Conversion of fatty aldehydes to alka(e)nes and formate by a cyanobacterial aldehyde decarbonylase: Cryptic redox by an unusual dimetal oxygenase

Article abstract of DOI:10.1021/ja2013517

Cyanobacterial aldehyde decarbonylase (AD) catalyzes conversion of fatty aldehydes (R-CHO) to alka(e)nes (R-H) and formate. Curiously, although this reaction appears to be redox-neutral and formally hydrolytic, AD has a ferritin-like protein architecture and a carboxylate-bridged dimetal cofactor that are both structurally similar to those found in di-iron oxidases and oxygenases. In addition, the in vitro activity of the AD from Nostoc punctiforme (Np) was shown to require a reducing system similar to the systems employed by these O₂-utilizing di-iron enzymes. Here, we resolve this conundrum by showing that aldehyde cleavage by the Np AD also requires dioxygen and results in incorporation of ¹⁸O from ¹⁸O₂ into the formate product. AD thus oxygenates, without oxidizing, its substrate. We posit that (i) O₂ adds to the reduced cofactor to generate a metal-bound peroxide nucleophile that attacks the substrate carbonyl and initiates a radical scission of the C1-C2 bond, and (ii) the reducing system delivers two electrons during aldehyde cleavage, ensuring a redox-neutral outcome, and two additional electrons to return an oxidized form of the cofactor back to the reduced, O₂-reactive form.

Full text of DOI:10.1021/ja2013517

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Conversion of Fatty Aldehydes to Alka(e)nes and Formate by a
Cyanobacterial Aldehyde Decarbonylase: Cryptic Redox by an
Unusual Dimetal Oxygenase
Ning Li,† Hanne Nørgaard,‡ Douglas M. Warui,‡ Squire J. Booker,*,†,‡ Carsten Krebs,*,†,‡ and
J. Martin Bollinger, Jr.*,†,‡
Departments of †Biochemistry and Molecular Biology and ‡Chemistry, The Pennsylvania State University, University Park,
Pennsylvania 16802, United States
S Supporting Information
b
punctiforme (Np) AD produced heterologously in Escherichia coli
(Ec) is formate rather than CO.4 On paper this reaction is
ABSTRACT: Cyanobacterial aldehyde decarbonylase
(AD) catalyzes conversion of fatty aldehydes (RꢀCHO)
formally hydrolytic (redox neutral). However, the structure of
to alka(e)nes (RꢀH) and formate. Curiously, although this
the Prochlorococcus marinus (Pm) MIT9313 ortholog showed
reaction appears to be redox-neutral and formally hydro-
lytic, AD has a ferritin-like protein architecture and a
carboxylate-bridged dimetal cofactor that are both structur-
ally similar to those found in di-iron oxidases and oxygen-
ases. In addition, the in vitro activity of the AD from Nostoc
punctiforme (Np) was shown to require a reducing system
similar to the systems employed by these O2-utilizing di-iron
enzymes. Here, we resolve this conundrum by showing that
aldehyde cleavage by the Np AD also requires dioxygen and
results in incorporation of 18O from 18O2 into the formate
product. AD thus oxygenates, without oxidizing, its sub-
strate. We posit that (i) O2 adds to the reduced cofactor to
generate a metal-bound peroxide nucleophile that attacks
the substrate carbonyl and initiates a radical scission of the
C1ꢀC2 bond, and (ii) the reducing system delivers two
electrons during aldehyde cleavage, ensuring a redox-neutral
outcome, and two additional electrons to return an oxidized
form of the cofactor back to the reduced, O2-reactive form.
