Regioselectivity of the oxidative C-S bond formation in ergothioneine and ovothiol biosyntheses

Article abstract of DOI:10.1021/ol402275t

Ergothioneine (5) and ovothiol (8) are two novel thiol-containing natural products. Their C-S bonds are formed by oxidative coupling reactions catalyzed by EgtB and OvoA enzymes, respectively. In this work, it was discovered that in addition to catalyzing the oxidative coupling between histidine and cysteine (1 → 6 conversion), OvoA can also catalyze a direct oxidative coupling between hercynine (2) and cysteine (2 → 4 conversion), which can shorten the ergothioneine biosynthetic pathway by two steps.

Full text of DOI:10.1021/ol402275t

ORGANIC
LETTERS
2
013
Vol. 15, No. 18
854–4857
Regioselectivity of the Oxidative CꢀS
Bond Formation in Ergothioneine and
Ovothiol Biosyntheses
4
Heng Song, Maureen Leninger, Norman Lee, and Pinghua Liu*
Department of Chemistry, Boston University, Boston, Massachusetts 02215, United States
pinghua@bu.edu
Received August 9, 2013
ABSTRACT
Ergothioneine (5) and ovothiol (8) are two novel thiol-containing natural products. Their CꢀS bonds are formed by oxidative coupling reactions
catalyzed by EgtB and OvoA enzymes, respectively. In this work, it was discovered that in addition to catalyzing the oxidative coupling between
histidine and cysteine (1 f 6 conversion), OvoA can also catalyze a direct oxidative coupling between hercynine (2) and cysteine (2 f 4
conversion), which can shorten the ergothioneine biosynthetic pathway by two steps.
Sulfur is an important functional group in both pri-
1
mary and secondary metabolites. Biological sulfur trans-
examples are the incorporation of the thiol functional
5
4
3a
groups into biotin, lipoate, thiamine pyrophosphate,
6
and molybdopterin cofactor, cofactor biogenesis in galac-
fer reactions can make use of either an ionic or radical
type of reaction mechanism. For example, the thioether
formation in lantibiotic biosynthesis is an ionic type of
reaction that is accomplished by having thiolates as the
7
tose oxidase, tRNA nucleotide thiolmethylation, and
8
thiazolidine ring formation catalyzed by isopenicillin N
9
synthase. For sulfur transfer reactions, two types of acti-
10
vated sulfur species have also been discovered, which are
2
direct nucleophiles. Radical type CꢀS bond formation
1
a,3
reactions are also well-documented.
Some of the
persulfides RꢀSꢀSH and thiocarboxylates RꢀCOꢀSH.
Ergothioneine (5) and ovothiol (8) are two thiolꢀ
imidazole-containing natural products. Humans obtain
ergothioneine from their diet and specifically enrich it in
some tissues (e.g., liver, kidney, central nervous system,
and red blood cells) by using an ergothioneine-specific
(
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Liu, H.-w. Chem. Soc. Rev. 2013, 42, 4377. (f) Parry, R. J. In Compre-
hensive Natural Products Chemistry I; Barton, D., ; Nakanishi, K.,
Meth-Cohn, O., Ed.; Pergamon: Oxford, U.K., 1999, Vol. 1, p 825. (g)
Wang, L.; Chen, S.; Xu, T.; Taghizadeh, K.; Wishnok, J.; Zhou, X.;
You, D.; Deng, Z.; Dedon, P. Nat. Chem. Biol. 2007, 3, 709. (h) Scharf,
D. H.; Remme, N.; Habel, A.; Chankhamjon, P.; Scherlach, K.; Heine-
kamp, T.; Hortschansky, p.; Brakhage, A. A.; Hertweck, C. J. Am. Chem.
Soc. 2011, 133, 12322. (i) Wang, Q.; Song, F.; Xiao, X.; Huang, P.;
Monte, A.; Abdel-Mageed, W. M.; Wang, J.; Guo, H.; He, W.; Xie, F.;
Dai, H.; Liu, M.; Chen, C.; Xu, H.; Liu, M.; Piggott, A. M.; Liu, X.;
Capon, R. J.; Zhang, L. Angew. Chem., Int. Ed. 2013, 52, 1231.
1
1
transporter. Ergothioneine’s beneficial effects to hu-
man health are due to its unusual redox properties, which
(5) Jurgenson, C. T.; Begley, T. P.; Ealick, S. E. Annu. Rev. Biochem.
2009, 78, 569.
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Biol. 2007, 11, 543. (b) Parry, R. J. Tetrahedron 1983, 39, 1215.
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Proteomics 2012, 1824, 1213.
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D. C.; Dean, D. R.; Smith, A. D.; Johnson, M. K. Annu. Rev. Biochem.
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6
(
(
1
0.1021/ol402275t r 2013 American Chemical Society
Published on Web 09/09/2013

