In Vitro Reconstitution of the Remaining Steps in Ovothiol A Biosynthesis: C-S Lyase and Methyltransferase Reactions

Article abstract of DOI:10.1021/acs.orglett.8b02332

Ovothiols are thiolhistidine derivatives. The first step of ovothiol biosynthesis is OvoA-catalyzed oxidative coupling between histidine and cysteine. In this report, the remaining steps of ovothiol A biosynthesis were reconstituted in vitro. ETA-14770 (OvoB) was reported as a PLP-dependent sulfoxide lyase, responsible for mercaptohistidine production. OvoA was found to be a bifunctional enzyme, which mediates both oxidative C-S bond formation and methylation of mercaptohistidine to afford ovothiol A. Besides reconstituting the whole biosynthetic pathway, two unique features proposed in the literature were also examined: A potential cysteine-recycling mechanism of the C-S lyase (OvoB) and the selectivity of the ?-N methyltransferase.

Full text of DOI:10.1021/acs.orglett.8b02332

Letter
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
pubs.acs.org/OrgLett
In Vitro Reconstitution of the Remaining Steps in Ovothiol A
Biosynthesis: C−S Lyase and Methyltransferase Reactions
†
,⊥
†,‡,⊥
§
†
§
†
Nathchar Naowarojna, Pei Huang,
Yujuan Cai, Heng Song, Lian Wu, Ronghai Cheng,
§
†
†
†
,§,∥
,†
Yan Li, Shu Wang, Huijue Lyu, Lixin Zhang, Jiahai Zhou,* and Pinghua Liu*
†
Department of Chemistry, Boston University, 590 Commonwealth Avenue, Boston, Massachusetts 02215, United States
‡
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai
2
§
00032, China
State Key Laboratory of Bioorganic and Natural Products Chemistry, Center for Excellence in Molecular Synthesis, Shanghai
Institute of Organic Chemistry, Chinese Academy of Sciences, 345 Lingling Road, Shanghai 200032, China
∥
Shaanxi Key Laboratory of Natural Products & Chemical Biology, College of Chemistry and Pharmacy, Northwest A&F University,
Taicheng Road, Yangling 712100, Shaanxi, China
3
*
S Supporting Information
ABSTRACT: Ovothiols are thiolhistidine derivatives. The first step of
ovothiol biosynthesis is OvoA-catalyzed oxidative coupling between
histidine and cysteine. In this report, the remaining steps of ovothiol A
biosynthesis were reconstituted in vitro. ETA_14770 (OvoB) was reported
as a PLP-dependent sulfoxide lyase, responsible for mercaptohistidine
production. OvoA was found to be a bifunctional enzyme, which mediates
both oxidative C−S bond formation and methylation of mercaptohistidine
to afford ovothiol A. Besides reconstituting the whole biosynthetic pathway,
two unique features proposed in the literature were also examined: a
potential cysteine-recycling mechanism of the C−S lyase (OvoB) and the selectivity of the π-N methyltransferase.
ulfur-containing natural products are widely distributed in
nature and include classes of compounds such as amino
Scheme 1. Two Aerobic Ergothioneine Biosynthetic
Pathways (A) and Proposed Ovothiol Biosynthesis (B)
S
acids, nucleotides, enzyme cofactors, and secondary metabo-
1
−7
lites.
derivatives with a sulfur substitution at the ε- and δ-carbons of
Ergothioneine 5 and ovothiols (8−10) are histidine
8
,9
the imidazole side chain, respectively. Ovothiol was first
isolated from the eggs and ovaries of sea urchins.
1
0−12
Depending on the degree of methylation at the amino group,
there are three forms of ovothiol: A (8, unmethylated), B (9,
monomethylated), and C (10, dimethylated) (Scheme 1B).
The pK of the ovothiol thiol group (∼1.4) is significantly
a
13−15
more acidic than other natural thiols.
