Stereochemical investigations reveal the mechanism of the bacterial activation of n-alkanes without oxygen

Article abstract of DOI:10.1002/anie.201106055

Anaerobic growth of the bacterium strain HxN1 with n-hexane gives nearly equal amounts of (2R,1 R)- and (2S,1 R)-(1-methylpentyl)succinate, which are formed by the radical addition of the hydrocarbon to fumarate (see scheme). The highly selective attack on the pro-S hydrogen atom at C2 of n-hexane is associated with inversion of the configuration at C2 during binding to fumarate and exhibits isotopic discrimination against a C-²H bond. Copyright

Full text of DOI:10.1002/anie.201106055

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DOI: 10.1002/anie.201106055
Alkane Activation
Stereochemical Investigations Reveal the Mechanism of
the Bacterial Activation of n-Alkanes without Oxygen**
Renꢀ Jarling, Masih Sadeghi, Marta Drozdowska, Sven Lahme, Wolfgang Buckel,
Ralf Rabus, Friedrich Widdel, Bernard T. Golding,* and Heinz Wilkes*
Dedicated to Professor Wittko Francke
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Certain heterotrophic bacteria conserve energy by coupling
the complete oxidation of n-alkanes to carbon dioxide with
the reduction of different electron acceptors. Aerobic n-
alkane-utilizing bacteria, which were first recognized at the
beginning of the last century, use dioxygen not only as the
terminal electron acceptor, but also as the co-substrate for
enzymatic reactions that transform these inert substrates to
oxygen-containing metabolites suitable for further degrada-
tion.[1] Owing to the specific function of dioxygen in the
activation of n-alkanes in aerobic bacteria, utilization of these
substrates by anaerobic bacteria under strictly anoxic con-
ditions has long been considered impossible. However, in the
last two decades numerous denitrifying, iron(III)-reducing,
and sulfate-reducing bacteria have been described that
oxidize n-alkanes and other hydrocarbons to CO2 in the
absence of molecular oxygen.[2] n-Alkanes contain exclusively
À
apolar C H s bonds, making homolytic mechanisms of
enzymatic activation most likely.[3] Accordingly, anaerobic
bacteria frequently employ glycyl radical enzymes to achieve
selective removal of a hydrogen atom from C2 of n-alkanes,
resulting in addition of an alk-2-yl radical to the double bond
of fumarate (2; Scheme 1).[4,5]
Mechanistically, this reaction appears to be similar to the
formation of benzylsuccinate from toluene catalyzed by the
glycyl radical enzyme benzylsuccinate synthase.[6] Indeed,
EPR spectroscopy provided strong evidence for the presence
of a glycyl radical enzyme in cells of the denitrifying
betaproteobacterium “Aromatoleum” strain HxN1 anaerobi-
cally grown with n-hexane.[4] A tentative (1-methylalkyl)suc-
cinate synthase similar to benzylsuccinate synthase has been
identified.[7] However, abstraction of a hydrogen atom from
Scheme 1. Initial steps of the anaerobic oxidation of n-hexane in the
denitrifying strain HxN1[9] including the proposed stereochemistry of
the reactions involved as elucidated in this study. a) (1-Methylalkyl)-
succinate synthase; b) (1-methylalkyl)succinate-CoA ligase; c) (1-meth-
ylalkyl)succinyl-CoA epimerase; d) (2-methylalkyl)malonyl-CoA mutase;
e) (2-methylalkyl)malonyl-CoA decarboxylase; f) 4-methylalkanoyl-CoA
dehydrogenase.
under anoxic conditions, we have studied the stereochemical
À
features of this defining example of C H activation.