while ovothiol’s thiol group is at its imidazole δ carbon. In
addition, EgtB and OvoA use different substrates. EgtB
catalyzes the oxidative coupling between hercynine (2)
and γ-Glu-Cys, while OvoA preferentially oxidatively
couples His and Cys. In this report, a key factor governing
OvoA-catalyzed regioselectivity was discovered. By systematic
modulation of the histidine methylation state, the OvoA
catalysis changes from an OvoA type of chemistry (1 f 6
conversion) to an EgtB type of chemistry (2 f 4 conversion).
Such a discovery can shorten the ergothioneine biosynthetic
pathway by two steps.
Scheme 1. Proposed Ergothioneine and Ovothiol Biosynthetic
Pathways
favor predominantly the thione form (5b, Scheme 1A)
and make ergothioneine much more stable than most
1
c,d,11g
other natural thiols.
from ergot by Tanret in 1909, and its biosynthetic gene
Ergothioneine was isolated
1
2
1
3
cluster was reported by Seebeck in 2010 (Scheme 1A).
Ergothioneine biosynthesis starts from histidine methyla-
tion to trimethylated histidine (hercynine, 2), which is
then oxidatively coupled with γ-glutamylcysteine (γ-Glu-
Cys) to 3. After glutamate is removed by hydrolysis, a
PLP-containing enzyme (EgtE) catalyzes the 4 f 5 con-
version. Ovothiol is another thiolꢀhistidine enriched in the
eggs of many marine species. Ovothiol is proposed to be
1
Figure 1. H NMR assay (chemical shifts of imidazole ring H
atoms). Reaction A: native EgtB reaction in ergothioneine
biosynthesis. The chemical shift assignments are as follows: 3,
the imidazole H atom of compound 3 (7.12 ppm); 2, the
hercynine imidazole H atoms (6.85, 7.58 ppm). Reaction D:
native OvoA reaction in ovothiol biosynthesis. The chemical
shift assignments are as follows: 6, the imidazole H atom of
compound 6 (7.83 ppm); 1, the imidazole H atoms of His (6.93,
7.70 ppm). Reaction F: a new OvoA reaction (oxidatively
coupling between hercynine and Cys). In this case, the oxidative
coupling product is compound 4, which has the EgtB type of
regioselectivity. Reaction list is shown in Scheme 2.
involved in H O scavenging and facilitation of the fertiliza-
2
2
14
tion process. For ovothiol biosynthesis (Scheme 1B), the
only known enzyme is OvoA, which catalyzes the oxidative
1
5
coupling between Cys and His (1 f 6 conversion). In
comparison to ergothioneine, the CꢀS bond in ovothiol is at
the δ instead of the ε position.
EgtB and OvoA were overexpressed in E. coli and purified
anaerobically using Strep-tavidin affinity chromatography
(Figure 1S, Supporting Information). The purified proteins
have close to a stoichiometric amount of iron. To examine
Both EgtB and OvoA are mononuclear nonheme iron
enzymes catalyzing four-electron oxidation processes
(
Scheme 1). However, EgtB and OvoA distinguish them-
1
selves from each other by their substrate preferences and
product CꢀS bond regioselectivity (Scheme 1). Ergothio-
neine’s thiol group is located at its imidazole ε carbon,
EgtB and OvoA substrate specificity, a H NMR assay was
1
utilized (Figure 1). In the H NMR spectrum, the chemical
shifts of the EgtB product (3) imidazole H atom, OvoA
product (6) imidazole H atom, and the substrate (histidine)
are well-separated from the rest of the reaction mixture
(
11) (a) Grundemann, D.; Harlfinger, S.; Golz, S.; Geerts, A.; Lazar,
A.; Berkels, R.; Jung, N.; Rubbert, A.; Schomig, E. Proc. Natl. Acad.
Sci. U.S.A. 2005, 102, 5256. (b) Melville, D. B.; Eich, S. J. Biol. Chem.
(Figure 1). Thus, the reaction mixture can be analyzed routin-
1
956, 218, 647. (c) Fahey, R. C.; Newton, G. L.; Dorian, R.; Kosower,
ely without the need for product separation (Figure 1). The
signals with chemical shifts of 6.93 and 7.70 ppm are assigned
to the histidine imidazole H atoms. The signal at 7.83 ppm is
from the imidazole H atom of ovothiol biosynthetic inter-
mediate 6 (reaction D, Figure 1), while for the oxidative
product 3in ergothioneine biosynthesis, its imidazole H atom
has a chemical shift of 7.12 ppm (reaction A, Figure 1).
E. M. Anal. Biochem. 1981, 111, 357. (d) Genghof, D. S.; Inamine, E.;
Kovalenko, V.; Melville, D. B. J. Biol. Chem. 1956, 223, 9. (e) Briggs, I.
J. Neurochem. 1972, 19, 27. (f) Epand, R. M.; Epand, R. F.; Wong, S. C.
J. Clin. Chem. Clin. Biochem. 1988, 26, 623. (g) Hartman, P. E. Methods
Enzymol. 1990, 186, 310.
(
(
(
12) Tanret, C. Compt. rend. 1909, 149, 222.
13) Seebeck, F. P. J. Am. Chem. Soc. 2010, 132, 6632.
14) Turner, E.; Klevit, R.; Hager, L. J.; Shapiro, B. M. Biochemistry.
1
987, 26, 4028.
15) Braunshausen, A.; Seebeck, F. P. J. Am. Chem. Soc. 2011, 133,
757.
1
Using the H NMR assay, we systematically examined
EgtB and OvoA substrate binding pocket flexibility
(
1
Org. Lett., Vol. 15, No. 18, 2013
4855