As a result, ovothiol
A exists predominantly in the thiolate form under physiological
conditions and functions as a potent radical and peroxide
1
6,17
scavenger.
Ovothiol-producing pathogens and marine
metazoan embryos utilize this property to protect themselves
1
8−20
from oxidative stress.
Ovothiol A has also been
demonstrated to induce autophagy in human liver carcinoma
cell lines, suggesting functions other than solely being a redox
agent.
intriguing cysteine recycling mechanism in the C−S lyase step
(6 → 7 transformation, Scheme S1). Recently, two aerobic
ergothioneine biosynthetic pathways were discovered (Scheme
2
1
About two decades ago, using partially fractionated cell
lysate from an ovothiol-producing strain Crithidia fasciculate,
Steenkamp and co-workers characterized ovothiol A biosyn-
thesis and proposed that this pathway (6 → 7 → 8) involves a
sulfoxide synthase and a PLP-dependent lyase, and a sulfur
1
A): the Mycobacterium smegmatis pathway (EgtA−EgtE,
24−31
Scheme 1A and Table S1)
and the fungal Neurospora
crassa pathway (Egt1, Egt2, and an unidentified methyltrans-
22,23
transfer process occurs prior to methylation (Scheme 1B).
In addition, Steenkamp et al. suggested the existence of an
Received: July 24, 2018
©
XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.8b02332
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
3
2,33
ferase (MTase)) (Scheme 1A and Table S1).
In these two
dithiothreitol (DTT) as a reductant, 5-mercaptohistidine 7 was
produced (Scheme 1B and Figure S6) along with pyruvate and
ammonia in a 1:1:1 ratio (Figures S7 and S8). In this reaction,
DTT most likely reduces a sulfenic acid intermediate to thiol 7
pathways, the two important steps are nonheme iron enzyme
catalyzed oxidative C−S bond formation (EgtB, Egt1) and
reductive C−S lyase reaction (EgtE, Egt2). Such a trans-
sulfuration strategy differs from all other trans-sulfuration
as reported in our ergothioneine C−S lyases EgtE/Egt2 studies
5
,34
31,33
reactions reported thus far.
Based on this ergothioneine
(Scheme S2).
Compound 7 was purified aerobically
biosynthetic information, Seebeck and co-workers identified a
nonheme iron enzyme OvoA in Erwinia tasmaniensis, which
catalyzes the oxidative coupling between L-His and L-Cys in the
and isolated in its stable disulfide form (compound 7a, Figures
9,44,45
S9 and S10).
ETA_14770 exhibited a catalytic efficiency of 167 mM
Using sulfoxide 6 as the substrate,
−
1
−1
s
32,35−39
ovothiol pathway (Scheme 1B and Table S1).
In this
(Table 1 and Figure S4), which is significantly better than any
other combinations listed in Table 1 (ETA_14770/sulfoxide 4,
Egt2/sulfoxide 4, and Egt2/sulfoxide 6, Table 1 and Figure
S11). Results from this comparative kinetic characterization
implied that ETA_14770 is most likely the ovothiol C−S lyase
(catalyzing 6 → 7 reaction), and we named this enzyme OvoB.
Due to the similarities between ergothioneine and ovothiol
biosynthesis, OvoB catalysis most likely follows a mechanistic
model similar to that of EgtE/Egt2 catalysis reported (Scheme
S2).
Compounds 4 and 6 are structurally distinct in several
aspects. First, the sulfoxide functionality is at the ε-carbon and
δ-carbon of the imidazole ring of compounds 4 and 6,
respectively. Second, in compound 4, the amine group is
trimethylated, while in compound 6, the primary amine group
is unmethylated. To understand the selectivity of the
ETA_14770 (OvoB)-catalyzed reaction, we obtained the
crystal structure of PLP-bound OvoB at 2.7 Å resolution
through a molecular replacement method (Figure 1 and Table
S2). The final model of OvoB is comprised of residues 11 to
report, we reconstituted the two remaining steps of the
ovothiol biosynthetic pathway: the C−S lyase and MTase.
