Insights into substrate and metal binding from the crystal structure of cyanobacterial aldehyde deformylating oxygenase with substrate bound

Article abstract of DOI:10.1021/cb500343j

The nonheme diiron enzyme cyanobacterial aldehyde deformylating oxygenase, cADO, catalyzes the highly unusual deformylation of aliphatic aldehydes to alkanes and formate. We have determined crystal structures for the enzyme with a long-chain water-soluble aldehyde and medium-chain carboxylic acid bound to the active site. These structures delineate a hydrophobic channel that connects the solvent with the deeply buried active site and reveal a mode of substrate binding that is different from previously determined structures with long-chain fatty acids bound. The structures also identify a water channel leading to the active site that could facilitate the entry of protons required in the reaction. NMR studies examining 1-[¹³C]-octanal binding to cADO indicate that the enzyme binds the aldehyde form rather than the hydrated form. Lastly, the fortuitous cocrystallization of the metal-free form of the protein with aldehyde bound has revealed protein conformation changes that are involved in binding iron.

Full text of DOI:10.1021/cb500343j

Articles
pubs.acs.org/acschemicalbiology
Insights into Substrate and Metal Binding from the Crystal Structure
of Cyanobacterial Aldehyde Deformylating Oxygenase with
Substrate Bound
†
†
†
‡
,†,§
Benjamin C. Buer, Bishwajit Paul, Debasis Das, Jeanne A. Stuckey, and E. Neil G. Marsh*
†
‡
§
Department of Chemistry, Life Sciences Institute, and Department of Biological Chemistry, University of Michigan, Ann Arbor,
Michigan 48109, United States
*
S Supporting Information
ABSTRACT: The nonheme diiron enzyme cyanobacterial
aldehyde deformylating oxygenase, cADO, catalyzes the highly
unusual deformylation of aliphatic aldehydes to alkanes and
formate. We have determined crystal structures for the enzyme
with a long-chain water-soluble aldehyde and medium-chain
carboxylic acid bound to the active site. These structures
delineate a hydrophobic channel that connects the solvent
with the deeply buried active site and reveal a mode of
substrate binding that is different from previously determined
structures with long-chain fatty acids bound. The structures also identify a water channel leading to the active site that could
13
facilitate the entry of protons required in the reaction. NMR studies examining 1-[ C]-octanal binding to cADO indicate that
the enzyme binds the aldehyde form rather than the hydrated form. Lastly, the fortuitous cocrystallization of the metal-free form
of the protein with aldehyde bound has revealed protein conformation changes that are involved in binding iron.
he biosynthesis of aliphatic hydrocarbons has recently
garnered much interest as drop in replacements for
that requires O and reducing equivalents, as shown in Scheme
2
1
7,18,22,23
T
1
.
1
−6
conventional transportation fuels.
In Nature, various
7
8
9
5
organisms such as plants, insects, birds, and microbes are
known to produce long-chain alkanes. These alkanes are
derived from fatty acyl-CoA esters in a two-step biosynthetic
pathway that involves a fatty-acyl-CoA reductase (FAR) that
first converts the fatty acyl-CoA ester to the corresponding
Scheme 1. Deformylation Reaction of Aldehydes Catalyzed
by cADO
1
0−13
aldehyde.
Next, an aldehyde decarbonylase (AD) cleaves
the C1−C2 (aldehyde carbon−α-carbon) bond of the aldehyde
Labeling studies have shown that the aldehyde proton is
to generate an alkane and, depending upon the organism,
17,18
1
4,15
8,16
17,18
retained in formate
derives from molecular oxygen, whereas the proton in the
product alkane derives from the solvent.
and that one of the oxygen atoms
CO,
CO2,
or HCO H.
22
2
Although alkanes are very simple molecules, their production
17,18
Spectroscopic
19
from aldehydes is a chemically difficult transformation, and
studies point to the initial formation of a diferric intermediate
20
the AD enzymes as a group remain very poorly understood.
24
in the cADO catalyzed reaction. Addition of a further electron
This is due in large part to the fact that most of these enzymes
are integral membrane proteins that have proved hard to purify
and express in recombinant form. Recently, however, a soluble
to this complex is proposed to lead to its breakdown and
scission of the C1−C2 bond. A radical mechanism for C1−C2
bond cleavage is supported by the observed ring-opening of
cyclopropyl aldehydes and oxiranyl aldehydes designed to act as
5
version of AD was discovered in cyanobacteria, which is
2
1
25,26
referred to as either cADO (cyanobacterial aldehyde
deformylating oxygenase) or in earlier reports as cAD
“radical clocks” during deformylation by cADO.
