeꢀsubstrate complex, and the enzymeꢀproduct
complex. The black lines represent the overall simulations.
18
18
ꢀ 229.1, traces 1ꢀ4). In the presence of ~ 75% OH , no
O
2
incorporation was detected (m/z = 231.1, traces 5ꢀ8). (c) Forꢀ
mation of 3 and the 5ꢀOH product (m/z 212.1 ꢀ 228.1) (top and
bottom panels) when 5 was reacted with IsnB. Due to poor reacꢀ
tivity of 5, traces are enlarged by 20 times (20X).
Product distributions for IsnB and AmbI3 reactions with 1, 2
and 5 were characterized using LCꢀMS. Analogous to SFꢀAbs
experiment condition, the IsnB (or AmbI3)•Fe(II)•2OG complex
was incubated with the substrate anaerobically, and then mixed
with equal volume of oxygenated buffer with final concentrations
of 0.12 mM enzyme, 0.1 mM Fe(II), 0.5 mM substrate, 1.0 mM
In conclusion, our results suggest that the catalytic strategy utiꢀ
lizing Fe(IV)ꢀoxo intermediate to trigger benzylic CꢀH bond actiꢀ
vation is operative in IsnB and AmbI3 catalysis. The following
steps diverge from the canonical OHꢀrebound pathway where
desaturation is likely to proceed through a pathway involving a
benzylic radical or a benzylic carbocation intermediate. An analoꢀ
gous pathway utilizing single electron transfer to the Fe(III)ꢀOH
species to produce a substrate cation or a diꢀradical intermediate
2
OG, and ~ 0.5 mM O . After 5 minutes, the reactions were subꢀ
2
jected to LCꢀMS. In addition to the desaturated products (3 for
IsnB and 4 for AmbI3), a minor peak corresponding to a hydroxꢀ
ylated 1 (1ꢀOH) in both reactions was detected (mass shift of +16,
m/z = 229.1, Fig. 3a and b). The ratio of 1ꢀOH to 3 or 4 is ~ 1.0 :
5
.6. We reasoned that 1ꢀOH could originate from either a hydroxꢀ
ylated intermediate prior to decarboxylation (e.g., as in Scheme
C, pathway i) or an offꢀpathway product generated by quenching
of a reactive intermediate, such as a benzylic cation or radical
Scheme 1C, pathway ii). To distinguish these possibilities, we
has been proposed in the H
zyme, OleT, and O dependent nonꢀheme iron enzyme, UndA,
which imply a common reaction mechanism for decarboxylaꢀ
tionꢀassisted olefination may exist among different types of
/H activating enzymes.
O dependent cytochrome Pꢀ450 enꢀ
2
2
1
5,
1
2
16, 22
(
18
carried out the reactions in buffer enriched with Oꢀwater (~ 75%
of OH ). In both reactions, the ratio of O/ O product is larger
O
2
O
2 2
18
16
18
2
than 99/1 (Fig. 3b). Thus, the oxygen atom of 1ꢀOH is likely deꢀ
ASSOCIATED CONTENT
rived from O , which is consistent with OHꢀrebound pathway.
2
Supporting Information
Although this observation disfavors the pathway where quenching
of reactive species yields 1ꢀOH, it cannot distinguish whether 1ꢀ
OH is the intermediate used for decarboxylation or a product reꢀ
sulting from residual hydroxylase activity.
The Supporting Information is available free of charge on the
ACS
Publications
website.
Methods, Figures S1ꢀS16, Tables S1ꢀS3, and references (PDF)
To probe whether the hydroxylated species is an onꢀpathway or
an offꢀpathway product, an amide analog 5 was prepared. By
replacing the carboxylate with an amide that cannot easily underꢀ
go hydroxylationꢀinduced decarboxylation, we anticipate that the
IsnB/AmbI3ꢀcatalyzed production of 3/4 would decrease with 5
and the hydroxylated intermediate would accumulate if the hyꢀ
droxylation/decarboxylation pathway operates. In contrast, if
desaturation does not proceed through this pathway, we would not
observe increased level of the hydroxylated product. When 5 was
incubated with IsnB, 3 was still produced. Additionally, a peak
with the m/z value corresponding to hydroxylated 5 (5ꢀOH) was
detected (m/z = 228.1), and the ratio of the 5ꢀOH to 3 is ~ 1 : 6
(Fig. 3c). In the absence of 2OG, neither 3 nor 5ꢀOH was formed.
When AmbI3 was tested, no obvious substrate consumption and
product formation could be detected. Although IsnB can catalyze
production of 3 and 5ꢀOH. The reactivity is only ~ 5% compared
to 1. We speculate that the binding affinity of 5 to IsnB and Amꢀ
bI3 is very poor. While the LCꢀMS results suggest that the hyꢀ
droxylated product likely originates from an OHꢀrebound pathꢀ
way, the lack of enhancement of hydroxylated product when 5
AUTHOR INFORMATION
Corresponding Author
Eꢀmail:
wchang6@ncsu.edu,
ysguo@andrew.cmu.edu,
xinyuliu@pitt.edu
&
These authors contribute equally.
ACKNOWLEDGMENT
This work was supported by grants from North Carolina State
University, Carnegie Mellon University, and NSF CHEꢀ1654060
(to Y. G.).
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