O-demethylations catalyzed by rieske nonheme iron monooxygenases involve the difficult oxidation of a saturated C-H bond

Article abstract of DOI:10.1021/cb400154a

Dicamba monooxygenase (DMO) catalyzes the O-demethylation of dicamba (3,6-dichloro-2-methoxybenzoate) to produce 3,6-dichlorosalicylate and formaldehyde. Recent crystallographic studies suggest that DMO catalyzes the challenging oxidation of a saturated C-H bond within the methyl group of dicamba to form a hemiacetal intermediate. Testing of this hypothesis was made possible by our development of two new independent techniques. As a novel method to allow use of ¹⁸O₂ to follow reaction products, bisulfite was used to trap newly formed ¹⁸O-formaldehyde in the stable adduct, hydroxymethanesulfonate (HMS^-), and thereby prevent the rapid exchange of ¹⁸O in formaldehyde with ¹⁶O in water. The second technique utilized unique properties of Pseudomonas putida formaldehyde dehydrogenase that allow rapid conversion of ¹⁸O-formaldehyde into stable and easily detectable ¹⁸O-formic acid. Experiments using these two new techniques provided compelling evidence for DMO-catalyzed oxidation of the methyl group of dicamba, thus validating a mechanism for DMO [and for vanillate monooxygenase, a related Rieske nonheme iron monooxygenase] that involves the difficult oxidation of a saturated C-H bond.

Full text of DOI:10.1021/cb400154a

Letters
pubs.acs.org/acschemicalbiology
O‑Demethylations Catalyzed by Rieske Nonheme Iron
Monooxygenases Involve the Difficult Oxidation of a Saturated C−H
Bond
Wenzhi Jiang, Mark A. Wilson, and Donald P. Weeks*
Department of Biochemistry, University of Nebraska-Lincoln, Lincoln, Nebraska 68588, United States
S
* Supporting Information
ABSTRACT: Dicamba monooxygenase (DMO) catalyzes the
O-demethylation of dicamba (3,6-dichloro-2-methoxyben-
zoate) to produce 3,6-dichlorosalicylate and formaldehyde.
Recent crystallographic studies suggest that DMO catalyzes
the challenging oxidation of a saturated C−H bond within the
methyl group of dicamba to form a hemiacetal intermediate.
Testing of this hypothesis was made possible by our
development of two new independent techniques. As a novel
method to allow use of 18O2 to follow reaction products,
bisulfite was used to trap newly formed 18O-formaldehyde in
the stable adduct, hydroxymethanesulfonate (HMS−), and
thereby prevent the rapid exchange of 18O in formaldehyde
with 16O in water. The second technique utilized unique
properties of Pseudomonas putida formaldehyde dehydrogenase that allow rapid conversion of 18O-formaldehyde into stable and
easily detectable 18O-formic acid. Experiments using these two new techniques provided compelling evidence for DMO-catalyzed
oxidation of the methyl group of dicamba, thus validating a mechanism for DMO [and for vanillate monooxygenase, a related
Rieske nonheme iron monooxygenase] that involves the difficult oxidation of a saturated C−H bond.
ieske nonheme iron monooxygenases are involved in the
enzyme. This and previous observations2 that the carbon
Rsynthesis of myriad natural organic compounds as well as closest to O2 is the most likely target for oxidation have
provided strong suggestive evidence that molecular oxygen,
activated by transfer of electrons from the enzyme’s
mononuclear iron atom, contributes one of its oxygen atoms
to break a C−H bond in the O-methyl group of dicamba with
no oxidation of the C2 carbon of the dicamba ring structure.