The crystal structure of the enzyme reveals it to possess an α-
5
,17,18,22,23
(
cyanobacterial aldehyde decarbonylase).
Although
helical structure with the di-iron center housed in a ferritin-like
5
,27
the physiological role of cADO in cyanobacteria remains
unknown, the amenability of the enzyme has stimulated
significant interest in understanding its mechanism and its
use in engineering new pathways to generate alkane-based
biofuels.
four-helix bundle at the core of the protein. The iron atoms
are coordinated by two histidine and four carboxylate residues,
as is seen in other di-iron oxygenases, such as methane
Received: May 2, 2014
cADO is a nonheme di-iron protein that catalyzes the
conversion of aldehydes to alkanes and formate in a reaction
Accepted: September 2, 2014
Published: September 15, 2014
©
2014 American Chemical Society
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Figure 1. Binding of 1-[ C]-octanal to cADO monitored by 13C NMR. Octanal (1 mM) was titrated with increasing concentrations of cADO and
the peaks correspond to the carbonyl (208 ppm) and hydrate forms (92 ppm) were monitored. At 125 μM cADO, the carbonyl peak is completely
lost indicating that the enzyme selectively binds this form.
13
9
monoxygenase, ribonucleotide reductase, stearoyl-ACP-Δ -
exhibits peaks at 208 ppm due to the carbonyl form and at 92
ppm due to the diol form. Addition of increasing concen-
trations of cADO to a 1 mM solution of octanal in assay buffer
resulted in a progressive reduction in intensity for the peak at
208 ppm. At 125 μM cADO, the 208 ppm peak could no longer
be detected, whereas the peak at 92 ppm retained significant
intensity. The loss of the 208 peak indicates that the enzyme
binds the aldehyde form. The peak is lost due to broadening
caused by chemical exchange with the enzyme-bound form on
the NMR time scale; some broadening may also be due to
paramagnetic relaxation associated with the iron center. Note
that the unbound aldehyde remains in chemical equilibrium
with the hydrated form but this equilibrium is slow on the
NMR time scale so that the peak at 92 ppm is not broadened.
Further increasing the enzyme concentration to be equimolar
with the substrate resulted in almost complete loss of the 92
ppm peak as well. This is due to the re-establishment of the
aldehyde-hydrate equilibrium in solution as more enzyme is
added and available to bind the aldehyde form. Eventually all
the substrate is converted to the aldehyde form and becomes
bound to the enzyme.
28−34
desaturase, and toluene-4-monooxygenase.
All cADO
structures solved so far include an aliphatic fatty acid that is
bound in a hydrophobic channel, with the carboxylate group
27,35
providing a bridging ligand to the two iron atoms.
The fatty
acid presumably mimics the binding of the aldehyde substrate;
however, it is unclear how the substrate gains access to the
active site because the hydrophobic channel is completely
enclosed by the protein.
Here, we address the question of how substrates and metal
ions gain access to the active site of cADO. We have solved
crystal structures of cADO with a long-chain, water-soluble
aldehyde bound and a medium-chain carboxylic bound that
reveal a new mode of substrate binding and protein
conformational changes involved in binding iron. We have
also undertaken NMR studies to establish whether the enzyme
binds the aldehyde or its hydrated form, which predominates in
solution.
RESULTS AND DISCUSSION
■
NMR Studies of Substrate Binding to cADO. In aqueous
solution, aldehydes exist in equilibrium with the hydrated
gem-diol) forms, which predominate. Mechanistic studies have
assumed that it is the aldehyde form that is reactive toward
deformylation. However, it is unclear which form initially binds
to the enzyme, as it would be plausible for the enzyme to bind
the hydrated aldehyde and for dehydration to occur at the
active site; this could be catalyzed by the Lewis acidic diferric
form of the enzyme. The gem-diol and carbonyl forms of the
aldehyde carbon resonate at very different frequencies in the
Synthesis and Activity of a Water-Soluble Long-Chain
Aldehyde Substrate. Previously solved crystal structures of
cADO all feature a long-chain fatty acid bound that was
presumed to occupy the binding site of the aldehyde
(
5
,27,35
substrate.
Recent studies have identified a mixture of
35
palmitic, stearic, and oleic acids as being bound, which most
likely are derived from E. coli during purification. Furthermore,
it appears that the fatty acids are necessary for crystallization as
even extensively lipid-depleted enzyme crystallizes with fatty
acids bound.
1
3
C NMR spectrum, making NMR an excellent technique to
probe which form binds to the enzyme.