Scheme 2. Examining EgtB and OvoA Substrate Specificities
(
Scheme 2). EgtB does not accept OvoA’s substrates as its
alternative substrates (reactions B and C, Scheme 2).
Interestingly, OvoA has much more flexibility in substrate
specificity. OvoA can accept γ-Glu-Cys as a substrate and
oxidatively couples it with His to form compound 15
Figure 2. HMBC-NMR analysis of compound 4. H-5 shows
1
13
Hꢀ C correlations between C-4 (orange circle) and C-6 (cyan
0
arrow). Additional cross-peaks between H-3 and C-6 (blue
circle), H-3 and C-4 (green circle), and H-3 and C-5 (pink circle)
are also shown.
(reaction E, Scheme 2). OvoA also can accept hercynine
(
the H NMR spectrum of this reaction gives a new signal at
2) as a substrate (reaction F, Scheme 2). More interestingly,
1
1
.12 ppm (reaction F, Figure 1). In comparison with the H
7
NMR spectra of the native EgtB reaction (reaction A,
Figure 1) and native OvoA reaction (reaction D, Figure 1),
the 7.12 ppm signal highly suggests that compound 4 is the
product from the OvoA-catalyzed oxidative coupling be-
tween Cys and hercynine (Scheme 2, reaction F in Figure 1
and Figure 11S (Supporting Information)). The formation
of 4 implies a change in OvoA regioselectivity from the
OvoA type (reaction D, Figure 1) to the EgtB type (reaction
F, Figure 1) upon the change of histidine to hercynine.
To provide further evidence to support the structural
assignment of compound 4, it was isolated and character-
Scheme 3. New OvoA Chemistries
1
ized by mass spectrometry and several NMR spectra ( H
1
NMR, C NMR, and 2D-NMR including COSY,
3
HMBC, and HMQC, Figure 2 and Figures 12Sꢀ17S
1
13
(
H-5 and C-4, C-6 in HMBC characterization support the
Supporting Information)). Hꢀ C correlations between
structural assignment of compound 4 (Figure 2). Addi-
0
tional cross-peaks (between H-3 and C-6 and between
Upon the change of histidine to hercynine, OvoA
changes its oxidative CꢀS bond formation regioselectiv-
ity. This result highly suggests that the OvoA binding
pocket for the histidine amino group plays a key role in
determining the imidazole ring binding orientation,
which in turn determines the oxidative CꢀS bond forma-
tion regioselectivity. To test this hypothesis, mono- and
dimethylhistidine were synthesized. Indeed, OvoA can
also make use of them as alternative substrates. Interestingly,
when monomethylhistidine (9) and cysteine are used as the
substrates, OvoA produces two oxidative coupling products,
compound 10 and compound 11, in a ratio of 2:3 (Scheme 3
H-3 and C-4, C-5, Figure 2) are also consistent with the
compound 4 structure. HMQC spectrum suggests that the
only proton in the compound 4 imidazole ring (δ 7.45 ppm)
is at the histidine δ carbon (122 ppm, Figure 16S
(
Supporting Information)), which provides additional
evidence for an OvoA-catalyzed direct 2 f 4 conversion.
In the spectra of Figure 2 and Figure 16S, in order to
resolve the resonances in the 3.0ꢀ4.5 ppm region, the
sample was acidified. As a result, the imidazole ring hydro-
gen chemical shift (Figure 2) is now at 7.45 ppm instead of
7.12 ppm in Figure 1.
4
856
Org. Lett., Vol. 15, No. 18, 2013