Analysis of metabolites present in cells of strain HxN1
anaerobically grown with n-hexane (1) had shown that the
formed (1-methylpentyl)succinate (3) consists of two diaste-
reoisomers,[4] indicating an apparent imperfect stereoselec-
tivity of the enzymatic reaction. The analogous formation of
benzylsuccinate from toluene by benzylsuccinate synthase
yields exclusively the R enantiomer.[10] Anaerobic incubation
of strain HxN1 with perdeuterated n-hexane revealed that the
hydrogen atom abstracted from C2 of the n-alkane is
transferred to C3 of the succinate moiety.[4] It has been
suggested that subsequent degradation of 3 by means of
activation as a coenzyme A thioester, intramolecular rear-
rangement to (2-methylhexyl)malonyl-CoA (5), and decar-
boxylation leads to 4-methyloctanoyl-CoA (6), which is then
further degraded by dehydrogenation and b-oxidation
(Scheme 1).[9]
À
any C H bond of an n-alkane is intrinsically more difficult
than such a process at the methyl group of toluene.[8] To better
understand the mechanism of n-alkane functionalization
[*] R. Jarling, Priv.-Doz. Dr. H. Wilkes
Organische Geochemie, Helmholtz-Zentrum Potsdam
Deutsches GeoForschungsZentrum GFZ
Haus B228, Telegrafenberg, 14473 Potsdam (Germany)
E-mail: wilkes@gfz-potsdam.de
M. Sadeghi, M. Drozdowska, Prof. Dr. B. T. Golding
School of Chemistry, Bedson Building
Newcastle University, NE1 7RU Newcastle upon Tyne (UK)
E-mail: bernard.golding@ncl.cac.uk
S. Lahme, Prof. Dr. R. Rabus
Institut fꢀr Chemie und Biologie des Meeres, Universitꢁt Oldenburg
Carl-von-Ossietzky Strasse 9-11, 26111 Oldenburg (Germany)
To elucidate the configuration at the newly formed
stereocenters we synthesized all four stereoisomers of 3 as
standards for comparison. This was achieved from racemic
and pure (R)- and (S)-hexan-2-ol through activation of the
hydroxy group and displacement with diethyl malonate,
followed by alkylation with ethyl bromoacetate, hydrolysis
of the ester groups, and decarboxylation (see the Supporting
Information). The mixtures of 3 resulting from (S)-hexan-2-ol
[(2R,1’R) and (2S,1’R) isomers 3a and 3d] and (R)-hexan-2-ol
[(2S,1’S) and (2R,1’S) isomers 3b and 3c] were used directly
for analysis, whereas the mixture from racemic hexan-2-ol was
separated into the (2R,1’R)/(2S,1’S) isomers (3a and 3b) and
(2R,1’S)/(2S,1’R) isomers (3c and 3d) by fractional recrystal-
S. Lahme, Prof. Dr. R. Rabus, Prof. Dr. F. Widdel
Max-Planck-Institut fꢀr Marine Mikrobiologie
Celsiusstrasse 1, 28359 Bremen (Germany)
Prof. Dr. W. Buckel
Max-Planck-Institut fꢀr Terrestrische Mikrobiologie
Karl-von-Frisch-Strasse 10, 35043 Marburg (Germany)
[**] We thank Gçran Braedt, Cornelia Karger, and Daniela Lange for
technical support. This project was funded by the Deutsche
Forschungsgemeinschaft (program SPP 1319; H.W., R.R., B.T.G.,
and W.B.), COST Action CM0603 (B.T.G), the Max-Planck-Gesell-
schaft (F.W. and W.B.), the University of Oldenburg (R.R.), and the
Helmholtz Gemeinschaft (H.W.).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201106055.
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Scheme 2. Synthesis of (2S,5S)-n-(2,5-2H2)hexane (10a). a) p-Toluene-
sulfonyl chloride, pyridine in dichloromethane (p-toluenesulfonyl=Ts) ;
b) LiAl2H4 in tetraglyme (see the Supporting Information for full
experimental details). Also shown are the structures of other dideuter-
ated n-hexanes used in this study.
Figure 1. Gas chromatograms showing the specific formation of the
(2R,1’R) and (2S,1’R) isomers of 3 during anaerobic growth of strain
HxN1 with 1. A) Synthetic mixture of all four stereoisomers; B) prod-
ucts formed by strain HxN1. For GC separation, the stereoisomers of
3 were derivatized with (R)-1-phenylethanamine (see the Supporting
Information).