To find the ovothiol C−S lyase, we analyzed the E.
tasmaniensis genome and generated a protein−protein family
40
network using Cytoscape 3.6.0 based on protein family
41
information from the pfam database. We identified 16 genes
belonging to aminotransferase class I and II (Figure S1) as the
3
1,32
C−S lyase candidates.
We used the String database to
further analyze the relationship between OvoA and these 16
candidates based on gene co-occurrence, fusion, gene
neighborhoods, and functional association information. The
String database analysis suggests that ETA_14770 is function-
ally related to OvoA in ovothiol biosynthesis (Figure S1). E.
tasmaniensis has not been reported as an ergothioneine
producer, even though ETA_14770 has been applied as an
ergothioneine C−S lyase, raising the question of the
ETA_14770 true identity. To address this issue, we purified
ETA_14770 (Figure S2) and measured the kinetic parameters
by coupling ETA_14770 catalysis using ovothiol/ergothio-
neine sulfoxide substrates with pyruvate reduction by lactate
42
2
4
3
89, with the N-terminal 10 residues disordered. Similar to our
43
dehydrogenase (Table 1).
33
recently reported ergothioneine C−S lyase Egt2, the OvoB
central catalytic domain (residues 94−256) has a classic seven-
stranded β-sheet wrapped within six α-helices, which is a
typical folding feature in type I PLP-dependent enzymes
Table 1. Kinetic Parameters for OvoB and Egt2
kcat (s−1
k /K (mM−1 s−1
)
cat m
enzyme
substrate
)
46
(
9
Figure 1A and Figure S12). The N-terminal (residues 11−
ETA_14770 (OvoB)
ETA_14770 (OvoB)
Egt2
Egt2
sulfoxide 4
sulfoxide 6
sulfoxide 4
sulfoxide 6
16.4 ± 0.6
56.4 ± 2.6
8.7 ± 0.1
23
167
56
3) and C-terminal domains (residues 257−389) form a lid
33
that shelters the PLP-binding pocket and the active site (Figure
S12). The active site is located at the dimer interface, formed
by the catalytic and C-terminal domains of one monomer
along with the N-terminal and C-terminal domains of the other
monomer (Figure S12). In the active site, an internal aldimine
between the PLP cofactor and K240 was observed. The PLP
pyridine ring and the aromatic ring of Y125 form a π−π
0.64 ± 0.02
0.4
Indeed, ETA_14770 can accept compound 4 as a substrate
31
(
Table 1, Figures S3 and S4). When compound 6 was used
36
as the ETA_14770 substrate (Figure S5) in the presence of
Figure 1. OvoB structural analysis. (A) Overall structure of OvoB. (B) The active site of OvoB shows the interaction network of residues around
the PLP cofactor (yellow stick). (C) Model of the OvoB•substrate 6 (blue stick) binary complex. (D) Model of the OvoB•sulfoxide 4 (blue stick)
binary complex. (E) Our reported structure of the Egt2•substrate 4 binary complex (salmon). (F) Model of the Egt2•substrate 6.
B
DOI: 10.1021/acs.orglett.8b02332
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
1
3
stacking interaction. The orientation of the PLP pyridine ring
is anchored by a side chain of N176, D204, and H207. The
phosphate group of PLP forms hydrogen bonds with side chain
of Y66 and the main chain amide of V100 and V101 (Figure
production of only [3- C]-pyruvate (Figure S13), without any
1
3
detectable [3- C]-Cys. This result suggested that OvoB does
not support cysteine regeneration, and the observed L-Cys
production in the Vogt report is most likely a result from other
enzymes in the cell lysate mixture. In our studies, we also
observed that PLP alone is capable of mediating the C−S lyase
reaction in the presence of DTT to afford thiol 7, pyruvate,
and ammonia in a 1:1:1 ratio (kcat = 0.31 ± 0.03 s , Figures
S8, S14, and S15). However, this activity is nearly two orders
of magnitude less active than that of OvoB.