We aimed to obtain a structure of the enzyme with an
aldehyde bound, which would be more mechanistically
informative. Preliminary screens with short-chain aldehydes in
the crystallization buffer failed to obtained crystals of cADO
and the cocrystallization of the enzyme with long-chain
1
3
We examined the binding of 1-[ C]-octanal to cADO
Figure 1). These experiments were conducted on the diferric
(
form of the enzyme in the absence of an auxiliary reducing
system so that turnover could not occur. 1-[ C]-octanal
1
3
2
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aldehydes, such as octadecanal, proved infeasible due to the
very low solubility of these compounds. Therefore, we
synthesized a water-soluble mimic of icosanal, 11-(2-(2-
ethoxyethoxy)ethoxy)undecanal (1) that incorporates three
oxygen atoms in the carbon chain (Scheme 2). This compound
and the structures were solved by molecular replacement to
resolutions ranging from 1.6 to 2.2 Å, with refinement statistics
given in Table 1.
Structure of cADO with Stearate Bound. To facilitate
our analysis of the enzyme, we redetermined the structure of
cADO with stearate bound at 1.60 Å resolution (PDB ID
4TW3). The enzyme comprises 8 α-helices (Figure 2A), of
Scheme 2. Structures of 11-(2-(2-
Ethoxyethoxy)ethoxy)undecanal, 1, and trans-2-
Nonylcyclopropane-1-carboxylic Acid, 2
was straightforwardly synthesized by coupling silyl-protected
1
1-bromoundecanol to 2-(2-ethoxyethoxy)ethanol followed by
deprotection and oxidation of the alcohol to the aldehyde, as
described in the Supporting Information.
Compound 1 was soluble at millimolar concentrations under
1
8,25
the buffer conditions typically used to assay cADO.
We
compared the activity of cADO in catalyzing the deformylation
of compound 1 (500 μM) and octadecanal (500 μM + 4%
DMSO to improve solubility) using previously described assay
conditions with NADH and PMS as the auxiliary reducing
Figure 2. Structure of cADO with stearate bound (PDB ID 4TW3).
(A) Ribbon diagram of cADO with helices colored from the N-
2
5,26
system.
cADO deformylated 1 to produce 1-(2-(2-
terminus: 1 (blue), 2 (cyan), 3 (cyan-green), 4 (green), 5 (yellow−
green), 6 (yellow), 7 (orange), and 8 (red); the iron atoms are brown
and stearate is shown in gray. (B) The region containing the surface of
helices 6, 7, and 8 shows a narrow hydrophobic cavity that is devoid of
crystallographically resolved water molecules; L194 is shown in stick
form at the center of the hydrophobic cavity.
−1
ethoxyethoxy)ethoxy)decane at a rate 0.049 ± 0.002 min , a
rate only ∼2-fold slower than the deformylation of octadecanal
to heptadecane under similar conditions.
Crystallization of cADO with Substrate Analogs
Bound. During the course of this investigation, we solved
the structure of cADO with the following substrate analogs
bound: stearate (i.e., enzyme as isolated from E. coli), PDB ID
which helices 1 (blue), 2 (cyan), 4 (green), and 5 (yellow-
green) make up the canonical antiparallel 4-helix bundle that
provides the coordinating protein ligands to the di-iron center.
Helix 3 (cyan−green), which is kinked in the middle, crosses
over helix 1 before connecting through a short loop to helix 4.
Helices 6 (yellow), 7 (orange), and 8 (red) form a subdomain
in which helices 6 and 7 pack against helices 1, 3, and 4,
4
TW3; with compound 1 bound, PDB ID 4PGK; and with
trans-2-nonylcyclopropane-1-carboxylate, 2, bound (Scheme 2),
PDB ID 4PG1. We also solved the structure of a mutant
enzyme, cADO-L194A, with 1 bound, PDB ID 4PGI. These
structures are discussed individually in detail in the following.