was greater than 98%. Previously, stereoisomeric n-(2,3-
2H2)butanes of relatively low isotopic purity were used to
study the aerobic oxidation of n-butane by the particulate
methane monooxygenase (p-MMO) from Methylococcus
capsulatus (Bath).[12]
Incubation experiments were performed with cultures of
strain HxN1 that had been adapted to n-heptane using a
mixture of n-heptane and a dideuterated n-hexane (8:1), to
avoid the formation of unlabeled 3. Rotational symmetry of
the n-(2,5-2H2)hexane isomers forces the enzyme to attack a
configurationally defined CHD center and simplifies the
product analysis. Complete transfer of a deuterium atom to
the succinate moiety of 3 was observed upon addition of 10a,
while essentially no deuterium transfer occurred with 10b
(Figure 2). This proves that in the initial step of the catalytic
cycle of the (1-methylpentyl)succinate-forming enzyme
exclusively the pro-S hydrogen atom is abstracted from C2
of 1. Subsequently, the secondary alkyl species binds with a
fumarate molecule on the face opposite to that from which
the hydrogen atom was abstracted, resulting in the 1’R confi-
guration of 3. Thus, an overall inversion of configuration at C2
of 1 takes place. The formation of 3 is completed by back
transfer of the originally abstracted hydrogen atom, which
regenerates the cysteinyl radical. The hydrogen abstraction
and addition to fumarate may even occur in a concerted
manner, similar to an SN2 reaction (Scheme 3). Such a
mechanism could explain how the high difference in bond
lization. As shown in Figure 1, strain HxN1 specifically forms
equal amounts of 3a and 3d during anaerobic growth with 1.
This demonstrates that both configurations at C2 in the
succinate moiety are present. However, the stereocenter at
C1’ in the products (C2 of the hydrocarbon substrate) has
exclusively the R configuration. Consequently, solely (R)-4-
methyloctanoyl-CoA (6) is formed as the product of subse-
quent steps in the degradation pathway as revealed by
comparison with synthetic authentic standards of (R)- and
(S)-4-methyloctanoic acid (see the Supporting Information).
The formation of a stereocenter at a secondary alkyl
moiety with complete stereocontrol suggested that the
abstraction of the hydrogen atom from the n-alkane must
also proceed in a stereoselective manner. To investigate this
point, we traced the fate of deuterium atoms in stereospe-
cifically labeled n-(2,5-2H2)hexane isomers 10a–c and in n-
(2,2-2H2)hexane (11) during anaerobic growth of strain HxN1.
For this purpose, a strategy for the synthesis of (R,R)-, (S,S)-,
and (meso)-n-(2,5-2H2)hexane from (S,S)-, (R,R)-, and
(meso)-hexane-2,5-diol, respectively, was developed; the syn-
thesis of 10a is representative of the sequence employed
(Scheme 2). Diol 8 was converted into di-p-toluenesulfonate
9, which was reduced by lithium aluminum deuteride in
tetraethyleneglycol dimethyl ether (tetraglyme) with inver-
sion of configuration (SN2) at each stereocenter.[11] We used
the high-boiling solvent tetraglyme (bp = 2768C) so that the
pure deuterated n-hexane (bp ꢀ 698C) could be removed
from the reaction mixture directly by distillation. The
stereochemical assignments for the n-(2,5-2H2)hexane iso-
mers were based on the known sense of chirality of the
precursor diols and confirmed by X-ray analysis of the
derived di-p-toluenesulfonates (see the Supporting Informa-
À
À
dissociation energy (BDE) between a thiol (RS H) and a C
H bond of a methylene group in an alkane (DBDE
ꢀ 40 kJmolÀ1)[8] could be overcome. Hence, the highly
energetic hex-2-yl radical may not exist as a single enzyme-
bound species. The only detectable radical in this reaction
would be that at C3 of the succinate moiety stabilized by the
adjacent carboxylate, similar to the radical detected in the
carbon-skeleton rearrangement catalyzed by glutamate
mutase.[13] With 10c and 11, mixtures of dideuterated
isotopologues of 3 were formed in which the main product
was formed by the transfer of the pro-S hydrogen atom from
C2 and C5, respectively (see the Supporting Information).
This indicates a significant primary kinetic isotope effect (ꢁ 3)
for the abstraction of the hydrogen atom.
1
tion). The deuterated n-hexanes were characterized by H,
2H, and 13C NMR spectroscopy and by electron ionization
mass spectrometry (EIMS; see the Supporting Information).
The estimated 2H content at each isotopically labeled position
Considering the proven stereoselectivity of benzylsucci-
nate synthase,[10] the formation of equal amounts of diaste-
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Scheme 3. Proposed mechanism of the (1-methylpentyl)succinate-
forming enzyme reaction. Please note that the active form of the
enzyme is generated by abstraction of a hydrogen atom from an
adjacent cysteine residue by the glycyl storage radical.[3]
ferases, representing the only known example of a CoA
racemase of this relatively new family.[15] Other members of
this superfamily are (E)-cinnamoyl-CoA:(R)-phenyllactate
CoA-transferase and succinyl-CoA:(R)-benzylsuccinate
CoA-transferase.[16] We thus speculate that in strain HxN1
activation of 3 and epimerization of 4 might be catalyzed by
one and the same enzyme.
