An Fe2+- and α-Ketoglutarate-Dependent Halogenase Acts on Nucleotide Substrates

Article abstract of DOI:10.1002/anie.201914994

While halogenated nucleosides are used as common anticancer and antiviral drugs, naturally occurring halogenated nucleosides are rare. Adechlorin (ade) is a 2′-chloro nucleoside natural product first identified from Actinomadura sp. ATCC 39365. However, the installation of chlorine in the ade biosynthetic pathway remains elusive. Reported herein is a Fe²⁺-α-ketoglutarate halogenase AdeV that can install a chlorine atom at the C2′ position of 2′-deoxyadenosine monophosphate to afford 2′-chloro-2′-deoxyadenosine monophosphate. Furthermore, 2′,3′-dideoxyadenosine-5′-monophosphate and 2′-deoxyinosine-5′-monophosphate can also be converted, albeit 20-fold and 2-fold, respectively, less efficiently relative to the conversion of 2′-deoxyadenosine monophosphate. AdeV represents the first example of a Fe²⁺-α-ketoglutarate-dependent halogenase that converts nucleotides into chlorinated analogues.

Full text of DOI:10.1002/anie.201914994

A Journal of the Gesellschaft Deutscher Chemiker
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Accepted Article
Title: An iron(II)- and α-ketoglutarate-dependent halogenase acts on
nucleotide substrates
Authors: Yonghui Zhang, Chunhua Zhao, Shan Yan, Qin Li, Hucheng
Zhu, Zhiyu Zhong, Ying Ye, and Zixin Deng
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10.1002/anie.201914994
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RESEARCH ARTICLE
An iron(II)- and α-ketoglutarate-dependent halogenase acts on
nucleotide substrates
Chunhua Zhao,[a] Shan Yan,[a] Qin Li,[a] Hucheng Zhu,[a] Zhiyu Zhong,[b] Ying Ye,[a] Zixin Deng,[b]Yonghui
Zhang *[a]
usually accept only substrates that have been tethered to an acyl
carrier protein (ACP) or a peptidyl carrier protein (PCP), with
[16]
[18]
Abstract: While halogenated nucleosides are used as common
anti-cancer and anti-viral drugs, naturally occurring halogenated
nucleosides are rare. Adechlorin (ade) is a 2’-chloro nucleoside
natural product first identified from Actinomadura sp. ATCC
39365. However, the installation of chlorine in ade biosynthetic
pathway still remains elusive. In this article, we report a Fe2+-α-
ketoglutarate halogenase AdeV that can install a chlorine atom
onto the C-2’ position of 2’-deoxyadenosine monophosphate to
WelO5[15]
and BesD[17]
being the very rare exceptions
known to act directly on stand-alone substrates.
Halogenated nucleosides represent an important class of
synthetic drugs used in anticancer and antiviral treatments,
including Cladribine (1, anti-leukaemia), Clofarabine (2, anti-
leukaemia), Fludaradine (3, anti-leukaemia), Fluorouracil (4,
antitumor), Gemcitabine (5, anticancer), and Sofosbuvir (6, anti-
Hepatitis C) (Figure 1A). In contrast, there are relatively few
known halogenated nucleoside natural products, with nucleocidin
afford
2’-chloro-2’-deoxyadenosine
monophosphate.
Furthermore, we found that 2’,3’-dideoxyadenosine-5’-
monophosphate and 2’-deoxyinosine-5’-monophosphate can
also be converted, less efficiently by 20-fold and 2-fold
respectively, relative to the conversion of 2’-deoxyadenosine
monophosphate. AdeV represents the first example of a Fe2+-α-
ketoglutarate-dependent halogenase that converts nucleotides
into chlorinated analogues.
[19]
(7a, fluorinated at C-4’ of ribose), AT-265 (7b,
dealanylascamycin, chlorinated at C-2 of adenosine)
[20]
and
adechlorin[21] (8, chlorinated at C-2’ of ribose) (Figure 1B) being
rare examples. Adechlorin is the 2’-chloro derivative of
pentostatin (9), a competitive inhibitor of adenosine deaminase
that is approved as a drug for leukaemia treatment [21]. While the
biosynthetic gene clusters for both adechlorin and pentostatin
have been characterized[22], it remains unclear how pentostatin is
chlorinated to give adechlorin. Here, we report the results of both
in vivo and in vitro studies on the halogenase AdeV involved in
the enzymatic chlorination step of adechlorin biosynthesis, and
the substrate scope of AdeV is also discussed.
