Oxidative Decarboxylase UndA Utilizes a Dinuclear Iron Cofactor

Article abstract of DOI:10.1021/jacs.9b02545

UndA is a nonheme iron enzyme that activates oxygen to catalyze the decarboxylation of dodecanoic acid to undecene and carbon dioxide. We report the first optical and M?ssbauer spectroscopic characterization of UndA, revealing that the enzyme harbors a coupled dinuclear iron cluster. Single turnover studies confirm that the reaction of the diferrous enzyme with dioxygen produces stoichiometric product per cluster. UndA is the first characterized example of a diiron decarboxylase, thus expanding the repertoire of reactions catalyzed by dinuclear iron enzymes.

Full text of DOI:10.1021/jacs.9b02545

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Communication
The oxidative decarboxylase UndA utilizes a dinuclear iron cofactor
Olivia M. Manley, Ruixi Fan, Yisong Guo, and Thomas M. Makris
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b02545 • Publication Date (Web): 14 May 2019
Downloaded from http://pubs.acs.org on May 14, 2019
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The oxidative decarboxylase UndA utilizes a dinuclear iron cofactor
Olivia M. Manley,† Ruixi Fan,‡ Yisong Guo,‡ Thomas M. Makris*,†
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†Department of Chemistry and Biochemistry, University of South Carolina, Columbia, South Carolina 29208, United States
‡Department of Chemistry, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, United States
Supporting Information Placeholder
carbon, a mechanism that is reiterated in the Fe(II)- and alpha-
ketoglutarate (αKG)-dependent decarboxylases IsnB and
AmbI3.19-20 A unifying feature of OleT and the Fe(II)/αKG
enzymes is the generation of a high-valent iron(IV)-oxo species for
HAT, highlighting the necessity of forming a potent oxidant to
carry out C–H abstraction at a largely unactivated carbon center.7,
19, 21-22 The two reducing equivalents necessary for O–O heterolysis
are either provided by H2O2 (OleT) or a combination of Fe(II) and
the αKG co-substrate (IsnB/AmbI3). On the basis of the
mononuclear assignment and inability of UndA to produce
undecene in the absence of exogenously added electrons, an
iron(III)-superoxo species was posited as the C–H bond-cleaving
intermediate. The equivalent species in CYPs is considered largely
unreactive and is instead a branchpoint between the formation of
Compound I and the unproductive release of superoxide.23
However, some non-heme enzymes such as isopenicillin synthase
(IPNS)24 and the diiron myoinositol oxygenase (MIOX)25 can
utilize Fe-superoxide species to catalyze HAT on C-H bonds that
are sufficiently activated by thiol or diol groups at adjacent carbon
centers.
ABSTRACT: UndA is a non-heme iron enzyme that activates
oxygen to catalyze the decarboxylation of dodecanoic acid to
undecene and carbon dioxide. We report the first optical and
Mössbauer spectroscopic characterization of UndA, revealing that
the enzyme harbors a coupled dinuclear iron cluster. Single
turnover studies confirm that the reaction of the diferrous enzyme
with dioxygen produces stoichiometric product per cluster. UndA
is the first characterized example of a diiron decarboxylase, thus
expanding the repertoire of reactions catalyzed by dinuclear iron
enzymes.
Driven by potential application as biocatalysts for drop-in
biofuel production, several enzymatic routes for hydrocarbon
(alkane and 1-alkene) biosynthesis have been recently clarified.1-6
A recurring theme in many of these enzymes is the involvement of
a redox-active iron cofactor that is tuned for the C–C cleavage of a
4, 7
fatty acid or aldehyde, producing either a one-carbon CO2
or
formate8 co-product. These transformations represent novel
reaction types for heme and non-heme iron enzymes.9 In efforts to
uncover the biochemical basis for 1-undecene production in many
Pseudomonads, Zhang and colleagues adopted an elegant genomic
screening strategy to identify the responsible gene (termed undA).4
The undA stratagem for 1-olefin production is widely distributed in
bacteria with over 1000 orthologs. This includes several important
human pathogens where undecene is the major component of
emitted volatile organic matter and can serve as an anti-fungal and
anti-microbial agent.10-12
The Basic Local Alignment Search Tool (BLAST) revealed
that UndA is similar (24 % identity) to the chlamydial virulence
factor CADD.26 The precise biochemical role of CADD is not yet
known, but it is strictly required in a pathway in Chlamydia for
generating the para-aminobenzoic acid component of folate.27
A
carboxylate-bridged dinuclear metal center, presumed to be iron
from metal analysis, is apparent in the CADD structure.