In each case, the protein crystallized in the space group, P4 2 2,
3
1
Table 1. Data Collection and Refinement Statistics for Crystal Structures
data set
PDB ID
cADO (stearate)
4TW3
P4 2 2
cADO-(1)
cADOL194A-(1)
4PGI
P4 2 2
cADO-(2)
4PGK
4PG1
space group
unit cell
P4 2 2
P4 2 2
3 1
3
1
3
1
3
1
a = b = 77.21; c = 116.57
α = β = γ = 90
0.97872
a = b = 77.34; c = 116.96
a = b = 77.07; c = 117.26
α = β = γ = 90
0.97872
a = b = 77.12; c = 116.20
α = β = γ = 90
1.0781
α = β = γ = 90
0.97856
wavelength (Å)
resolution (Å)
Rsym (%)
1.60 (1.60−1.63)
6.6 (71.9)
2.17 (2.17−2.21)
8.5 (92.3)
20 (5)
2.08 (2.08−2.12)
8.7 (57.0)
1.70 (1.70−1.73)
7.3 (86.4)
{
I/σI}
20 (3)
20 (3)
20 (3)
completeness (%)
redundancy
100.0 (100.0)
14.4 (14.3)
100.0 (100.0)
14.4 (14.7)
Refinement Statistics
2.17 (22.13−2.17)
19.4
100.0 (100.0)
14.3 (14.6)
99.9 (100.0)
14.2 (14.3)
resolution (Å)
R-factor (%)
Rfree (%)
1.60 (38.61−1.60)
17.3
2.08 (22.48−2.08)
19.9
1.70 (36.60−1.70)
18.6
18.9
21.3
22.8
20.5
protein atoms
water molecules
unique reflections rmsd
bonds
1815
306
1907
1886
148
1783
204
227
47 171
0.010
0.89
19 412
21 862
0.010
0.91
39 033
0.010
0.85
0.010
angles
0.97
MolProbity score
clash score
0.95
1.19
1.26
0.64
1.91
2.37
4.47
0.28
2
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whereas the shorter helix 8 packs at an angle of ∼45 across the
interface of helices 6 and 7. Close inspection of the structure
revealed a shallow hydrophobic cavity on the surface composed
of side chains from helices 6, 7, and 8. This region of the
surface is notably devoid of crystallographically resolved water
molecules, as illustrated in Figure 2B. Although crystallographic
symmetry places identical hydrophobic patches from neighbor-
ing protein molecules in close proximity, there are no
intermolecular van der Waals contacts between hydrophobic
residues of different proteins and the protein in solution is a
monomer.
At the center of the hydrophobic cavity is L194, which is
located on helix 6 at the interface between the aqueous protein
exterior and hydrophobic protein interior. Interestingly, this
residue appeared highly mobile as evidenced by the poor
electron density and high temperature factors of L194 side
chain carbon atoms. This degree of side chain mobility is not
observed for surrounding side chains in the hydrophobic patch
or buried hydrophobic side chains elsewhere in the protein.
These observations suggested that L194 might serve as a
gateway for substrate entry. Therefore, to assess the extent to
which L194 might constrict access to the substrate-binding
channel, we mutated this residue to alanine. However, this
mutant, referred to as cADO-L194A, had kinetic properties
very similar to the wild-type enzyme (data not shown)
suggesting that L194 does not play a kinetically significant
role in limiting substrate access to the active site.
Figure 3. Electron density 2F − F map contoured at 1σ for cADO-
0
c
L194A (PDB ID 4PGI) complexed with 1. The substrate can be
modeled in two conformations to fit the T-shaped region of electron
density, suggesting that at least two conformations of the substrate
binding are possible. The electron density map also indicates that the
region encompassing E157 and H160 also exists in two conformations:
one in which these residues coordinate the metal; the other in which
helix 5 is partially unfolded. The carboxylate group of E157 is
disordered in both conformations and has therefore not been modeled
in the structure.
whereas the other represents the metal-free protein in which
helix 5 is partially unfolded and the substrate binds in a similar
conformation to stearate. These conformations are shown in
Figure 4. To support this conclusion, each conformation was
fitted and refined independently. The resulting electron density
maps produced density for the alternate conformation in both
the F −F maps (contoured at 3σ) and in the 2F −F maps
Structure of cADO with 1 Bound. We were next able to
solve the structures of cADO and cADO-L194A with 1 bound
at 2.17 and 2.08 Å resolution, respectively (PDB IDs 4PGK and
4PGI, respectively). These structures were essentially identical,
differing only in minor alterations to side chain conformations
of residues in the immediate vicinity of the mutation. However,
because the L194A mutant structure is at higher resolution, we
have used this structure in our discussion of 1 binding to cADO
in complex as described in the following.
o
c
o
c
(contoured at 1σ). A figure showing the alternate positions of
His160 and Glu157 overlaid with a map calculated from the
refined B conformation of the helix is available in the
Supporting Information (Figure S10). The low metal content
of the crystals is not surprising because, as purified from E. coli,
only 30−35% of the active sites contain metal, which we have
The initial inspection of the electron density 2F − F map
0
c
contoured at 1σ (Figure 3 and Supporting Information Figure
S10) for the complex of cADO-L194A with 1 revealed several
interesting features. First, where the substrate was expected to
bind, there was T-shaped region of electron density extending
from the metal center with one fork of the T occupying the
cavity previously observed to bind stearic acid. The other fork
exited the protein structure adjacent to L194 (A194 in the
mutant cADO), supporting the view that this is the substrate
entry point. The overall shape and the relatively poor definition
of this electron density made it apparent that 1 must bind to
the enzyme in two quite different, and somewhat mobile,
conformations. A second area of ambiguity concerned the
electron density of helix 5, which appeared partially unfolded
between residues 154 and 165. Notably, this region
encompasses E157 and H160, which supply ligands to Fe2.