Introduction
Organic compounds containing one or more halogen atoms are
extremely common and structurally diverse; many of these are
highly important medicinal, agricultural and industrial materials[1].
While most are produced synthetically, nature also produces a
rich library of halogenated natural products that are widespread
in the biosphere, especially from marine territory. Biological
halogenations are known to take place by one of three established
Results and Discussion
Identification of an iron(II)- and α-ketoglutarate-dependent
halogenase gene adeV from the adechlorin gene cluster
mechanisms[2] [3]: 1) SN2 nucleophilic substitution, exemplified by
[5]
the S-adenosyl methionine (SAM)-dependent fluorinase[4]
,
chlorinase and halide methyltransferase[7]; 2) via an oxidative
[6]
[22a]
Previous work by Gao et al.
revealed that the adechlorin
strategy exemplified by the flavin adenine dinucleotide (FAD)-
biosynthetic gene cluster (ade BGC) was composed of 13 genes
(adeA-adeM), but none of these were identified to encode the
required chlorinase (Figure 2A). We then obtained Actinomadura
sp. ATCC 39365 from the American Type Culture Collection
(ATCC) and found that it indeed produces both adechlorin (8),
pentostatin (9) and 2’-amino deoxyadenosine (2’-amino dA, 10,
Figure 1C) in our laboratory conditions (Figure 2B). To pursue the
chlorinase involved in adechlorin biosynthesis, we re-sequenced
the genome of Actinomadura and discovered that eight genes
downstream of the ade BGC lies a gene annotated as an Fe(II)-
α-ketoglutarate-dependent oxygenase (for adechlorin gene
cluster organization and annotation, see Figure 2A and
Supplementary 1), which exhibits only 15% amino acid similarity
to the previously identified Fe(II)-α-ketoglutarate-dependent
chlorinase WelO5 (for gene and protein sequence, see
Supplementary 2; for BLAST analysis, see Supplementary 3). We
therefore took an interest in this gene and its encoded protein as
a candidate chlorinase enzyme and designated this gene as adeV.
[8] [9] [10]
dependent halogenase
via iron(II)- and
and haloperoxidases [11]; and 3)
α-ketoglutarate
(α-KG)-dependent
[12] [13]
metalloenzymes utilizing halogen radical (X•) equivalents
[14]
.
Intriguingly, iron(II) (Fe2+)-α-KG-dependent halogenases
[a]
Dr. Chunhua Zhao, Dr. Shan Yan, Qin Li, Dr. Hucheng Zhu, Dr. Ying
Ye, Prof. Zhang Yonghui
Hubei Key Laboratory of Natural Medicinal Chemistry and Resource
Evaluation, School of Pharmacy, Tongji Medical College, Huazhong
University of Science and Technology, Wuhan 430030, China.
E-mail: zhangyh@mails.tjmu.edu.cn
[b]
Mr. Zhiyu Zhong, Prof. Zixin Deng
Key Laboratory of Combinatorial Biosynthesis and Drug Discovery,
Ministry of Education, School of Pharmaceutical Sciences, Wuhan
University, Wuhan 430071, PR China
Supporting information for this article is given via a link at the end of
the document.
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10.1002/anie.201914994
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To verify our hypothesis whether adeV is indeed the chlorinase
involved in the biosynthesis of adechlorin, gene inactivation was
carried out (See Experimental Section). HPLC analysis of the
metabolites from the ΔadeV mutant could not detect any
adechlorin (8) being produced, while the yield of pentostatin (9)
increased five-fold when compared to that of the wild-type strain
(concentration of 6μg/mL vs 1.2μg/mL in fermentation broth)
(Figure 2B), thus strongly suggesting that adeV is responsible for
the chlorination during the adechlorin biosynthesis (Scheme 1).