Intriguingly, all of the metal-coordinating ligands of CADD are
retained in UndA (Figure 1A and S1). We thus hypothesized that
UndA may also accommodate two irons, which would provide the
two electrons necessary to form a high-valent species. Indeed, such
potent high-valent intermediates are well-known to be generated by
dinuclear iron enzymes, best typified by soluble methane
monooxygenase (sMMO), that performs one of the most difficult
oxidation reactions in nature.28
We selected an ortholog from P. syringae pv. tomato DC3000
(PsUndA) that is 79% identical to the structurally solved UndA
from P. aeruginosa (PaUndA) (Fig. S1). All potential active-site
metal ligating residues that could form a diiron site in PsUndA are
conserved with both PaUndA and CADD, and the protein was
previously crystallized in an apo-form through structural genomics
efforts (PDB: 3OQL) showing identical placement of putative
metal-ligands. PsUndA was heterologously expressed in E. coli
and purified to homogeneity (Fig. S2) by metal affinity
chromatography as described in the SI. When purified from
bacterial cultures grown in LB medium with added FeCl3, the iron
content of PsUndA is <0.2 molar equivalents (Table S1), similar to
the low metal yields reported for PaUndA.4 Fortuitously, the
expression of PsUndA in minimal medium and FeCl3 enhanced the
extent of iron-loading to as high as 1.1 molar equivalents per
protein. Inductively coupled plasma mass spectrometry revealed
The in vitro reconstitution of UndA activity has shown that the
decarboxylation of lauric acid to form undecene and a CO2 co-
product strictly requires Fe2+ and dioxygen.4 The medium chain-
length (CL) specificity and co-substrate requirement differ from the
cytochrome P450 (CYP) fatty acid decarboxylase OleT. These
variations offer discrete advantages for developing an in vivo
platform for hydrocarbon production. For example, the preferred
physiological substrate for OleT is a long CL (Cn = 20) fatty acid
(FA),2 and the metabolism of more biotechnologically attractive
substrates (e.g. Cn = 12 – 16) often compromises chemoselectivity,
producing unwanted hydroxy (typically at C3) fatty acids that
accompany the Cn-1 olefin.13-14 Despite numerous efforts to
circumvent the H2O2 requirement by reconstituting OleT with
biological redox chains and O2,15-17 the sluggish rates of electron
transfer and inability of OleT to mediate proton delivery for O–O
heterolysis appear to make the use of peroxide obligatory for
efficient turnover.18
UndA was purported to utilize a mononuclear iron(II) cofactor
based on metal-counting procedures and the presence of a single
metal ion visible in the crystal structure of the reconstituted protein.
4 Based on analogy to OleT, FA decarboxylation would very likely
involve hydrogen atom transfer (HAT) from the fatty acid β-
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that the only other metal found in appreciable quantity was Ni,
presumably an artifact from purification.
being in a magnetically isolated mononuclear ferric state,37 likely
arising from adventitiously bound iron or partially-formed cluster.
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Figure 1: Structural alignment of PaUndA (PDB: 4WWZ, green)
with CADD (PDB: 1RCW, grey) (A). The iron atoms of CADD are
shown in orange and that of mononuclear UndA is in brown. The
optical spectrum of 75 μM UndA (iron-loaded) is shown as-isolated
(red) and in the presence of 2 M sodium azide (black) (B). Titration
of UndA with sodium dithionite results in conversion to the
reduced state (blue) requiring ~2 reducing equivalents per cluster
(C).