that the ADs have a ferritin-like protein architecture and cofactor
site that are similar to those found in nonheme di-iron oxygen-
ases and oxidases,3 including bacterial multicomponent mono-
oxygenases (e.g., soluble methane monooxygenase)9,10 and plant
fatty acyl-ACP desaturases.11 Moreover, the in vitro activity of the
Np AD was observed (by both Schirmer et al.3 and us4) to require
a reducing system (fulfilled by NADPH and spinach ferredoxin
and ferredoxin reductase; hereafter N/F/FR), just as these di-
iron oxidases and oxygenases require reducing systems to con-
III/III
vert the Fe2
“resting” forms of their cofactors to the O2-
reactive Fe2II/II states during each reaction cycle.9ꢀ11 It appears
that these analogies to known di-iron oxidases and oxygenases
led to the incorrect depiction of the C1-derived coproduct as
CO2 (which would be an oxidative outcome) and the designation
of the cyanobacterial ADs as “decarboxylative monooxygenases”
in a patent application from the company Joule Unlimited
(Boston, MA).6 The available data thus created a puzzle: how
does an enzyme that looks and acts like an O2-utilizing oxido-
reductase effect a formally hydrolytic reaction?
We suggested two possible resolutions to this conundrum.4,12
First, the Np AD might emerge from expression and purification
with its cofactor in an inactive oxidation state, requiring reduc-
tion of the cofactor and reaction with O2 to regenerate the active
state (Scheme 1A). This idea was founded on our under-
standing of class I ribonucleotide reductases, which (i) are
structurally similar to the ADs,13,14 (ii) contain stable, cataly-
tically essential oxidants (a tyrosyl radical in class Ia15ꢀ17 and
Ib18,19 and the MnIV ion of a Mn/Fe cluster in class Ic20ꢀ22),
(iii) can be inactivated by reduction of their essential
oxidants,15,23,24 and (iv) in the case of the class Ia enzymes,
can then be reactivated by reduction of their Fe2III/III clusters
and subsequent reaction of the Fe2II/II forms with O2.25 Second,
O2 might be an obligatory cosubstrate in some sort of (hereto-
fore unprecedented) cryptically redox pathway for the formally
hydrolytic aldehyde cleavage. This case would require, for redox
balance, that four electrons be delivered in each turnover by the
reducing system (Scheme 1B).
yanobacteria use light to “fix” CO2 into energy-rich biomol-
C
ecules, including fatty acids.1,2 A two-step pathway recently
identified by Schirmer et al. allows these organisms also to
produce alkanes and alkenes from abundant saturated and
unsaturated fatty acids.3 Together, these two pathways could
potentially be harnessed for a bioprocess that would effectively
harvest solar energy, store it as a fungible fuel, and consume an
important greenhouse gas.2 The incalculable potential value of
such a process has motivated a flurry of scientific,3,4 intellectual-
property,5,6 and investment activity.7 A deeper understanding of
the cyanobacterial alkane-biosynthetic pathway might facilitate
efforts to develop such a process.
The second step in the pathway is catalyzed by the enzyme
aldehyde decarbonylase (AD8). Schirmer et al. suggested that
AD converts the Cn fatty aldehyde product of the first enzyme
into the corresponding Cnꢀ1 alkane or alkene and carbon mon-
oxide (CO),3 but we recently showed that the C1-derived co-
13
product from the in vitro conversion of octadecanal (Rꢀ CHO,
Received: February 12, 2011
Published: April 04, 2011
where R = n-C17H35) to heptadecane (RꢀH) by the Nostoc
r
2011 American Chemical Society
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Scheme 1. Two Alternative Explanations for the Similarity
of Np AD to Di-iron Oxidases and Oxygenases and its
Requirement for a Reducing System to Promote an
Apparently Hydrolytic Reactiona
Figure 1. Reconstructed mass spectra illustrating the catalytic require-
ment for O2 and the incorporation of 18O from 18O2 into the formate
product in the Np AD reaction. (A) Reactions in the continuous
presence of O2 (under air atmosphere, black bars), the absence of O2
(gray), and the presence of O2 during preincubation with the N/F/FR
system (without substrate) but the absence of O2 during the reaction
(orange). (B) Reactions under an atmosphere of natural-abundance O2
(red) or 18O2 (99% isotopic purity; blue). (C) Control reactions in
which the 2NPH derivative of either natural-abundance formate (green)
or [13C]-formate (purple) was generated in H218O (70% enrichment) to
quantify the extent of exchange of the oxygen atoms during the coupling
reaction. The reactions in A were carried out at 23 °C for 20 h and
contained, in a final volume of 0.40 mL, 0.10 mM Np AD, 0.5 mM
13
Rꢀ CHO substrate, 2 mM NADPH, and 100 μg/mL each of spinach
a (A) The reducing system provides n electrons (eꢀ), and the most
reduced form of the cofactor reacts with O2 to generate the active state,
which is more oxidized than the as-isolated form by 4 ꢀ n units (x þ y ꢀ
i ꢀ j = 4 ꢀ n); (B) O2 is reduced in every cycle by four electrons from the
reducing system.