Lastly, the electron density for the metal ions in the structure
refined to an occupancy of only ∼60%, which is considerably
lower than that of any fatty acid-containing cADO structure.
Taken together, these observations led to the conclusion that
18
previously characterized as being mainly iron and zinc, and no
additional divalent metal ions were present in the crystallization
buffer.
Structure of Metal-Bound cADO. In this structure, the
coordinating ligands surrounding the two metal atoms adopt a
similar geometry to that seen in the structures of the cADO/
stearate complex and other dinuclear metalloenzymes that
possess the canonical ‘4-carboxylate, 2-histidine’ binding motif.
As shown in Figure 4B, for Fe1, the carboxylate of E45 from
helix 1 provides a bidentate ligand, whereas E73 and H76 from
helix 2 provide monodentate ligands. Similarly, for Fe2, E128
from helix 4 provides a bidentate ligand, and H160 from helix 5
provides a monodentate ligand. The carboxylate of E157, which
should provide the final protein ligand, was not resolved in the
electron density maps. This suggests that it is conformationally
labile and because of this we do not include it in the model.
The fifth coordination site to Fe1 is occupied by water, whereas
it is the aldehyde oxygen that supplies the fifth ligand to Fe2,
with an Fe−O distance of 2.5 Å.
(at least) two structural forms of cADO were represented
within the crystal. Although there is some ambiguity associated
with interpreting electron density maps in which residues and
ligands appear to adopt more than one conformation, we
consider that, as discussed below, the most reasonable
interpretation is that one form represents the metal-containing
protein in which the substrate enters the protein at L194,
In the metal-bound form of cADO, there is insufficient space
available within the hydrophobic cavity for compound 1 to bind
in the same conformation as stearate. We therefore modeled
the aldehyde chain to fit the electron density that exits the
protein between helices 6 and 7 (Figure 4B). To accommodate
2
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Figure 4. Structure of metal-bound (purple) and metal-free (yellow) cADO-L194A in complex with 1 (PDB ID 4PGI). (A) Overlay of the two
conformations of cADO; metal-bound form in purple; metal-free form in yellow, in which helix 5 is partially unfolded between the residues 154 and
165. (B) Details of the active site geometry for the metal-bound form. (C) Detail of the substrate entry channel showing conformational changes in
L194 and F130 with the structure of the stearate-bound form of cADO superimposed for comparison. (D) Details of the active site geometry in the
metal-free form in which the substrate intrudes into the metal-binding site. The carboxylate group of E157 is disordered in both conformations and
has therefore not been modeled in the structure.
1
, the side chain of L194 is displaced by ∼2 Å, (measured with
Structure of Metal-Free cADO. The cocrystallization of
the metal-free form of the enzyme was unexpected and has
provided insight into the pathway by which metals gain access
to the active site of cADO. Several attempts to crystallize apo-
cADO in the presence of 1 failed to yield crystals, presumably
due to the conformational lability of the protein. Therefore, we
conclude that the fortuitous presence of an approximately
50:50 mixture of apo- and metallo-enzyme, as isolated from E.
coli, allowed the apoenzyme to cocrystallize with the metallo-
enzyme and thereby be trapped for crystallographic analysis.
respect to Cγ) compared to the structure of the stearate-
binding enzyme. V197 on the consecutive helical turn
accommodates the rotomeric shift of L194. The phenyl ring
of F130 undergoes a χ2 rotation of 50 degrees so the face of the
ring now lines the channel that terminates at the protein
exterior as illustrated in Figure 4C. Attempts to crystallize the
enzyme in the fully metalated form by including Fe2 or Zn
ions in the crystallization buffer together with compound 1
were unsuccessful.
+
2+
2
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Figure 5. Structure of cADO in complex with cyclopropyl carboxylic acid, 2 (PDB ID 4PG1). (A) The structure of cADO shows two molecules of 2
bound to the enzyme; one molecule directly coordinates to the di-iron center by the carboxyl group, other molecule assumes a “reverse” orientation
where the carboxyl group extends into the solvent. (B) Overlay of structures of cADO with stearate bound (tan) and 2 bound (orange) illustrating
large scale movement of helix 8 (for clarity the stearate molecule has been omitted from the figure).
