Dibenzothiophene Catabolism Proceeds via a Flavin-N5-oxide Intermediate

Article abstract of DOI:10.1021/jacs.6b00583

The dibenzothiophene catabolic pathway converts dibenzothiophene to 2-hydroxybiphenyl and sulfite. The third step of the pathway, involving the conversion of dibenzothiophene sulfone to 2-(2-hydroxyphenyl)-benzenesulfinic acid, is catalyzed by a unique flavoenzyme DszA. Mechanistic studies on this reaction suggest that the C2 hydroperoxide of dibenzothiophene sulfone reacts with flavin to form a flavin-N5-oxide. The intermediacy of the flavin-N5-oxide was confirmed by LC-MS analysis, a co-elution experiment with chemically synthesized FMN-N5-oxide and ¹⁸O₂ labeling studies.

Full text of DOI:10.1021/jacs.6b00583

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Communication
Dibenzothiophene Catabolism proceeds via a Flavin-N5-oxide Intermediate.
Sanjoy Adak, and Tadhg P. Begley
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.6b00583 • Publication Date (Web): 27 Apr 2016
Downloaded from http://pubs.acs.org on April 28, 2016
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Dibenzothiophene Catabolism proceeds via a Flavin-N5-oxide
Intermediate
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Sanjoy Adak and Tadhg P. Begley*
Department of Chemistry, Texas A&M University, 3255 TAMU, College Station, TX 77843, USA.
Supporting Information Placeholder
gives 9. Ring closure and electron transfer to the Flavin
ABSTRACT: The dibenzothiophene catabolic pathway
converts dibenzothiophene to 2-hydroxybiphenyl and sulfite.
The third step of the pathway, involving the conversion of
semiquinone gives 11. Hydrolysis of 11 completes the reac-
tion. While the initial electron transfer to form 6 is likely to be
unfavorable, rapid rearrangement of 6 to 10 could drive the
reaction.
dibenzothiophene
sulfone
to
2-(2-hydroxyphenyl)-
benzenesulfinic acid, is catalyzed by a unique flavoenzyme
DszA. Mechanistic studies on this reaction suggest that the
C2 hydroperoxide of dibenzothiophene sulfone reacts with
flavin to form a flavin-N5-oxide. The intermediacy of the
flavin-N5-oxide was confirmed by LC-MS analysis, a coelu-
tion experiment with chemically synthesized FMN-N5-oxide
and 18O2 labeling studies.
DszD/NADH
DszC/FMN
DszD/NADH
DszC/FMN
S
S
S
1
2
3
O
O
O
HO
DszD/NADH
DszA/FMN
HO
DszB
S
OH
5
4
Acid rain is formed from sulfur dioxide generated during
fossil fuel combustion. The removal of sulfur from these
fuels is an important step in pollution control. Metal-
catalyzed hydrodesulfurization is only partially successful
because it is inefficient with sulfur-containing heterocyclic
compounds. Dibenzothiophene is the major sulfur-
containing heterocycle in crude petroleum.1 The possibility
of removing this compound by microbial degradation has
led to the discovery of its catabolic pathway in Rhodococcus
erythropolis (Figure 1).2 The first two steps3 of this pathway
involve well-precedented flavin-hydroperoxide-mediated
thioether oxidation4 to sulfone 3 via sulfoxide 2 and the last
step5 most likely involves bisulfite loss in an ipso substitu-
tion reaction. The mechanism of the third step, involving
conversion of sulfone 3 to sulfinic acid 4, is not obvious and
does not conform to any of the well-established motifs in
flavoenzymology. This communication describes studies to
elucidate the mechanism of this reaction.
O
Figure 1: The four-step dibenzothiophene catabolic path-
way in Rhodococcus erythropolis.
S
S
6
3
O
O
O
O
R
N
R
N
S
H
N
H
N
FlH
8
O
O
O
O
9
NH
NH
N
N
FlH-
FlH
8
O
O
7
HO
DszA was overexpressed in E.coli BL21(DE3) and puri-
fied by Ni-affinity chromatography. The protein overex-
pressed well (6 mg /liter) and was purified to homogeneity
(Figure S1). The DszA-catalyzed reaction was reconstituted
by incubating sulfone (3), DszA, E.coli flavin reductase (to
replace DszD), NADH and FMN at 25 ˚C. The formation of
sulfinic acid 4 was monitored by HPLC analysis (Figure S2).
FlH
8
S
O
10
FlH-
FlH-
S
O
11
S
OH
4
O
O
O
7
7
Figure 2: The SRN1 mechanistic proposal for the DszA-
catalyzed reaction.
The previously reported SRN1 reaction of dibenzothio-
phene sulfone 3 with alkoxides to form the corresponding
alkylated analog of 4 served as our first model for the en-
zymatic reaction6 and the corresponding SRN1 proposal7 for
DszA is shown in Figure 2. In this proposal, electron trans-
fer from FlH- to the electron deficient sulfone 3 gives the
sulfone radical anion 6. Fragmentation of the C-S bond
This proposal predicts that the DszA-catalyzed reaction is
oxygen independent. To test this, the DszA reaction was
run under anaerobic conditions. No product was formed.