This oxygenation is proposed to form an unstable hemiacetal
intermediate that would likely decompose to release form-
aldehyde and DCSA (Figure 1a and b). A possible mechanism
for this oxidation in DMO is illustrated in Supporting Figure
S1. Furthermore, 4-toluene sulfonate methyl monooxygenase, a
homologue of DMO that acts on a substrate that cannot be O-
demethylated as can dicamba, produces 4-sulfobenzyl alcohol as
a product.13 This stable primary alcohol product of 4-toluene
sulfonate methyl monooxygenase is similar to the proposed
hemiacetal intermediate of DMO and suggests that hemiacetal
formation is plausible in related Rieske O-demethylases. The
comparative difficulty of inserting an oxygen atom into a
saturated C−H bond suggests that either a high valency iron-
oxo species such as HO-Fe(V)O or an isoelectronic oxygen
radical species are likely involved in DMO-catalyzed oxygen-
ation.14,15 However, to date no direct evidence for the attack of
in the degradation of these (and xenobiotic) compounds by
bacteria and other microorganisms in the environment.1−4 One
such organism is the soil bacterium Stenotrophomonas (formerly
Pseudomonas) maltophilia, strain DI-6, that is capable of
completely degrading the herbicide dicamba (2-methoxy-3,6-
dichlorobenzoic acid) to CO2, H2O, and Cl−.5,6 The first step in
this degradation pathway is catalyzed by dicamba O-
demethylase (DOD), which converts the herbicide dicamba
to 3,6-dichlorosalicylic acid (DCSA) through the sequential
action of three separate proteins.7,8 In this reaction, electrons
originating from NADH are transferred to FAD in a reductase,
which then reduces a ferredoxin that, in turn, reduces the
terminal monooxygenase (dicamba monooxygenase, DMO).
DMO is a nonheme iron Rieske oxygenase that catalyzes the
two-electron oxidation of dicamba to produce DCSA and
formaldehyde.8,9 The ability of DMO to inactivate dicamba has
been used to create dicamba-resistant transgenic crop plants
that soon will be commercially available to farmers.10,11
Although Rieske nonheme iron monooxygenases are known
to catalyze the O-demethylation of several aromatic com-
pounds, the mechanisms by which oxygen is activated and
attacks the enzyme substrate remains uncertain. The recent
determination of the crystal structure of DMO12 has revealed
that the carbon atom of dicamba’s O-methyl group is the
portion of the substrate closest to O2 in the active site of the
Received: March 4, 2013
Accepted: May 29, 2013
Published: May 29, 2013
© 2013 American Chemical Society
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Figure 1. Proposed mechanism for DMO-catalyzed conversion of dicamba to DCSA and formaldehyde and methods for trapping formaldehyde as a
reaction product. (a) The proposed mechanism for the reaction catalyzed by DMO. Atoms contributed by molecular oxygen are labeled in red, and
the flow of electrons in the spontaneous decomposition of the proposed hemiacetal intermediate is indicated by curved arrows. (b) Structural
analysis of dicamba-bound DMO supports oxygenation at the exocyclic methyl group. The 2.1 Å resolution crystal structure of dicamba-bound
DMO (PDB 3GL2) is shown with key residues in the mononuclear iron active site labeled. The water coordinating the iron atom was observed to be
substituted by dioxygen in higher resolution structures of DMO alone and bound to product.12 Therefore its position is a reliable indicator of the
binding site for molecular oxygen. The distances between the water and the two possible target carbon atoms of dicamba are indicated with dotted
lines, with distances given in angstroms. The exocyclic methyl group of dicamba is well-positioned for attack by reduced oxygen, while the aromatic
carbon atom appears too distant. (c) Scheme for trapping formaldehyde with bisulfite and the formation of the formaldehyde-sodium bisulfite
adduct, CH2(OH)SO3− (hydroxymethane sulfonate, HMS−). (d) Scheme for trapping formaldehyde as formic acid in the presence of P. putida C83
formaldehyde dehydrogenase (FDH) with tightly bound NAD+/NADH as a cofactor.
an oxygen atom on a C−H bond in the O-methyl group of
dicamba or of the resulting hemiacetal intermediate is available,
and the partitioning of the atoms derived from molecular
oxygen into the products is unknown.