In the apo enzyme, residues 154−165, which in the
metalated enzyme are contained within helix 5 directly adjacent
to the diiron center, unfold to form a solvent-exposed loop
helices in RNR R2 and MMOH are similarly the more solvent
3
7,38
exposed of the helices housing the di-iron center.
cADO is
smaller than either RNR R2 or MMOH and so may require
metal coordination to stabilize the folded structure, whereas for
RNR and MMO more extensive tertiary interactions with other
helices in the protein presumably stabilize the metal-free
structure. This suggests that, although RNR R2 and MMOH
remain folded in the absence of metals, the same localized
unfolding may provide a mechanism for metalation in these and
other enzymes of this structural class.
In the metal-free structure, the electron density due to the
substrate, 1, extends into the region that would be occupied by
Fe2 (Figures 4D and 3). It is possible that the intrusion of the
substrate into the metal-binding site may have helped stabilize
the open conformation of helix 5, allowing this conformation to
be trapped in the crystal. The additional space available to the
substrate in the metal-free structure, allows the tail end of 1 to
be accommodated completely within the protein in a
conformation very similar to that seen for cADO with stearate
bound.
(
Figure 4D). This region of the protein includes E157 and
H160, which coordinate Fe2. A less dramatic change in the
conformation of the other ligand to Fe2, E128, is also seen;
small changes in the positions of Y52 and Q123 also occur,
which adopt alternate hydrogen bonding arrangements within
the protein. In contrast, the positions of the ligands to Fe1 are
very little changed; interactions with surrounding residues
appear to maintain their metal-binding conformations in the
absence of metal.
The conformation of helix 5 does not appear to be influenced
by crystal packing effects in either conformation. None of the
residues in helix 5 make contacts with other symmetry related
protein molecules within the crystal lattice. The side-chain of
the closest residue Tyr163 is makes the closest approach, 4.4 Å.
Helical residues N-terminal to Y163 are completely solvent
exposed whereas residues C-terminal to Y163 are greater than
5.3 Å from any symmetry related molecule.
Helix 5 is one of the two more solvent exposed helices
Although we chose to model the substrate in two
conformations associated with metal-bound and metal-free
forms of cADO, we do not rule out the possibility that the
substrate may bind in other orientations. The conformational
mobility of the long aliphatic chain, combined with the lack of
any hydrogen-bonding residues in the substrate-binding
channel that might serve as “anchor points” for the substrate,
suggest that many energetically similar configurations may be
possible. In particular, it is possible to model the substrate in a
nonproductive binding mode in which the aliphatic chain
stretches across the top of the T-shaped channel, with the
aldehyde carbonyl extended into the solvent rather than
binding to the metal.
housing the di-iron center and its partial unfolding suggests a
mechanism by which metal ions can enter the active site. The
metal-free structures of other structurally related di-iron
enzymes have been described previously, notably Class I
36
ribonucleotide reductase R2 subunit (RNR R2) and methane
3
7
monooxygenase hydroxylase subunit (MMOH). In both
these enzymes, the tertiary structure remains intact and the
metal-chelating ligands adopt conformations that are close to
their metal-bound conformations. In contrast, in cADO, one
metal binding site remains intact, but the localized unfolding of
helix 5 between residues 154 and 165 completely dissembles
the second metal-binding site. Interestingly, the corresponding
2
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Structure of cADO with trans-2-Nonylcyclopropane-
-Carboxylate (2) Bound. Because the electron density map
N51, and Q123 and could potentially form a proton relay to the
solvent. The three closest potential proton donors, a water
molecule, the side-chain of Q123 and an iron-bound water/
hydroxide, are each about 4.8 Å from the α-carbon of 2.
Although this is too far for them to act as proton donors, one
may imagine that in the catalytically active enzyme relatively
small movements in the active site may bring any of these
donors within range. Furthermore, recently published solvent
isotope effect measurements have provided evidence that the
proton is most likely transferred from the iron-bound water
1
associated with 1 bound to cADO left some ambiguity as to its
mode of binding, we also attempted to cocrystallize cADO with
other aldehydes. We were able to obtain highly diffracting
crystals in the presence of trans-2-nonylcyclopropane-1-
carbaldehyde, which allowed the structure to be solved at 1.7
Å resolution, PDB ID 4PG1.
During refinement it became apparent that the aldehyde
carbon had oxidized to the carboxylic acid, to give compound 2
Scheme 2), a finding confirmed by chromatographic analysis
39
molecule.