We also observed that the production of 2’-amino
deoxyadenosine (2’-amino dA) (10) improved sharply (about ten
fold) (concentration of 50μg/mL vs 5μg/mL in fermentation broth)
in contrast to ATCC 39365 (Figure 2B). It is then believed that 8
and 10 may share the same precursor possibly from the primary
metabolite pathway. The semi-purified fractions containing 8, 9
and 10 are subjected to the analysis of high resolution mass
spectrometry (HRMS) with the mass-to-charge ratio (m/z) of
303.0837, 269.1244 and 267.1168, respectively, fully consistent
separated from the mixture by semi-preparative HPLC, and the
fractions containing 13 were combined and lyophilized, affording
1.9 mg of 13 as a dry powder for proton NMR analysis. we also
compared the differences of
H chemical shifts between
compounds 12 and 13 as shown in Figure S7 as compound 12 is
a well-characterized molecule. Two H signals at C2’ (2.74 ppm (m,
1H, 2’-H), 2.32 ppm (m, 1H, 2’-H)) of 12 disappeared in 13,
instead, a new H signal at 4.21 ppm (m, 1H, 2’-H) emerged. Due
to electronegative chlorine installed at C2’, other Hs in different
carbons (C3’ and C4’) also move to upfield, i.e. 4.43 ppm vs 5.14
ppm (m, 1H, 4’-H), 4.01 ppm vs 4.44 ppm (m, 1H, 3’-H). Taken
together with our HR-MS and NMR analyses, 13 was confirmed
to be 2’-chloro-2’deoxyadenosine.
with the previous results. [22a]
.
To determine the effect of incubation time on the enzymatic
reaction, we incubated 2’-dAMP (12), Fe2+ and α-KG at room
temperature and terminated the reaction after 1 h, 2 h, and 3 h by
adding an equal volume of methanol to denature the protein.
HPLC profile analysis showed that the amount of conversion
increased over time (Figure 2C). When α-KG was absent from the
reaction system, no product could be detected, while when Fe2+
was not supplemented into the full reaction system, the amount of
conversion sharply decreased, which could be accounted for by a
trace amount of ferron ions from the purified protein. These results
is consistent with our hypothesis that AdeV is an iron(II)-α-
ketoglutarate-dependent halogenase (Supplementary 12).
Biochemical assay of AdeV
To confirm whether AdeV indeed catalyses the chlorination
reaction, we set up in vitro enzymatic assays. Overexpression of
the entire adeV gene, which encodes 310 amino acids, in E. coli
BL21 (DE3) allowed us to isolate and purify the recombinant
protein to near homogeneity with the estimated molecular weight
of 34 kDa as observed by SDS-PAGE analysis (Supplementary
4).
Enzyme kinetic parameters of AdeV toward compound 12
To determine the substrates of AdeV, we incubated purified AdeV
protein with 9, Fe2+ and α-KG, but found that 9 was not converted
into 8 (Supplementary 5), demonstrating that 9 is not the direct
substrate of AdeV. This result also suggested that 9 is not the last
intermediate of the biosynthesis of 8. We then considered the
possibility that the halogenation step occurs in the early stage of
the biosynthesis of 8. To this end, we incubated 2’-
A detailed steady-state kinetic analysis was carried out on AdeV
toward 12. A series of biochemical reactions were performed with
a range of concentrations of 12 (0.12 mM, 0.25 mM, 0.5 mM, 1
mM, 2 mM, 4 mM, 8 mM) in duplicate. These reactions were then
performed at 25°C for 1 hr and terminated by heat inactivation at
65°C. HPLC analysis was carried out as described in
Experimental Section. The concentrations of the products were
determined based on the standard curve of what correlated with
HPLC peaks. The Michaelis-Menten equation was least-squares
fitted to plots of the initial formation rate of products to extract the
Km and kcat parameters (Supplementary 8). Finally, we calculated
that apparent kinetic parameters for AdeV are as followed:
Km=2.5*10-3 M, kcat=0.6 min-1, kcat/Km=240 min-1.M-1. In contrast,
deoxyadenosine
(2’-dA,
11)
and
2’-deoxyadenosine
monophosphate (2’-dAMP, 12), both of which are earlier
proposed intermediates from the pathway with AdeV. We found
that, while AdeV showed activity not to 2’-dA (Supplementary 5),
a new peak (labelled compound 13) emerged in the UV trace of
our HPLC analyses at a retention time of 20.8 min when 2’-dAMP
(12, retention time = 12.7 min) was employed as a substrate
(Scheme 2A and Figure 2C). HR-MS analysis of this peak is
consistent with 2’-chloro-2’-deoxyadenosine monophosphate (2’-
Cl-2’-dAMP, 13) (calculated [M+H+] 366.03988; observed [M+H+]
366.03586), which also exhibited a characteristic isotopic pattern
of mono substitution of chlorine (Figure Supplementary 6B).