After successfully reconstituting the OvoB activity in vitro,
we sought to identify the remaining π-N-MTase in ovothiol A
biosynthesis. Our bioinformatic analysis of the E. tasmaniensis
genome garnered 52 MTases; however, none of these is
functionally related to OvoA based on String database analysis.
Interestingly, pfam analysis revealed that OvoA has three
domains: the iron-binding DinB_2 and formylglycine (FGE)-
sulfatase domain for sulfoxidation activity and the uncharac-
terized MTase domain. Recently, Seebeck and co-workers
suggested the possibility of having the OvoA MTase domain
being responsible for π-N-methylation (7 → 8, Scheme 1B).
In ergothioneine biosynthesis, the fungal N. crassa Egt1 gene
also encodes three domains, a DinB_2 and FGE-sulfatase
domains for C−S bond formation and a MTase domain.
Similarly, the Egt1 gene was proposed as a bifunctional
enzyme, responsible for both sulfoxidation and methylation in
1
B).
We also sought to obtain the structure of the OvoB•sub-
strate 6 binary complex. This effort was unsuccessful, however,
due to the high turnover number of OvoB. To probe the
substrate selectivity of OvoB, we modeled substrates 4 and 6
separately into the OvoB structure (Figure 1C and D) and
compared them with the reported structure of ergothioneine
C−S lyase Egt2•compound 4 binary complex and Egt2•com-
−
1
3
3
pound 6 docking model (Figure 1E and F). In the
OvoB•compound 6 docking model, the histidine carboxyl
group of substrate 6 forms a salt bridge with R364 and a
hydrogen bond with S352, while the amino moiety forms a
hydrogen bond with the side chain of D356. The orientation of
the histidine imidazole ring is loosely defined through a
hydrogen bond with R364. The sulfoxide of substrate 6 forms a
hydrogen bond with the side chain of R364, and this position
is further strengthened through a hydrogen bond with the
main-chain amide of A41. The amino group of the cysteine
moiety forms hydrogen bonds with the side chains of Y66 and
Y125 and the PLP phosphate group. To explain substrate
preference of OvoB for sulfoxide 6 over sulfoxide 4, we
modeled sulfoxide 4 into the OvoB structure (Figure 1D). In
this model, the cysteine amino group maintains hydrogen
bonds with the side chain of Y66 and Y125 and the PLP
phosphate group. However, due to the difference in C−S bond
locations (ε-position in 4 vs δ-position in 6), most of the
interactions predicted for the histidine portion in the OvoB•6
complex are absent in the OvoB•4 complex. The sulfoxide
forms only one hydrogen bond with the main-chain amide of
A41, while the interactions between the R364 side chain to
both sulfoxide and histidine imidazole ring predicted in the
OvoB•6 complex are not observed in the OvoB•4 complex. In
32
48
49
ergothioneine biosynthesis. However, the proposed MTase
activity in Egt1 was not observed in our in vitro Egt1
32
characterization. To test whether OvoA functions as a
bifunctional enzyme, we examined OvoA MTase activity using
thiol 7 and S-adenosylmethionine (SAM) as the substrates.
OvoA indeed methylates compound 7 to afford ovothiol A.
The kinetic parameters of OvoA MTase activity were
measured using coupled assays commercially available from
Promega (MTaseGlo, Promega). In this coupled assay, the
side-product S-adenosylhomocysteine (SAH) was converted to
ATP through a series of enzymatic transformations (Scheme
The amount of ATP was then quantified by
luminescence intensity generated from ATP-involved luciferase
catalysis. Through this assay, OvoA kinetic parameters were
measured: kcat of 3.05 ± 0.17 min and K of 7.81 ± 1.18 μM
for SAM at saturating concentration of compound 7 and k of
3
3
addition, in our reported Egt2•4 binary complex, the N-
trimethylamine of sulfoxide 4 forms a cation−π interaction
with F304 (Figure 1E), and this interaction is not observed in
OvoB either (Figure 1C and D). We also generated the model
of the Egt2•substrate 6 complex (Figure 1F). Similar to the
OvoB case, the difference in C−S bond location in the
substrate dictates its interaction with Egt2. The histidine
imidazole of compound 4 forms hydrogen bonds with Y134
and H276, while these interactions are absent in the model
Egt2•substrate 6 complex. These docking results are consistent
with the selectivity of OvoB for compound 6 and Egt2 toward
compound 4, respectively. Notably, OvoB exhibits a larger
5
0
S3).