ferredoxin and ferredoxin reductase in air-saturated 100 mM HEPES
buffer, pH 7.4, containing 0.2% Triton X-100. The reactions in B had the
same composition but were carried out for 2 h.
By contrast, spectra of the samples prepared and incubated in the
absence of O2 have much weaker peaks at m/z = 181 (Figure 1A,
gray bars), implying that much less formate (9 μM) was
produced in these reactions. This result demonstrates that the
Np AD requires O2 in some capacity. To assess whether O2 is
required for activation (Scheme 1A) or catalysis (Scheme 1B), an
additional matched sample was prepared by removing the
solution of the N/F/FR system and Np AD from the anoxic
chamber and exposing it to air for 30 min, with periodic mixing to
ensure permeation of O2 into the enzyme solution. The air-
exposed (N/F/FR þ Np AD) solution was then returned to the
anoxic chamber and mixed therein with 9 equivalent volumes of
an O2-free solution of substrate and detergent. The sample was
placed in a sealed vessel, incubated for 20 h, and analyzed as
above. The weak peak at m/z = 181 (Figure 1A, orange bars)
implies that prior exposure of the Np AD to O2 in the presence of
the reducing system does not activate it for subsequent O2-
independent turnover. These results suggest that O2 must be
continuously present for activity.
In addition to scission of the C1ꢀC2 bond and formation of a
new C2ꢀH bond, a second O-atom is added to C1 in conversion
of RꢀCHO to RꢀH and HCO2ꢀ. The requirement for O2
raised the possibility that it could be the source of this O-atom, as
occurs in other oxygenase reactions. Because substrate oxygena-
tion without net oxidation is, to the best of our knowledge,
unprecedented, we considered this possibility to be unlikely
but tested for it nevertheless. Surprisingly, the mass spectrum
of a sample in which the reaction was carried out under an
atmosphere of 18O2 (Figure 1B, blue bars) exhibits a strong peak
at m/z = 183 (an increase of two mass units) that is absent in the
spectrum of a control sample prepared identically but with
natural-abundance O2 (red bars). It is important to note here
that the analysis for formate, which involves its prior conversion
to formyl-2NPH, leads to loss of one of two equivalent oxygen
atoms, and so quantitative incorporation of a single 18O atom
into the formate product should result in precisely 50% of the
derivative having m/z = 183 (the other 50% having m/z = 181).
Both potential resolutions imply that O2 should be required
for AD activity, a prediction not tested to date. More diagnos-
tically, if the N/F/FR reducing system and possibly O2 should be
required only for reactivation of the AD, but not for turnover,
then it might be possible to observe activity after first exposing
the enzyme to both in the absence of substrate and then
removing one or both prior to initiating the reaction by addition
of the substrate. By contrast, the second scenario would imply
that omission of O2 or any component of the reducing system
should prevent turnover, irrespective of any pretreatment. To
clarify the nature and mechanism of the novel AD reaction, we
experimentally evaluated these predictions.