Tandem mass spectrometry (MS2) analysis further confirmed the
fragmentation pattern is also fully consistent with the predicted
one (for detailed raw data and analysis, see Supplementary 6).
another stand-alone halogenase WelO5 halogenates its preferred
[23]
substrate 12-epi-fischerindole U at 1.8±0.2 min-1
.
Biochemical assay of AdeV on different substrates
AdeV can convert 2’-dAMP (12) rather than 2’-dA (11) or
pentostatin (9) hints that the 5’-phosphate moiety in 2’-
deoxyadenosine scaffold is essential for enzyme activity. We also
tested whether 2’-deoxyadenosine-5’-diphosphate (2’-dADP, 14)
or 2’-deoxyadenosine-5’-triphosphate (2’-dATP, 15) (Figure 3A)
could be utilized as substrate of AdeV, and HRMS analysis
showed that no chlorinated counterpart could be detected, but
To further affirm the chemical identity of 13, we carried out a
scale-up biochemical conversion. The reaction was composed of
40 mg of AdeV protein purified from a 2 L culture of E. coli, 0.5
mg Fe2+, 1.5 mg α-KG and 20 mg 2’-dAMP (12) in 10 ml of
reaction buffer (see Experimental Section). Compound 13 was
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new UV peaks with molecular weights of 366.03 and 368.03
emerged, which could be considered halogenation of 2’-dAMP
which comes from minor hydrolysis of 2’-dADP (14) or 2’-dATP
(15) (Supplementary 9).
Site-directed mutagenesis of active amino acids of AdeV
AdeV showed very weak similarity to the previously identified iron-
α-KG-dependent halogenase WelO5 (Supplementary 3). Like
most other enzymes of this family, AdeV has a conserved
H*A***H motif (two histidine 170 and 251), a common feature of
iron-α-KG halogenases compared to the conserved ones for iron-
α-KG-dependent hydroxylases (H*D/E***H) (Supplementary 2).
To check whether chemical alteration of the ribose ring of the
nucleotide will affect the activity of AdeV, 2’, 3’-dideoxyadenosine
monophosphate (16) (Figure 3A) was used as substrate, and a
new UV peak at a retention time of 29.8 min was observed
(Scheme 2B and Figure 3B). HRMS analysis of this peak is
consistent with 2’-chloro-2’,3’-dideoxyadenosine monophosphate
(2’-Cl-2’,3’-ddAMP, 22) (calculated [M+H+] 350.0421; observed
[M+H+] 350.0418), which also exhibited a characteristic Cl
isotopic pattern (Supplementary 11BC). Tandem mass
H251 was believed to coordinate iron(II) to facilitate
a
halogenation reaction. To this end, we changed this histidine
residue into phenylalanine by site-directed mutagenesis [25]. The
new protein variant (H251F) was tested for its activity toward
compound 12, and its halogenation activity was completely
abolished (Figure 2D). These results are fully consistent with
those of the previously reported enzyme WelO5 [15]. Thus, we
proposed the mechanism of AdeV and the key reaction
intermediates involved in the chlorination of 12 (Supplementary
12).
spectrometry (MS2) analysis further confirmed
a
typical
fragmentation pattern [MS2 136.0], fully consistent with the
predicted fragmentation pathway (Supplementary 11DE).