It is notable that exclusively the fumarate- rather than the
n-hexane-derived hydrogen atom at C3 of 3 migrates during
the carbon-skeleton rearrangement of 4 to 5 (Scheme 1).[9]
Further, the n-hexane-derived hydrogen atom at C2 and the
fumarate-derived hydrogen atom at C3 of 6 are exclusively
removed during the dehydrogenation to 4-methyloct-2-enoyl-
CoA (7). This observation is in accord with the expected
stereochemistry of hydrogen removal during the first step of
b-oxidation catalyzed by acyl-CoA dehydrogenase.[17] Con-
sidering that the analogous decarboxylation/transcarboxyla-
tion of methylmalonyl-CoA and the intramolecular rear-
rangement catalyzed by methylmalonyl-CoA mutase proceed
with retention of configuration,[18] we propose the overall
stereochemical course of the (1-methylpentyl)succinate-
forming reaction and subsequent steps leading to 7 shown in
Scheme 1. This requires a syn addition of 1 to 2, which is in
agreement with the proposed mechanism of benzylsuccinate
synthase.[19] The only difference from the conversion of
succinyl-CoA via (R)- and (S)-methylmalonyl-CoA to pro-
pionyl-CoA is the postulated decarboxylation/transcarboxy-
lation of (2R,2’R)-(2-methylhexyl)malonyl-CoA (5) rather
than the (2S,2’R) isomer (for details see the Supporting
Information). This would require a second epimerization,
which is not consistent with retention of the n-hexane-derived
hydrogen at C2 of 6.
Figure 2. Mass spectra showing the labeling patterns of deuterated
isotopologues of 3 (as dimethyl esters after derivatization with diazo-
methane) formed during anaerobic growth of strain HxN1 in the
presence of stereospecifically deuterated n-hexanes. A) 3 formed from
1. B) (1’,4’-2H2)-3 formed from 10b; C) (3,4’-2H2)-3 formed from 10a.
Depicted are the mass spectra of the (2R,1’R) isomers; essentially
identical mass spectra were obtained for the corresponding (2S,1’R)
isomers. The fragment ion at m/z 199 ([MÀ31]+ resulting from loss of
-OCH3 from one of the methyl ester moieties) in (A) is shifted to m/z
201 in (B) and (C), indicating the presence of two deuterium atoms.
The fragment ion at m/z 157 in (A) is shifted to m/z 159 and m/z 158
in (B) and (C), respectively. The fragment ions at m/z 114 and m/z 146
in (A) are shifted to m/z 115 and m/z 147, respectively, in (C), while
no shift is observed in (B). Thus exclusively the pro-S hydrogen atom
is abstracted from the n-alkane.
reoisomers 3a and 3d is unlikely to be the result of a non-
stereoselective addition of the hex-2-yl radical to fumarate.
Rather, the complete exchange with external hydrogen of the
deuterium atom at C2 of 3 derived from (2,3-2H2)fumarate[4]
points to an epimerization. A precedent for such a reaction is
that catalyzed by a-methylacyl-CoA racemase,[14] an enzyme
required for the metabolism of certain 2-methyl fatty acids
with R configuration, as only the S isomers are substrates for
b-oxidation. We therefore suggest that the initially formed
isomer of 3, as the CoA-thioester 4, is epimerized to generate
the diastereoisomer required by the putative mutase. Notably,
the homologue of a-methylacyl-CoA racemase from Myco-
bacterium tuberculosis belongs to the family III CoA-trans-
Overall, our results document a highly stereospecific
À
anaerobic C H activation of an n-alkane with stereochemical
features in complete contrast to the aerobic oxidations of
methylene groups by cytochrome P450 monooxygenases and
other monooxygenases for which a retention pathway is
preferred.[12,20] Further investigations to confirm the proposed
epimerization of an initially formed diastereoisomer of 3 with
defined configuration at C2 are underway.
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[13] H. Bothe, D. J. Darley, S. P. J. Albracht, G. J. Gerfen, B. T.
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Received: August 26, 2011
Published online: November 30, 2011
À
Keywords: alkanes · C H activation · enzyme mechanisms ·
radical enzymes · stereochemistry
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