three components of dicamba O-demethylase (DOD) and
dicamba, reactions were carried out in the absence of bisulfite
and in the presence of 0.5 mM and 2 mM bisulfite (Supporting
Figure S2). Although a small peak of m/z = 111, the expected
mass of the HMS− ion, was observed in the control reaction
containing no bisulfite (Supporting Figure S2a), addition of
increasing amounts of bisulfite resulted in the production of
increasing amounts of the m/z = 111 ion, suggesting that
bisulfite was reacting with formaldehyde produced by the DOD
reaction to form HMS−. Further verification of the formation of
HMS− in the complete DOD reaction containing 2 mM
bisulfite was obtained in reactions performed either in the
absence of added 13C-labeled formaldehyde or in the presence
of 0.5 mM and 1 mM 13CH2O (Supporting Figure S3a, b, and
c, respectively). In addition to the expected HMS− ion (m/z =
111) formed with formaldehyde produced during the O-
demethylation of dicamba, an ion of m/z = 112 was observed
when exogenous 13CH2O was provided in the reaction mixture,
verifying that the HMS adduct is being formed between
formaldehyde and bisulfite in the reaction mixture.
Here we describe development and use of two new methods
to follow the fate of 18O2 in the DMO-catalyzed monooxyge-
nation of dicamba. The first approach takes advantage of the
ability of bisulfite to rapidly bind with formaldehyde to form
the stable adduct hydroxymethanesulfonate (HMS−)16 (Figure
1c). In our unique application of this reaction, HMS− acts as a
trap for 18O incorporated into formaldehyde by preventing
rapid exchange of 18O in formaldehyde with 16O in water, a
process that occurs through the reversible formation of
methanediol [methylene glycol, H2C(OH)2]. The second
independent technique used to document 18O incorporation
into formaldehyde exploited the unique ability of Pseudomonas
putida formaldehyde dehydrogenase (FDH)17,18 (Figure 1d) to
rapidly convert newly formed 18O−formaldehyde to a 1:1
combination of 18O-methanol and 18O-formic acid, with the
latter being easily detected by standard mass spectrometry
(MS) procedures. Results using these new techniques are
presented and discussed in regard to the mechanism of the
DMO catalyzed O-demethylation of dicamba and the substrate
specificity of activated oxygen in this reaction. These techniques
have provided evidence for the existence of a hemiacetal
intermediate in the oxidative demethylation of dicamba by
DMO as well as in the structurally similar enzyme vanillate
monooxygenase (VMO).
To determine if added bisulfite inhibited the DOD-catalyzed
conversion of dicamba to DCSA, we carried out the reaction in
the absence of bisulfite and in the presence of 0.5 mM and 2
mM bisulfite (Supporting Figure S4a, b, and c, respectively)
and with a reaction time that allowed for the detection of both
substrate and product. In all cases approximately equal
proportions of the two dicamba ions [m/z = 219 (Cl isotope
35) and m/z = 221 (Cl isotope 37)] and DCSA ions (m/z =
205 and m/z = 208) were produced, indicating no obvious
inhibition by bisulfite under these conditions.
Trapping of Formaldehyde with Sodium Bisulfite. To
demonstrate the detection of formaldehyde using sodium
bisulfite to form the adduct hydroxymethane sulfonate (HMS−,
Detection of 18O-Labeled Formaldehyde, but No
Detection of 18O-Labeled DCSA. If our model of substrate
−
CH2(OH)SO3 ) in a complete reaction mixture containing the
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oxygenation based on the DMO crystal structure is correct, the
DOD reaction carried out in the presence of 18O2 should yield
formaldehyde with a mass of 32 and a subsequent HMS− ion
with m/z = 113. During MS analysis of products from a DOD
reaction performed in the presence of 18O2 but lacking
dicamba, a “background” ion of m/z = 113 was detected
(Figure 2a). [This background compound was determined to
in the presence of dicamba apparently resulted in slightly more
of the m/z = 113 peak (Figure 2b). To determine if either ion
with (m/z = 113) contained 18O-labeled HMS−, the m/z = 113
peak was subjected to MS/MS (Figure 2, lower panels).