−
1
m
cat
−
1
3.37 ± 0.19 min with K of 7.03 ± 1.15 μM for compound 7
m
at saturating SAM concentration (Figures S16 and S17). To
examine the regioselectivity of the methylation reaction,
compound 8 was isolated and characterized (Figure S18).
Similar to thiol 7, ovothiol A was isolated as a dimer. Upon the
addition of DTT, the reduced form of ovothiol A was obtained
(Figure S19). The methylation of ovothiol A 8 on π-N position
instead of the τ-N position was confirmed by 2D COSY NMR
spectroscopy (Figure S20). This OvoA-catalyzed N-methyl-
ation reaction is among the very few reported cases of histidine
side-chain N-methylation. Thus, far, carnosine N-methyltrans-
ferase 1 is the only reported structure on such a type of
47
active site (∼700 Å calculated by Dogsite) relative to that of
3
3
Egt2 (∼600 Å).
In 2001, using cell lysate of C. fasciculate, an ovothiol-
producing strain, as the catalytic system and in the presence of
DTT as the reductant, Vogt et al. reported that L-Cys was
observed as one of the products in ovothiol A C−S lyase
reaction, and a mechanistic model was offered to explain this
51
enzymes. The identification of OvoA as an N-MTase could
pave the way for future structure−functional studies on this
subclass of MTases.
In summary, we have biochemically reconstituted the
remaining two steps in ovothiol A biosynthesis (C−S lyase
and MTase) and established the ovothiol A pathway in vitro,
which could facilitate future mechanistic studies of these
enzymes and ovothiol A production through metabolic
engineering.
2
2
unexpected result (Scheme S1). Our OvoB structural
characterization indicated that the active site of OvoB is
slightly larger than that of Egt2, suggesting a possibility of
accommodating additional molecules in the OvoB active site
for the proposed cysteine regeneration activity. With the
establishment of the in vitro catalytic system, we repeated the
13
OvoB reaction using [3′- C]-labeled sulfoxide 6a (structural
information in Figure S13). 13C NMR analysis revealed the
C
DOI: 10.1021/acs.orglett.8b02332
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
(
16) Holler, T. P.; Hopkins, P. B. Biochemistry 1990, 29, 1953−
ASSOCIATED CONTENT
Supporting Information
■
1
961.
*
S
(
17) Turner, E.; Klevit, R.; Hager, L. J.; Shapiro, B. M. Biochemistry
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.8b02332.
1987, 26, 4028−4036.
(18) Spies, H. S. C.; Steenkamp, D. J. Eur. J. Biochem. 1994, 224,
203−213.
(19) Steenkamp, D. J.; Spies, H. S. C. Eur. J. Biochem. 1994, 223,
Experimental details, NMR spectra of products, and
kinetic study results (PDF)
4
(
3−50.
20) Castellano, I.; Migliaccio, O.; D’Aniello, S.; Merlino, A.;
Napolitano, A.; Palumbo, A. Sci. Rep. 2016, DOI: 10.1038/srep21506.
(21) Russo, G. L.; Russo, M.; Castellano, I.; Napolitano, A.;
Palumbo, A. Mar. Drugs 2014, 12, 4069−4085.
AUTHOR INFORMATION
Corresponding Authors
■
*
*
(
22) Vogt, R. N.; Spies, H. S. C.; Steenkamp, D. J. Eur. J. Biochem.
E-mail: pinghua@bu.edu.