Figure 2: The 4.2-K Mössbauer spectra of UndA in the as-isolated
state measured under zero field (A) or 7 T applied field (B), the
dithionite-reduced form (C), and the reduced state in the presence
of C12 fatty acid (D) with experimental data shown in vertical black
bar and the spectral simulation shown in red. The arrows in B
indicate the outer-most lines of a spectral feature that originates
from a mononuclear ferric species; the corresponding simulation
using a generic S = 5/2 ferric species is shown in the black solid
line. The grey dashed line and the red line in B are the simulation
assuming an S = 0 species and an exchange coupled species,
respectively (see discussion in SI).
As-isolated, UndA exhibits a broad absorption band from 300-
450 nm (ε340 ≈ 4 mM-1 cm-1 assuming a cluster) (Fig. 1B, red trace).
The absorption is significantly more intense than that expected for
non-heme, Fe2+-dependent enzymes (e.g. 29-30). Titration of the
enzyme with sodium dithionite results in bleaching of the
chromophore and requires the addition of ~1 electron per iron (or
two per cluster) to reach saturation, consistent with the presence of
an Fe3+ center (Fig. 1C). The di-metal assignment was substantiated
through the addition of azide to the enzyme, a technique that has
been used to successfully predict the cofactor in several
carboxylate-bridged diiron enzymes.31-32 Upon incubation with
2 M sodium azide, a protein-bound chromophore forms with
absorption maxima at 350 and 440 nm (Fig. 1B, black trace), owing
to terminal (μ1,1) or bridging (μ1,3) ligation to one or both irons.33
Mössbauer spectroscopy of PsUndA unambiguously
demonstrates that a magnetically coupled dinuclear iron center is
present and is the predominant form of the iron-loaded enzyme.
The zero-field, 4.2-K spectrum of PsUndA (Figures 2A and S3) is
best simulated by two nested quadrupole doublets with parameters
indicative of high-spin (S = 5/2) ferric iron (Tables 1 and S3),
consistent with those of other enzymes containing magnetically
coupled diferric centers (see SI for additional analysis).31-32, 34-35
The major feature obtained in a spectrum measured under an
applied magnetic field of 7 T parallel to the gamma radiation
cannot be simulated as a strict diamagnetic species (S = 0) (Figure
2B, grey dashed line). A more reasonable simulation requires a
small isotropic antiferromagnetic exchange interaction (J ≤ 15 cm-
1) between the ferric centers with the inclusion of an antisymmetric
exchange interaction (Figure 2B, red line, Figs. S4-S6). A very
similar situation was encountered in characterization of the diferric
state of sMMO.36 The appearance of minor absorption peaks at ~
8 mm/s in the high-field spectrum of oxidized UndA can be
attributed to a small component (~15%) of the iron in the sample
Upon reduction with sodium dithionite, PsUndA produces a
Mössbauer spectrum (Figure 2C) that can also be simulated with
two quadrupole doublets (Table 1, Fig. S7). The larger isomer shifts
of ~1.24 mm/s are consistent with high-spin ferrous iron. The
requirement of two doublets to simulate both the diferric and
diferrous states indicates that there are either two different types of
clusters or that each iron in the cluster is distinguishable. Although
neither of these possibilities can be rigorously ruled out, we
currently favor the latter interpretation due to the equal areas and
predicted asymmetry of the ligation. Nonetheless, the addition of
excess dodecanoic acid to the diferrous state results in a significant
perturbation of the spectrum (Figs. 2D, S7-S8, Table 1). This
structural alteration is very likely to arise from the direct ligation of
FA, as suggested by co-crystal structures of mononuclear UndA
with several FA substrate analogs.4 The fact that most of the
clusters are bound suggests a relatively high affinity of the enzyme
for the C12 substrate, consistent with the single turnover studies that
follow.
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Table 1. Parameters used to simulate the Mössbauer spectra of
UndA in the as-isolated state, reduced by dithionite, and reduced in
the presence of dodecanoic acid substrate.