Collision induced dissociation of the m/z = 113 ion from the
reaction lacking dicamba produced an abundance of m/z = 63
and m/z = 83 ions from the background compound and a small
peak of m/z = 81 (Figure 2a). In contrast, the reaction
containing dicamba produced a significantly greater abundance
of ion m/z = 81 (Figure 2b) (P < 0.01, Supporting Figure S6),
suggesting the reaction conducted in the presence of dicamba
produced CH218O, as predicted. Conversely, when similar
reactions were conducted in the presence of 18O2 (Supporting
Figure S7a) or in the presence of air (16O2) (Supporting Figure
S7b), there were no differences in the masses (m/z = 205 and
m/z = 207) of the product, DCSA, verifying that oxygen is not
incorporated into the ring structure of dicamba during dicamba
O-demethylation.
Independent Verification of Incorporation of 18O into
Formaldehyde, but Not DCSA. As an independent method
for verifying that the reaction of activated 18O with the carbon
atom of the dicamba O-methyl group produced 18O-labeled
formaldehyde, we utilized formaldehyde dehydrogenase (FDH)
from Pseudomonas putida C83.17,18 Each subunit of this enzyme
has the unique characteristic of tightly binding a single oxidized
nictotinamide adenine dinucleotide molecule (NAD+) and
converting it as a cofactor (not as a coenzyme) to NADH
during dismutation of formaldehyde to formic acid (an
irreversible reaction). Once NADH is formed, the enzyme is
then capable of converting the next incoming formaldehyde
molecule to methanol and simultaneously converting NADH to
NAD+, without the release of the cofactor17,18 (Figure 1d).
Because the DOD reaction must be conducted in the presence
of high levels of NADH (2.5 mM) and this concentration
drives the conversion of formaldehyde to methanol in reactions
catalyzed by most other types of FDHs,19,20 the P. putida FDH
offered an opportunity to capture at least a portion of 18O-
formaldehyde as formic acid that, unlike formaldehyde, does
not readily exchange oxygen with water.
Figure 2. Mass spectrometry MS and MS/MS analyses of 18O-labeled
formaldehyde-bisulfite adduct (HMS, m/z = 113) formed in a DOD
reaction (a) without dicamba and (b) with dicamba. A mixture of 21%
18O2 and 79% of N2 was injected into the reaction mixture in an
evacuated reaction vessel along with bisulfite (2 mM final concn) to
trap 18O-labeled formaldehyde as 18O-HMS with a m/z of 113. Upper
scans, MS analyses of reaction contents and products with m/z
between 99 and 121. Lower scans, MS/MS analyses of products from
bombardment of the m/z 113 ion selected from the initial MS ion
−
separations resulting in detection of HSO3 (m/z 81), a product
derived from bombardment of 18O-HMS (m/z = 113) and the release
−
of H2C18O (m/z 32) and HSO3 from the formaldehyde/sulfite
adduct formed in the DOD reaction containing dicamba. cps: counts
To confirm that P. putida FDH could be used successfully to
detect formaldehyde when included in a DOD reaction
mixture, we first performed reactions containing 13C-labeled
formaldehyde. If the reaction was conducted in the absence of
H213CO, no ion corresponding to m/z = 46 was observed
(Supporting Figure S8a). When increasing amounts of 13C-
labeled formaldehyde (i.e., 0.1 mM and 0.5 mM) were added to
the reaction, increasing amounts of ions of m/z = 46 were
per second.
be a consistent contaminant in the acetonitrile used as a solvent
for MS samples and when subjected to MS/MS analysis
produced two product ions of m/z = 69 and m/z = 83 and,
importantly, no observable m/z = 81 corresponding to HMS−
(Supporting Figure S5)]. The same DOD reaction carried out
Figure 3. Mass spectrometry detection of 18O-labeled formic acid (HC18OOH, m/z = 47) formed in DOD reaction mixtures having both 2.5 mM
NADH and 2.5 mM NAD and carried out in the presence of 21% 18O2 and 79% N2 . Reaction mixtures contained (a) dicamba plus 0.1 unit of FDH,
(b) FDH but no dicamba, and (c) dicamba but no FDH.