E-mail: jiahai@mail.sioc.ac.cn.
ORCID
2001, 268, 5229−5241.
(23) Steenkamp, D. J.; Weldrick, D.; Spies, H. S. C. Eur. J. Biochem.
1
(
996, 242, 557−566.
24) Seebeck, F. P. J. Am. Chem. Soc. 2010, 132, 6632−6633.
Pinghua Liu: 0000-0002-9768-559X
Author Contributions
(25) Vit, A.; Mashabela, G. T.; Blankenfeldt, W.; Seebeck, F. P.
ChemBioChem 2015, 16, 1490−1496.
(
26) Vit, A.; Misson, L.; Blankenfeldt, W.; Seebeck, F. P.
⊥
N.N. and P.H. contributed equally.
Notes
ChemBioChem 2015, 16, 119−125.
(27) Goncharenko, K. V.; Seebeck, F. P. Chem. Commun. 2016, 52,
1
(
945−1948.
The authors declare no competing financial interest.
28) Goncharenko, K. V.; Vit, A.; Blankenfeldt, W.; Seebeck, F. P.
Angew. Chem., Int. Ed. 2015, 54, 2821−4.
ACKNOWLEDGMENTS
■
(29) Faponle, A. S.; Seebeck, F. P.; de Visser, S. P. J. Am. Chem. Soc.
2017, 139, 9259−9270.
The invention related to this work was made with Government
Support under Contract No. CHE-1309148 awarded by the
National Science Foundation to P.L., and the government has
certain rights in the invention. The work here is also partially
supported by the Strategic Priority Research Program (B) of
the CAS (XDB20000000 to J. Z.), National Science
Foundation of China (31430002 and 31320103911 to L.Z.),
and Shanghai Science and Technology Committee
(30) Burn, R.; Misson, L.; Meury, M.; Seebeck, F. P. Angew. Chem.,
Int. Ed. 2017, 56, 12508−12511.
(31) Song, H.; Hu, W.; Naowarojna, N.; Her, A. S.; Wang, S.; Desai,
R.; Qin, L.; Chen, X.; Liu, P. Sci. Rep. 2015, DOI: 10.1038/
srep11870.
(32) Hu, W.; Song, H.; Sae Her, A.; Bak, D. W.; Naowarojna, N.;
Elliott, S. J.; Qin, L.; Chen, X.; Liu, P. Org. Lett. 2014, 16, 5382−5385.
(
(
33) Irani, S.; et al. Cell Chem. Biol. 2018, 25, 519−529.
34) Naowarojna, N.; Cheng, R.; Chen, L.; Quill, M.; Xu, M.; Zhao,
(
17DZ1205402). N.N. is partially supported by the Warren−
McLoed Fellowship from the Boston University Marine
Program. P.H. is supported by a fellowship from the China
Scholarship Council. We thank the staff of the beamline
BL19U1 of the National Center for Protein Science (Shanghai,
China) for X-ray data collection.
C.; Liu, P. Biochemistry 2018, 57, 3309−3325.
(35) Braunshausen, A.; Seebeck, F. P. J. Am. Chem. Soc. 2011, 133,
1757−1759.
(36) Song, H.; Leninger, M.; Lee, N.; Liu, P. Org. Lett. 2013, 15,
4
(
7
(
854−4857.
37) Mashabela, G. T. M.; Seebeck, F. P. Chem. Commun. 2013, 49,
714−7716.
38) Chen, L.; Naowarojna, N.; Song, H.; Wang, S.; Wang, J.; Deng,
Z.; Zhao, C.; Liu, P. J. Am. Chem. Soc. 2018, 140, 4604−4612.
39) Song, H.; Her, A. S.; Raso, F.; Zhen, Z.; Huo, Y.; Liu, P. Org.
Lett. 2014, 16, 2122−2125.