(
The structures of cADO with 1 and 2 bound have
illuminated the likely entry point for the substrate, which was
not apparent from previous structures with stearate or palmitate
bound. The structures also demonstrate that for long-chain
aldehydes several modes of binding are possible and that for
short-chain aldehydes more than one molecule can bind at a
time. These observations suggest that nonproductive modes of
substrate binding are possible and may contribute to the low
rates of turnover measured for cADO. For example, substrate
inhibition might occur with short aldehydes if a second
substrate molecule is bound in the channel preventing product
release. Likewise, if a long-chain aldehyde bound across the top
of the ‘T’-shaped substrate channel, instead of in the channel
leading to the metal center, this would effectively inhibit the
enzyme complex. This suggests that protein engineering could
be used to occlude the “dead-end” branch of the substrate-
binding channel and possibly improve catalytic efficiency.
A final intriguing observation is that the entry point for the
aldehyde is in the center of a hydrophobic depression on the
enzyme’s surface that is flanked by several positively charged
residues. This suggests that cADO may interact with either
another protein for the delivery of these very hydrophobic
molecules, or possibly the cell membrane. No interaction
partners for cADO have been identified and the enzyme does
not appear to be membrane associated; however, it seems
unlikely that cytosolic long-chain aldehydes would be the
physiological substrates for cADO because of their extremely
low solubilities. Indeed, it is unclear why some species of
cyanobacteria biosynthesize alkanes, and it remains possible
that the physiological substrates for cADO are other hydro-
phobic molecules.
of the mother liquor. This is not especially surprising given that
aldehydes are sensitive to oxidation by oxygen in the presence
of metal ions, and no attempt was made to rigorously exclude
oxygen from the crystallization trays. Nevertheless, the
structure is instructive because it reveals two molecules of 2
bound to the protein (Figure 5), which clearly delineate the
entry point for the substrate. The high quality of the electron
density map allowed the cyclopropyl ring to be clearly resolved,
removing any ambiguity about the identity and orientation of
the ligands. Interestingly, although the aldehyde of 2 was
synthesized in racemic form, it is the 1R,2R enantiomer of 2
that cocrystallizes with the enzyme in both positions.
As shown in Figure 5, one molecule of 2 is coordinated to
the metal center through the carboxylate group, the other
molecule binds to the protein in the substrate entry channel in
a “reverse” orientation and extends into the solvent, with the
carboxylate group making a hydrogen bond with the guanidine
group of R191 and last three carbon atoms of 2 contacting
L194. As seen with 1, access to the protein interior is provided
through a displacement of the L194 side chain to accommodate
the aliphatic tail of 2. The binding of 2 also results in a large-
scale movement of helix 8 (Figure 5B), which pivots about its
N-terminus resulting in a displacement of the C-terminus of the
helix of ∼4.3 Å with respect to the stearate-bound structure.
This movement is accompanied by the formation of additional
hydrophobic contacts between 2 and residues in helix 8.
The structure of cADO with 2 bound also provided insight
into another aspect of the reactionthe pathway by which the
proton that is introduced into the product alkane is shuttled
between the solvent and the interior of the protein. A chain of
water molecules is seen in the structure that enters the protein
between helices 1 and 3 close to the metal center (Figure 6).
These waters are stabilized by hydrogen bonds to Q108, E47,
CONCLUSION
■
The deformylation reaction catalyzed by cADO is chemically
complex and unlike that of other nonheme iron oxygenases in
that O is completely reduced to water during the catalytic
2
cycle. This reaction requires that, in addition to the aldehyde
substrate and O , further electrons and a proton be delivered to
2
1
7,18,22,23
the active site during the reaction.
aldehydes extremely slowly,
fastest substrates not exceeding ∼1 min . We had considered
that the improved solubility of 1 and its product might result in
faster turnover; however, the sluggish rate at which 1 is
deformylated indicates that the solubility of the substrate is not
a limiting factor in the rate of turnover.
cADO deformylates
1
7,18,21,23,35
with k for even the
cat
−1
A key step in understanding both the mechanism of the
enzyme and its possible function in vivo is to understand how
the substrates gain access to the active site. The NMR studies
presented here indicate that the enzyme selectively binds the
aldehyde form from solution, rather than the hydrate. The
selectivity may arise because the hydrated form is too
hydrophilic to enter the hydrophobic substrate-binding
Figure 6. Water molecules within the structure of cADO with 2 bound
(
PDB ID 4PG1). A chain of water molecules were resolved, which
enter the enzyme in between helices 1 and 3 and are hydrogen bonded
to Q108, E47, N51, and Q123. The channel potentially provides a
conduit to supply the proton necessary for alkane formation.