The DszA reaction was also run in H2O18 buffer to look for
the oxygen incorporation expected for the hydrolysis of 11
to 4. No oxygen incorporation was detected (Figure 3A). In
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contrast, when the enzymatic assay was carried out in the
Villiger oxidation of ketones8 and RutA-catalysed uracil ring
presence of 18O2, a 2 Da increase in the mass of the prod-
uct was observed consistent with the incorporation of a sin-
gle oxygen atom from molecular oxygen (Figure 3B). Oxy-
gen incorporation into the phenol rather than the sulfinate
was established by demonstrating retention of label when
[18O1]-4 was converted to 5 using DszB (Figure S3). These
experiments eliminate the possibility that the DszA reaction
proceeds by an SRN1 mechanism.
opening reaction.9 In this proposal (Figure 4), addition of the
flavin hydroperoxide to dibenzothiophene sulfone gives the
sulfone-stabilized carbanion 13. Protonation, followed by
flavin elimination gives hydroperoxide 14. NADH mediated
reduction of this hydroperoxide, as previously observed for
the RutA system, gives 16, which is converted to product by
a ring opening/tautomerization/bond rotation sequence. A
Baeyer Villiger-like mechanism, involving flavin peroxide
attack on the sulfone sulfur, results in the formation of a
sulfonic rather than a sulfinic acid and is therefore not pos-
sible. The normal mechanism of flavoenzyme mediated
peroxide cleavage10 is also not possible because DszA is
cysteine free.
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A)
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This mechanism predicts that it should be possible to
trap hydroperoxide 14 if the DszA reaction is run in the ab-
sence of excess reducing agent. These reaction conditions
were achieved by initial photoreduction of the flavin using
EDTA and by running the reaction in phosphate buffer using
stoichiometric amounts of photoreduced FMN, DszA and
dibenzothiophene sulfone. The reaction mixture did not con-
tain DTT, protein thiol, free FMN or NADH and the photo-
generated glyoxylic acid was not consumed (Figure S5). It
was prepared under anaerobic conditions in a glove box
and then exposed to air to allow formation of the enzyme
bound flavin hydroperoxide. Under these reaction condi-
tions, sulfinic acid 4 was the major reaction product and
peroxide 14 was not detected raising the question as to how
peroxide 14 is reduced.
B)
In the absence of any obvious reducing agent in the
reaction buffer, the possibility of a flavin-N5-oxide was con-
sidered (Mechanism 3, Figure 4). This new flavin oxidation
state was recently identified in the EncM-catalyzed Favor-
skii rearrangement involved in enterocin biosynthesis.11-13
HPLC analysis of the DszA reaction mixture (containing
stoichiometric amounts of photoreduced FMN, DszA and
dibenzothiophene sulfone) demonstrated the consumption
of FMN and the formation of a new species eluting after
20.3 minutes (Figure 5A). This species comigrates with and
has an identical UV-Visible spectrum to a synthetic sample
of FMN-N5-oxide (Figures 5B and 5C). MS analysis of I20.3
is also consistent with the FMN-N5-oxide structure (Figure
Figure 3: LC-MS analysis of the DszA reaction run in [18O]-
H2O/16O2 buffer or H2O/18O2. Panel A shows that oxygen
from the buffer is not incorporated into the reaction product
4. Panel B shows the incorporation of a single oxygen atom
from molecular oxygen. m/z for 4 and [18O1]-4 are 235 Da
and 237 Da respectively.
We next considered the possibility of a nucleophilic addition
of flavin hydroperoxide 12 to 3. The nucleophilicity of 12 is
well established in the flavoenzyme-mediated Baeyer-
Figure 4: The proposed DszA-catalyzed dibenzothiophene sulfone ring opening via a flavin hydroperoxide intermediate.
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Figure 5: Characterization of a new intermediate in the DszA-catalyzed reaction. (A) Partial HPLC chromatogram of the
DszA/photoreduced FMN reaction mixture showing the new intermediate (I20.3) eluting at 20.3 min. The peak eluting at 20.1
min corresponds to an impurity present in commercial FMN. (B) Co-elution of I20.3 with synthesized FMN-N5-oxide. (C) UV-
Visible spectra of FMN and FMN-N5-oxide. The I20.3 spectrum was identical to that of FMN-N5-oxide. D) Extracted ion
chromatograms for [M+H] = 473.1 Da demonstrate that I20.3 is present only in the full reaction mixture. E) Exact mass of
I20.3 consistent with the mass expected for FMN-N5-oxide ([M+H] = 473.11 Da). F) Exact mass of I20.3 generated using 18O2
consistent with the mass expected for [18O1]-FMN-N5-oxide ([M+H] = 475.11 Da).