40) Shannon, P.; Markiel, A.; Ozier, O.; Baliga, N. S.; Wang, J. T.;
Ramage, D.; Amin, N.; Schwikowski, B.; Ideker, T. Genome Res. 2003,
REFERENCES
■
(
(
1) Fahey, R. C. Annu. Rev. Microbiol. 2001, 55, 333−356.
2) Fontecave, M.; Ollagnier-de-Choudens, S.; Mulliez, E. Chem.
(
Rev. 2003, 103, 2149−2166.
(
(
(
(
3) Hand, C. E.; Honek, J. F. J. Nat. Prod. 2005, 68, 293−308.
(
4) Jacob, C. Nat. Prod. Rep. 2006, 23, 851−863.
5) Kessler, D. FEMS Microbiol. Rev. 2006, 30, 825−840.
6) Lin, C.-I.; McCarty, R. M.; Liu, H.-w. Chem. Soc. Rev. 2013, 42,
1
(
(
(
(
3, 2498−2504.
41) Finn, R. D.; et al. Nucleic Acids Res. 2016, 44, D279−D285.
42) Szklarczyk, D.; et al. Nucleic Acids Res. 2011, 39, D561−568.
43) Ghatge, M. S.; et al. PLoS One 2012, 7, e41680.
4
(
377−4407.
7) Wang, L.; Chen, S.; Xu, T.; Taghizadeh, K.; Wishnok, J. S.;
Zhou, X.; You, D.; Deng, Z.; Dedon, P. C. Nat. Chem. Biol. 2007, 3,
44) Weaver, K. H.; Rabenstein, D. L. J. Org. Chem. 1995, 60, 1904−
7
(
(
09−710.
1907.
8) Tanret, C. CR Acad. Sci., Ser. IIc: Chim. 1909, 149, 222−224.
9) Turner, E.; Klevit, R.; Hopkins, P.; Shapiro, B. J. Biol. Chem.
(
(
45) Turner, E.; Hager, L. J.; Shapiro, B. M. Science 1988, 242, 939.
46) Schneider, G.; Kack, H.; Lindqvist, Y. Structure 2000, 8, R1−
R6.
47) Volkamer, A.; Kuhn, D.; Rippmann, F.; Rarey, M.
̈
1
986, 261, 13056−13063.
10) Rossi, F.; Nardi, G.; Palumbo, A.; Prota, G. Comp. Biochem.
Physiol. B: Biochem. Mol. Biol. 1985, 80, 843−845.
11) Palumbo, A.; Misuraca, G.; d’Ischia, M.; Donaudy, F.; Prota, G.
Comp. Biochem. Physiol. B: Biochem. Mol. Biol. 1984, 78, 81−83.
12) Palumbo, A.; d’Ischia, M.; Misuraca, G.; Prota, G. Tetrahedron
Lett. 1982, 23, 3207−3208.
13) Marjanovic, B.; Simic, M. G.; Jovanovic, S. V. Free Radical Biol.
Med. 1995, 18, 679−685.
14) Holler, T. P.; Hopkins, P. B. J. Am. Chem. Soc. 1988, 110,
837−4838.
15) Mirzahosseini, A.; Orgovan, G.; Toth, G.; Hosztafi, S.; Noszal,
B. J. Pharm. Biomed. Anal. 2015, 107, 209−216.
(
(
Bioinformatics 2012, 28, 2074−2075.
(
(
(
48) Liao, C.; Seebeck, F. P. ChemBioChem 2017, 18, 2115−2118.
49) Bello, M. H.; Barrera-Perez, V.; Morin, D.; Epstein, L. Fungal
(
Genet. Biol. 2012, 49, 160−172.
50) Hsiao, K.; Zegzouti, H.; Goueli, S. A. Epigenomics 2016, 8,
21−339.
51) Cao, R.; Zhang, X.; Liu, X.; Li, Y.; Li, H. Cell Res. 2018, 28,
94−496.
(
3
(
4
(
(
4
(
D
DOI: 10.1021/acs.orglett.8b02332
Org. Lett. XXXX, XXX, XXX−XXX