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Figure 4. Mass spectrometry detection of 18O-labeled formic acid (HC18OOH, m/z = 47; HC18O18OH, m/z = 49;) formed in the DOD enzyme
reaction system containing H218O, H216O, and 18O2 and in the presence or absence of dicamba and the presence or absence of FDH. The reactions
were carried out in a 1:1 mixture of H218O and H216O in the reaction buffer and with an atmosphere of 21% 18O2 and 79% N2. (a) Reaction
containing dicamba plus FDH, (b) reaction containing no dicamba, and (c) reaction containing no FDH.
observed (Supporting Figure S8b and c, respectively),
indicating successful conversion of formaldehyde to formic
acid in the presence of P. putida FDH. Next, we added FDH to
a complete DOD reaction mixture containing dicamba and
18O2. This reaction produced a product with m/z = 47, the
expected mass of formic acid containing a single 18O atom (i.e.,
HC18OOH) (Figure 3a), whereas reactions lacking dicamba or
FDH (Figure 3b and c, respectively) lacked such an ion. Similar
reactions conducted in the presence of 18O2 and in the presence
of FDH (Supporting Figure S9a) or in the absence of FDH
(Supporting Figure S9b) display no shifts in the masses of ions
associated with the reaction product, DCSA, again confirming
the lack of an 18O atom addition to the DCSA ring structure. In
a modification of these experiments aimed at confirming the
identity of the ion with a mass m/z = 47 as 18O-labeled formic
acid, similar reactions were conducted in an buffer containing a
1:1 mixture of H218O and H216O in an atmosphere of 18O2
(Figure 4a). In this reaction two ions with m/z = 47 and m/z =
49 corresponding to the expected masses of HC18O16OH and
HC18O18OH were produced. Reactions lacking dicamba or
FDH (Figure 4b and c, respectively) produced no detectable
formic acid ions.
S12b). These results suggest the VMO and DMO mono-
oxygenase reactions proceed by similar, if not identical,
mechanisms.
Details of the DMO Reaction Mechanism As Related
to DMO Structure. The 1.75 Å crystal structure of DMO
showed evidence of a diatomic species bound near the catalytic
mononuclear iron site that was consistent with oxygen.12 The
proximity of this species to the exocyclic methyl group of
dicamba (Figure 1b) suggested that activated oxygen attacks a
saturated C−H bond in this moiety, which has been confirmed
in this work. The oxygenation of saturated hydrocarbons is
known to be one of the most chemically difficult reactions in
nature but is catalyzed by at least two other classes of enzymes:
methane monooxygenases24 and the cytochrome P450s.25 In
both cases, highly reactive Fe(IV)- or Fe(V)-oxo species are
proposed to be the active oxygenating species. The ability of
the Rieske nonheme iron oxygenases to catalyze a similar
chemistry suggests that a high valency iron-oxo species may
also be the oxygenating species for at least some of these
enzymes. This is consistent with recent proposals by
Chakrabarty et al.14 and van der Donk et al.15
Implications Regarding the DMO and VMO Enzyme
Mechanism to That of Other Rieske Monooxygenases.
DMO and VMO are members of a larger group of Rieske
oxygenases that includes 4-methoxybenzoate monooxygenase22
4-toluene sulfonate methyl monooxygenase26 and stachydrine
demethylase.27 Our results suggest that other enzymes
catalyzing similar reactions may produce the same partitioning
of isotopically labeled oxygen into products akin to those
generated by DMO and VMO. Although the structures of the
related VMO and 4-toluene sulfonate methyl monooxygenase
enzymes are not available, a recent structural and spectroscopic
study of the more distantly related stachydrine demethylase
features a proposed mechanism that is similar to the one we
have proposed for DMO, including the production of an
intermediate resembling the hemiacetal in the DMO
mechanism.27 The techniques developed in the present studies
should facilitate future experiments to determine if these and
other Rieske nonheme monooxygenases share a common
mechanism. More broadly, these methods should be of
significant value to biologists and biochemists analyzing any
of the myriad enzyme mechanisms involving formation of
formaldehyde.