4
5
6
|ΔEQ|
(mm/s)
Linewidth
(mm/s) a
Area
(%)
UndA
δ (mm/s)
7
8
0.51
0.50
0.88
0.53
0.32
0.34
43b
43b
As-isolated
9
Dithionite-
reduced
1.25
1.23
2.93
1.87
-0.5
-0.55
48
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Reduced
+ C12 FA
1.26
1.24
3.01
2.05
-0.5
-0.5
65
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a
A negative linewidth in the simulation represents a Voigt
lineshape that is a convolution of a Gaussian and Lorentzian in 1:1
b
ratio. A mono-ferric species accounts for the rest of absorption
Figure 3: Proposed mechanism for the decarboxylation of fatty
acids by UndA. The curved line represents bridging ligand(s). The
remainder of the acyl chain of the fatty acid is abbreviated by a
curved line.
area.
The catalytic activity of dinuclear PsUndA was tested in
single-turnover studies. The diferric enzyme was anaerobically
reduced in the presence of 1.1 molar equivalents dodecanoic acid
(relative to Fe-loaded protein) and subsequently exposed to excess
O2. Gas chromatography revealed the formation of undecene as the
sole product of the reaction. The identity of the product was verified
through comparison to an authentic standard by mass spectrometry
(Fig. S5). Product was not detected in reactions where the
reductant, protein, or FA were omitted (Table S2). Based on the
amount of cluster determined by Mössbauer, the amount of product
formed is stoichiometric (0.92 ± 0.12) to cluster. Thus, the
dinuclear form is catalytically active and no accessory effector
proteins nor additional reducing equivalents are required for
turnover.
The 3-His/3 to 4 carboxylate ligand-set used by UndA appears
to be
a flexible scaffold capable of diverse chemical
transformations. In addition to FA decarboxylation by UndA and
the cryptic oxidation that leads to pABA generation by CADD, a
very similar coordination motif is employed by arylamine N-
42-43
oxygenases
and likely common to enzymes that catalyze the
halogenation of acyl carrier protein (ACP)-linked FAs,44 N-
nitrosation,45 and the biosynthesis of alkene amino acids (Fig.
46
S10). Thus, an intriguing question is what structural factors lead to
differences in O2-activation and elicit such dramatic reaction
diversification.
In summary, the spectroscopic and activity studies provide a
firm basis for reassignment of UndA as a diiron enzyme. Oxidative
decarboxylation represents
ASSOCIATED CONTENT
Supporting Information
a new chemical outcome for
Description of experimental procedures, Tables S1-S5 and Figures
S1–S10. The Supporting Information is available free of charge on
the ACS Publications website.
carboxylate-bridged diiron enzymes. By analogy to the
aforementioned decarboxylases and established sMMO chemistry,
a proposed mechanism for UndA is shown in Figure 3. The
catalytic cycle proceeds from reaction of the diferrous FA-ligated
ternary complex with O2, consistent with the single turnover and
Mössbauer studies presented here. The direct coordination of the
FA to the cluster may be envisioned to serve a role in positioning
the Cβ–H in such a way as to ensure HAT regiospecificity and for
reinforcing C–C scission by thwarting oxygen rebound. It is
enticing to invoke formation of a bis-Fe(IV) “Q” intermediate as
the species responsible for Cβ HAT following O–O bond cleavage.
AUTHOR INFORMATION
Corresponding Author
*makrist@mailbox.sc.edu
ORCID
Thomas M. Makris: 0000-0001-7927-620X
Yisong Guo: 0000-0002-4132-3565
Both closed-38 and open-core formulations are shown, either of
39
Notes
which would be predicted to have ample oxidizing power to
perform such a reaction. Further oxidation of the substrate through
single electron transfer (SET) by an FeIIIFeIV species would
produce a carbocation or substrate-biradical that could rearrange to
form CO2 and undecene and restore the diferric resting state.
Through analogy to the X intermediate of the R2 subunit of
ribonucleotide reductase40 and the consensus dehydrogenation
mechanism for desaturases,41 an FeIIIFeIV species should be able to
satisfy such a role.
The authors declare no competing financial interests.
ACKNOWLEDGMENTS
This work was funded by the University of South Carolina Office
of Research through the ASPIRE program and an NSF CAREER
grant (CHE 155066) to TMM. Y. G. acknowledges the financial
support from Carnegie Mellon University for this work.
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