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5D and 5E) and running the DszA reaction in the presence
terial is available free of charge via the Internet at
of 18O2 showed the expected 2 Da mass increase (Figure
http://pubs.acs.org
5F). Finally, the ratio of FMN consumed to sulfinic acid
formed in a reaction containing stoichiometric amounts of
photoreduced FMN, DszA and dibenzothiophene sulfone
was 1:0.9 consistent with the stoichiometric conversion of
FMN to FMN-N5-oxide during the course of the DszA cata-
lysed reaction (Figure S4).
AUTHOR INFORMATION
Corresponding Author
begley@chem.tamu.edu
Our studies on DszA suggest a mechanism involving
initial formation of flavin hydroperoxide from reduced flavin.
This adds to 3 to form the sulfone-stabilized carbanion 13.
Protonation followed by flavin elimination gives 14 and 15.
Flavin mediated peroxide cleavage14,15 would form 16 and
17. The catalytic cycle is then completed by the conversion
of 16 to 4 and 17 to 15 (Figure 4).
ACKNOWLEDGMENT
This research was supported by the Robert A. Welch Foun-
dation (A-0034 to TPB) and by a grant from the National
Institutes of Health (DK44083).
REFERENCES
(1) Soleimani, M.; Bassi, A.; Margaritis, A. Biotechnology
advances 2007, 25, 570.
(2) Gray, K. A.; Pogrebinsky, O. S.; Mrachko, G. T.; Xi, L.;
Monticello, D. J.; Squires, C. H. Nature biotechnology 1996,
14, 1705.
(3) Liu, S.; Zhang, C.; Su, T.; Wei, T.; Zhu, D.; Wang, K.;
Huang, Y.; Dong, Y.; Yin, K.; Xu, S.; Xu, P.; Gu, L. Proteins:
Struct., Funct., Bioinf. 2014, 82, 1708.
(4) Ryerson, C. C.; Ballou, D. P.; Walsh, C. Biochemistry
1982, 21, 2644.
(5) Lee, W. C.; Ohshiro, T.; Matsubara, T.; Izumi, Y.;
Tanokura, M. J. Biol.Chem. 2006, 281, 32534.
(6) Aida, T.; Squires, T. G.; Venier, C. G. Tetrahedron Lett.
1983, 24, 3543.
(7) Rossi, R. A.; Pierini, A. B.; Penenory, A. B. Chem. Rev.
(Washington, DC, U. S.) 2003, 103, 71.
Decades ago flavin-N5-oxide 17 was considered as an
intermediate in flavin-dependent oxygenase chemistry but
was almost entirely replaced by the flavin hydroperoxide
mechanistic paradigm.11,12 Since FMN-N5-oxide and organ-
ic peroxides are easily reduced by widely used biochemical
reducing agents, (DTT, TCEP, NADH etc.)16, it is easy to
miss this intermediate and a systematic search for other
FMN-N5-oxide mediated reactions is merited.
ASSOCIATED CONTENT
Supporting Information
Detailed experimental procedures for the syntheses of 4
and 17, DszA and DszB overexpression and purification,
enzymatic assays, and NMR and LC-MS analysis.This ma-
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(8) Walsh, C. T.; Chen, Y. C. J. Angew. Chem. 1988, 100,
342.
1
(9) Mukherjee, T.; Zhang, Y.; Abdelwahed, S.; Ealick, S. E.;
Begley, T. P. J. Am. Chem. Soc. 2010, 132, 5550.
(10) Hall, A.; Karplus, P. A.; Poole, L. B. The FEBS journal
2009, 276, 2469.
(11) Teufel, R.; Stull, F.; Meehan, M. J.; Michaudel, Q.;
Dorrestein, P. C.; Palfey, B.; Moore, B. S. J. Am. Chem.
Soc. 2015, 137, 8078.
2
3
4
5
6
7
8
9
(12) Teufel, R.; Miyanaga, A.; Michaudel, Q.; Stull, F.;
Louie, G.; Noel, J. P.; Baran, P. S.; Palfey, B.; Moore, B. S.
Nature (London, U. K.) 2013, 503, 552.
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11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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30
31
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48
49
50
51
52
53
54
55
56
57
58
59
60
(13) Teufel, R.; Agarwal, V.; Moore, B. S. Curr. Opin. Chem.
Biol. 2016, 31, 31.
(14) Litvak, Z. I.; Berezovskii, V. M. Zh. Obshch. Khim.
1981, 51, 464.
(15) Yoneda, F.; Sakuma, Y.; Ichiba, M.; Shinomura, K. J.
Am. Chem. Soc. 1976, 98, 830.
(16) Lavilla, R.; Baron, X.; Coll, O.; Gullon, F.; Masdeu, C.;
Bosch, J. J. Org. Chem. 1998, 63, 10001.
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HO
Flavin
Flavin-4a-OOH
S
S
H
O
S
OH
O
O
O
O
OH
O
Flavin
Flavin-N5-oxide
5
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