2
590
dx.doi.org/10.1021/cb500343j | ACS Chem. Biol. 2014, 9, 2584−2593

ACS Chemical Biology
Articles
4
2
channel. The crystal structures of cADO with substrate analogs
bound have identified local protein conformational changes that
allow iron to enter the active site of cADO and a pathway by
which the proton required in the reaction may enter the active
site. Notably, the substrate-binding channel identified in these
structures is different from that inferred for previous structures
containing fatty acids. These observations should facilitate
protein engineering to improve the efficiency of the enzyme.
suite. Smiles for the compounds were created using Molinspiration
WebME Editor 3.81 (Dr. Peter Ertl, http://www.molinspiration/
docu/webme/index.html) while restraints were produced using
4
3,44
mogul=qm option in grade.
rigid body refinement and restrained refinement using Buster. Side
chains were visualized and adjusted using Coot with 2F −F and F −
F electron density maps from Buster. Structures were validated using
Molprobity. All residues are in allowed regions of the Ramachandran
plot. Data refinement statistics are given in Table 1.
Protein models were refined by
44
45
0
c
0
c
4
6
METHODS
ASSOCIATED CONTENT
■
■
Materials. Phenazine methosulfate (PMS) and ferrous ammonium
*
S
Supporting Information
The procedures for the synthesis and characterization of 11-(2-
2-ethoxyethoxy)ethoxy)undecanal (1) and trans-2-nonylcyclo-
propane-1-carbaldehyde (2). This material is available free of
sulfate were from Sigma-Aldrich; NADH was obtained from Acros
13
Organics; 1-[ C]-octanal was obtained from Cambridge Isotope
(
Laboratories.
Synthesis of 11-(2-(2-Ethoxyethoxy)ethoxy)undecanal, 1,
and trans-2-Nonylcyclopropane-1-Carbaldehyde (Aldehyde
of Compound 2). Details of the synthesis and characterization of
compound 1 and the aldehyde of compound 2 are described in the
Supporting Information.
Enzyme Purification and Reconstitution. The purification of
recombinant P marinus cADO from Escherichia coli was performed as
described previously.
were performed as described previously.
Preparation of Samples for NMR. NMR samples of the diferric
form of cADO were prepared in 50 mM potassium phosphate buffer
pH 7.2) and 20 mM CHAPS (500 μL volume). 1-[ C]-octanal was
charge via the Internet at http://pubs.acs.org.
Accession Codes
Coordinates of the protein structures discussed in this
manuscript have been deposited in the Protein Data Bank
with IDs 4TW3, 4PGK, 4PGI, and 4PG1.
18,23
Activity assays of cADO with compound 1
25,26
AUTHOR INFORMATION
■
Corresponding Author
*Email: nmarsh@umich.edu.
13
(
dissolved in d -DMSO for a stock concentration of 100 mM and added
Notes
6
to the proteins solution to give the desired final concentration. Spectra
were acquired using a Varian VNMRS 700 MHz spectrometer with a 5
mm PFG AutoX broadband probe at 4 °C using 2000 scans.
Crystallization and Structure Determination. For crystalliza-
tion of cADO or cADOL194A with 1, the compound was added
directly to a solution of 396 μM cADO in 50 mM HEPES, pH 7.4 for a
final concentration of 10 mM 1. Samples were incubated for 16 h at 4
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was supported in part by grants from the
National Science Foundation, CHE 1152055, CBET 1336636,
the National Institutes of Health, GM 093088, and the
European Union, FP-7 256808. Use of the Advanced Photon
Source was supported by the U.S. Department of Energy,
Office of Basic Energy Sciences, Contract No. DE-AC02-
°C immediately prior to crystallization screening. The protein samples
used for these experiments did not undergo removal/replacement of
endogenous metals and cADO retained the metal content as isolated
from E. coli. The crystals of cADO and cADOL194A with 1, from
which the highest resolution data were collected and were crystallized
against 20% PEG 3350, 0.3 M NaCl and 20% PEG 3350, 0.2 M
sodium tartrate, respectively, with drops containing equal volumes of
protein sample and precipitant.
0
6CH11357. Use of the LS-CAT Sector 21 was supported by
the Michigan Economic Development Corporation and the
Michigan Technology Tri-Corridor (Grant 085P1000817). We
thank Dr. Jennifer Meagher for her assistance with crystal-
lization and Dr. David Smith for his help with data collection at
LS-CAT.
For crystallization of cADO with 2, the sample was prepared by
adding 2.0 equiv of Fe(II) in the form of ferrous ammonium sulfate to
400 μM apo cADO in 50 mM HEPES buffer, pH 7.4 with 2% glycerol
under a microaerobic environment. Protein solutions were oxidized for
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