We first assessed whether O2 is required for activity. One set of
reaction samples (see Figure 1 legend for their composition) was
constituted with O2-free components inside an anoxic chamber
(MBraun, Peabody, MA) by adding a premixed solution of the
N/F/FR reducing system and the Np AD (the affinity-tagged
enzyme used in our previous study and purified from Ec as
13
described therein4) to a solution containing the Rꢀ CHO
substrate and Triton X-100 detergent (0.2% in the complete
sample). A matched set of samples was prepared by removing the
premixed solution of N/F/FR reducing system and Np AD from
the anoxic chamber and adding it under an air atmosphere to an
air-saturated solution of the substrate and detergent. These two
sets of samples were incubated at 23 ( 1 °C for between 1.5 and
20 h and then analyzed for [13C]-formate by conversion to its
2-nitrophenylhydrazide (2NPH) derivative and quantification by
liquid chromatography and mass spectrometry (LC-MS), as
previously described.4 Mass spectra of the samples incubated
under air exhibit intense peaks at m/z = 181 (Figure 1A, black
bars), indicating that, as expected, formate was produced in these
reactions (145 μM in Figure 1A). Note that the peak at m/z =
180 in the mass spectra of all samples arises from contaminating
environmental formate, which also contributes weakly at m/z =
181 due to the presence of natural-abundance M þ 1 isotopic
species (∼8.9% of the m/z = 180 species) in its 2NPH derivative.
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Integration of the peaks in Figure 1B and analogous spectra from
two other trials gave only (34 ( 3)% (mean and standard
deviation) of the m/z = 183 species. However, abortive coupling
in the derivatization (i.e., carboxylate attack on the activating
carbodiimide, EDC, followed by hydrolysis of the adduct) would
lead to further loss of 18O and diminution of the m/z = 183 peak
relative to the m/z = 181 peak. To assess whether the loss of
∼16% of the m/z = 183 species in Figure 1B can be explained this
way or reflects partial exchange of the O2-derived atom with
solvent during the AD reaction, control samples were constituted
in H218O (70% enrichment), subjected to the 2NPH coupling
procedure, and analyzed by LC-MS (Figure 1C). They contained
all reaction components except the substrate and were amended
with either natural-abundance formate or [13C]-formate. Promi-
nent peaks at m/z = 182 for the sample containing natural-
abundance formate (green bars) and m/z = 183 for the sample
with H13CO2ꢀ (purple bars) demonstrate that, as expected, the
coupling reaction does result in partial exchange of the formate
O-atoms with solvent. Comparison of the integrated intensities
of the peaks corresponding to the 16O- and 18O-containing formyl-
2NPH isotopologues gave 15% of the latter species, which
corresponds to 20% when the 18O content of 70% is taken into
account. Within the expected limits of error of the two measure-
ments, this extent of exchange accounts for the diminution of the
heavier isotope from the theoretical maximum of 50% in the 18O2
experiments in Figure 1B. These results show that (i) O2 is the
source of the incorporated oxygen and (ii) to the limit of our
analysis, there is no demonstrable exchange with solvent during
the enzyme reaction. The AD reaction is thus the first example
(of which we are aware) of substrate oxygenation without net
oxidation.
Scheme 2. Hypothetical Mechanism for the Np AD Reaction
product. One possibility, shown in Scheme 2, is that the reducing
system would deliver an electron during cleavage of the OꢀO
bond, forming a gem-diolyl radical and (μ-oxo)-Fe2III/III cluster
(D). The gem-diolyl intermediate could undergo radical frag-
mentation of the C1ꢀC2 bond, generating formate and the R•
radical (E). Transfer of a hydrogen atom, either from the cofactor
(as depicted) or from an amino acid in the active site, would
produce RꢀH and either a (μ-oxo)-Fe2III/IV complex (analogous
to the X intermediate that generates the tyrosyl radical in a class I
RNR41,42) or an amino acid radical. The reducing system would
then complete the reaction by quenching this remaining oxidiz-
ing equivalent and subsequently deliver two more electrons to
convert the Fe2III/III “product” form of the cofactor (F) back to
the O2-reactive Fe2II/II “reactant” state (A) in preparation for the
next turnover.
The metal ions in the AD cofactor have not yet been identified.