Vanillate Monooxygenase Reaction Also Produces
18O-Labeled Formaldehyde, but Not 18O-Labeled Proto-
catechuate. Similarity of the structural components of DMO
and vanillate monooxygenase (VMO) and their reaction
products21−23 suggests that their reaction mechanisms may be
analogous. To determine if VMO directs an activated oxygen
atom to the carbon atom of the O-methyl group of vanillate
during its conversion to protocatechuic acid (PCA) or,
alternatively, to the ring structure of vanillate, we conducted
the VMO reaction in the presence of 18O2. At the start of the
reaction (Supporting Figure 10a), a major ion of m/z = 167
corresponding to the vanillate ion was present. After 2.5 h of
reaction, a new ion (m/z = 153) corresponding to nonisotopi-
cally labeled PCA was detected (Supporting Figure S10b).
When the same reaction was conducted in the presence of
bisulfite, an ion with the expected mass m/z = 113 of 18O-
containing HMS− was present both at zero time and after 2.5 h
of reaction (Supporting Figure S11a and b). When the m/z =
113 ion from the zero time reaction was subjected to MS/MS
analysis, the parent HMS− ion of m/z = 113 was seen as well as
a minor ion of m/z = 81 (Supporting Figure S12a). However,
in the reaction lasting 2.5 h, the parent (m/z = 113) ion was
degraded during MS/MS bombardment to produce substantial
−
quantities of a m/z = 81 ion, the expected mass of an HSO3
ion resulting from the release of CH218O from the m/z = 113
METHODS
■
HMS− adduct of H2C18O and HSO3 (Supporting Figure
Methods are available in Supporting Information.
−
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Letters
nonheme oxygenase that catalyzes oxidative demethylation. J. Mol.
Biol. 392, 498−510.
(13) Locher, H. H., Leisinger, T., and Cook, A. M. (1991) 4-Toluene
sulfonate methyl-monooxygenase from Comamonas testosteroni T-2:
purification and some properties of the oxygenase component. J.
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(14) Chakrabarty, S., Austin, R. N., Deng, D., Groves, J. T., and
Lipscomb, J. D. (2007) Radical intermediates in monooxygenase
reactions of rieske dioxygenases. J. Am. Chem. Soc. 129, 3514−3525.
(15) van der Donk, W. A., Krebs, C., and Bollinger, J. M., Jr. (2010)
Substrate activation by iron superoxo intermediates. Opin. Struct. Biol.
20, 673−683.
ASSOCIATED CONTENT
■
S
* Supporting Information
Methods and Supporting Figures S1−S12. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: dweeks1@unl.edu.
Notes
The authors declare the following competing financial
interest(s): The University of Nebraska has licensed dicamba
resistance gene technology for commercial development and
D.P.W. receives royalties from that licensing.
■
(16) Rocha, F. R., Coelho, L. H., Lopes, M. L., Carvalho, L. R., da
Silva, J. A., do Lago, C. L., and Gutz, I. G. (2008) Environmental
formaldehyde analysis by active diffusive sampling with a bundle of
polypropylene porous capillaries followed by capillary zone electro-
phoretic separation and contactless conductivity detection. Talanta 76,
271−275.
ACKNOWLEDGMENTS
■
We thank J. Adamec for helpful comments regarding mass
spectrometry analyses in the manuscript. This work was
supported by a grant from the USDA (Hatch Multistate NC-
1168) to D.P.W.
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(17) Oppenheimer, N. J., Henehan, G. T., Huete-Perez, J. A., and Ito,
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K. T. (2002) Crystal structure of formaldehyde dehydrogenase from
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