regioselectivity of OvoA catalysis changes from the OvoA
type to the EgtB type (Scheme 3). Due to many of
1c,16
ergothioneine’s beneficial roles in human health,
Scheme 4. Two OvoA Mechanistic Routes
there is a longstanding interest in developing more effi-
1
7
cient methods for its production. The discovery of
this unique OvoA chemistry (a direct 2 f 4 conversion)
suggests that such a chemistry may be further explored for
future ergothioneine production through metabolic engi-
neering. This transformation not only shortens the ergothio-
neine biosynthetic pathway by two steps (elimination of
EgtA and EgtC steps; see Scheme 1) but also eliminates the
competition between ergothioneine and glutathione bio-
syntheses because γ-Glu-Cys is a substrate in both the EgtB
reaction and glutathione biosynthesis.
Several mechanistic models have been proposed for
oxidative CꢀS bond formation in EgtB and OvoA
1
5,18
and Figure 19S (Supporting Information)). In the case of
dimethylhistidine (12), compound 13 is the dominant pro-
catalysis since their discovery.
bond formation in OvoA and EgtB seems to be distinct
The oxidative CꢀS
1
duct and compound 14 is barely detectable in our H NMR
from currently known biological CꢀS bond formation
1a,c,d,8
mechanisms.
assay (Scheme 3 and Figure 20S (Supporting Information)).
Results from these studies indicate that the OvoA substrate
binding pocket for the histidine amino group plays a key role
in orienting substrates in the enzyme active site, which in turn
determines the oxidative CꢀS bond formation regioselectiv-
ity (Scheme 3).
Thus far, mechanistic evidence is not yet
available for differentiating among the proposed mechanistic
options in the literature. Both EgtB and OvoA catalyze four-
electron oxidation processes, and two different functional
groups are constructed in these reactions (sulfoxide and CꢀS
bond, Scheme 4). Thus, the first mechanistic issue to be
addressed in OvoA and EgtB catalysis is whether the sulfenic
acid formation (model A) or the CꢀS bond formation
(model B) is the first step. This mechanistic question is
currently under investigation in our laboratory.
In air-saturated HEPES buffer (∼250 μM of oxygen),
these new OvoA reactions were characterized kinetically
by monitoring the oxygen consumption rate using the
NeoFoxy oxygen electrode. (A) When histidine and
cysteine are the substrates, the kinetic parameters are
ꢀ
1
kobs = 572 ( 20 min and K = 420 ( 31 μM for His
m
Acknowledgment. This work was partially supported
by an NSF award (CHE-1309148) and an NIH award
and Km = 300 ( 34 μM for Cys. (B) When Cys and
monomethylhistidine (9) are the substrates, the kinetic
(GM093903) to P.L. We thank the NSF (CHE0619339,
ꢀ
1
parameters are kobs = 527 (10 min and K of 466 (32 μM
m
CHE 0443618) for the 500 MHz NMR and high-resolution
mass spectrometers used in this work. We also thank the
NSF (DBI0959666) for the Perkin-Elmer Elan 6100 DRC
instrument and Drs. Robyn Hannigan, Alan Christian,
and Bryanna J. Broadaway (University of Massachusetts
Boston) for Fe analyses. We thank Prof. John Snyder for
help in NMR analysis.
for monomethylhistidine and Km = 0.99 ( 0.05 mM
for Cys. (C) When Cys and dimethylhistidine (12) are the
substrates, the kinetic parameters are k
ꢀ
= 367 ( 8
obs
1
min
and Km = 466 ( 38 μM for hercynine and
Km = 1.61 ( 0.12 mM for Cys. (D) When Cys and
hercynine (2) are the substrates, the kinetic parameters
ꢀ1
are kobs = 270 ( 5 min and Km = 395 ( 30 μM for
hercynine and K = 3.19 ( 0.41 mM for Cys.
m
Supporting Information Available. Text and figures
giving experimental procedures, characterization data,
and H and C NMR spectra of the synthesized com-
pounds 2, 4, and 6. This material is available free of
charge via the Internet at http://pubs.acs.org.
In summary, our studies here revealed that OvoA has a
very relaxed substrate binding pocket and can accept
EgtB substrates as alternatives. Moreover, by modulation
of the histidine amino group methylation state, the
1
13
(
16) (a) Weaver, K. H.; Rabenstein, D. L. J. Org. Chem. 1995, 60,
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Erdelmeier, I.; Daunay, S.; Lebel, R.; Farescour, L.; Yadan, J. C. Green
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The authors declare no competing financial interest.
Org. Lett., Vol. 15, No. 18, 2013
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