Nevertheless, the vast majority of proteins in this structural
The mechanism in Scheme 2 accounts for all the available data
of which we are aware, including the requirement for a reducing
system and the continuous presence of O2, the identity of the C1-
derived coproduct (formate),4 the origin of the new O-atom in
the formate, and the origins of the hydrogen atoms in both the
formate and RꢀH products.4 It also predicts an NADPH:for-
mate (NADPH:RꢀH) reaction stoichiometry of 2:1, in order to
account for the four electrons needed in each turnover for the
complete reduction of O2 (Scheme 1B). We tested this predic-
tion by spectrophotometric determination of NADPH consump-
tion and parallel quantification of formate at two reaction times
(Figure S1). Experimental NADPH:formate ratios of 5.6 after 1 h
and 6.7 after 2 h were calculated from the results. These ratios
are sufficiently large to be consistent with the mechanism of
Scheme 2 and also imply considerable uncoupling of NADPH
oxidation from RꢀCHO cleavage. This uncoupling is un-
surprising, given that the reaction is so slow and that we are
employing a heterologous reducing system (ferredoxin and ferre-
doxin reductase from spinach) to transfer the electrons from the
reduced nicotinamide to the AD cofactor. It should be more
informative to redetermine this ratio with the native reducing
system, once it has been identified.
family that have been studied to date have di-iron clusters. In the
III/III
reactions of several of these enzymes, peroxo-Fe2
inter-
mediates have been identified.26ꢀ31 These intermediates either
III/IV
undergo OꢀO-bond cleavage to give high-valent (Fe2
or
Fe2IV/IV) complexes that effect oxidation reactions, or they react
directly as electrophiles (e.g., via attack of the π-electrons of an
olefinic or aromatic substrate or the nitrogen of an amine or
hydroxylamine), also leading to oxidative outcomes. However,
studies of heme enzymes (cytochromes P-450) and inorganic
complexes have shown that metal-bound peroxides can also act
as nucleophiles to attack (among other electrophiles) aldehydes,
leading to production of formate and oxidized coproducts.32ꢀ38
Moreover, recent computational studies by Hirao and Moroku-
ma on myo-inositol oxygenase and hydroxyethylphosphonate
dioxygenase suggest that, in each reaction, the nucleophilic attack
of an Fe-coordinated peroxide on the carbonyl of a reaction inter-
mediate may be a key step in effecting CꢀC-bond cleavage.39,40
These reactions would provide precedent for the early steps of
the hypothetical AD mechanism shown in Scheme 2. It involves
II/II
addition of O2 to the reduced Fe2
cofactor (A) to form a
peroxide intermediate (B) and attack of the peroxide on the C1
carbonyl of the substrate to form a peroxyhemiacetal complex
(C). Breakdown of this intermediate leading to OꢀO and
C1ꢀC2 scission would explain the production of formate with
one O-atom from O2. The intriguing and (to date) unprece-
dented aspect of this hypothetical mechanism would be the
breakdown of the peroxyhemiacetal intermediate to convert C2
of the substrate into a fully reduced (methyl) rather than partially
oxidized (e.g., alcohol or olefinic methylene) center in the RꢀH
The mechanism of Scheme 2 implies that the cyanobacterial
AD cofactor could indeed be an Fe2 cluster, as originally sugges-
ted by Schirmer et al.3 However, the unprecedented aspects of
the mechanism leave room for the possibility that a different
transition metal might be present at one or both sites. Such a
substitution might, for example, permit formation of a peroxy-
hemiacetal intermediate less disposed toward an oxidative
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Journal of the American Chemical Society
COMMUNICATION
outcome than the depicted Fe2 complex is expected to be on the
basis of the cited precedents. Similarly, mechanisms in which a
metal-bound superoxide attacks C1 can also be formulated. In
this case, a mixed-metal cluster might disfavor further reduction
of the O2 unit to the peroxide state and give the superoxo
complex sufficient time to add to the carbonyl. Clearly, identi-
fication of the metals in the cofactor is urgently needed for a
deeper understanding of this novel reaction.
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’ ASSOCIATED CONTENT
S
Supporting Information. Figure illustrating determina-
b
tion of the ratio of NADPH oxidized to formate produced in the
Np AD reaction. This material is available free of charge via the
Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
squire@psu.edu; ckrebs@psu.edu; jmb21@psu.edu
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’ ACKNOWLEDGMENT
This work was supported by the National Institutes of Health
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