To check whether different nucleosides will affect the activity of
AdeV, four 2’-deoxy nucleoside monophosphates that are
Phylogenetic tree analysis and cross comparison of KG-
dependent enzymes
common
primary
(2’-dGMP,
(2’-dUMP,
metabolites
18),
19),
(2’-deoxyguanine-5’-
2’-deoxyuracil-5’-
monophosphate
monophosphate
2’-deoxythymine-5’-
A phylogenetic tree was also generated between function-known
α-KG-dependent oxygenases and halogenases. Interestingly, the
reported substrate ACP-bound halogenases are clustered
(highlighted in dark green) (Figure 4). However, AdeV (nucleotide
halogenases) together with other ACP-independent halogenases
(WelO5/AmbO5 [23] and BesD) are of more evolutionary relevance
with oxygenases such as GloF (L-proline hydroxylase) and VldW
(aminoglycoside hydroxylase). We also carried out a detailed
comparison among three halogenases that act on free-standing
substrates along with the classical substrate ACP-tethered
halogenase SyrB2 (Scheme 3 and Table 1).
monophosphate (2’-dTMP, 20) and 2’-deoxycytosine-5’-
monophosphate (2’-dCMP, 21)) (Figure 3A) were used as
substrates. no new UV peak was detected, indicating that these
four compounds are not the substrates of AdeV (Supplementary
10). With 2’-deoxyinosine-5’-monophosphate (2’-dIMP, 17), in
which adenine had been replaced by inosine, a new UV peak at
a retention time of 26.2 min was observed (Scheme 2C and
Figure 3B). HRMS analysis of this peak is consistent with 2’-
chloro-2’-deoxyinosine monophosphate (2’-Cl-2’-dIMP, 23)
(calculated [M+H+] 367.0210; observed [M+H+] 367.0200), which
exhibited a characteristic isotopic pattern of monosubstituted
chlorine atom (Supplementary 11FG). Tandem mass
spectrometry (MS2) analysis further confirmed
a
typical
fragmentation pattern [MS2 137.0], fully consistent with the
predicted fragmentation pathway (Supplementary 11HI).
Since the above three compounds 12, 16 and 17 can all be
converted by AdeV, we then examined the substrate specificity of
[24]
AdeV toward them. A competition experiment
with equimolar
12 and 16 (0.2 mM each) was carried out with AdeV (20 μM).
Subsequent HPLC analysis revealed that the conversion of 12 to
13 under these experimental conditions is approximately 20 times
higher than the conversion of 16 to 22 (Figure 3C). Additionally, a
similar competition experiment with equimolar 12 and 17 (0.5 mM
each) was carried out with AdeV (20 μM). The conversion of 12
to 13 is approximately 2 times higher than that of 17 to 23 (Figure
3C). These results clearly demonstrated that 12 is the preferred
substrate for AdeV. These data are reasonable since 12
originates from the primary metabolite pathway and may act as a
native substrate of AdeV, which is abundant among cell
metabolites.
SCHEMES
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Scheme 1. Schematic representation of the adechlorin biosynthetic pathway by Gao et al. in a previous study.
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Scheme 2. A) AdeV catalyses the conversion from 2’-dAMP (12) to 2’-
Cl-2’-dAMP (13). B) AdeV catalyses the conversion from 2’,3’-ddAMP
(16) and to 2’-Cl-2’,3’-ddAMP (22). C) AdeV catalyses the conversion
from 2’-dIMP (17) to 2’-Cl-2’-dIMP (23).
Scheme 3. Schematic representation of three iron-α-ketoglutarate-
dependent halogenases that act on ACP (PCP)-free substrates. A)
WelO5 acts upon alkaloid as a late-stage modification, B) BesD
halogenate lysine to initiate the natural compound ethylyneserine
biosynthetic pathway, and C) AdeV halogenate 2’-dAMP (12) to initiate
adechlorin biosynthesis.
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Figure 1. Selected nucleosides: A）Top-selling halogenated nucleosides for
clinical use. B) Natural halogenated nucleosides. C) 2’-amino-dA produced by
ATCC 39365.
Figure 2. A) Gene organization of the adechlorin biosynthetic pathway. B)
HPLC profiles of metabolites. From upper to bottom: pentostatin authentic
sample; ATCC 39365; ΔadeV. C) HPLC analysis of enzymatic reactions
catalysed by AdeV. i) dAMP; ii) full reaction (AdeV, 2’-dAMP, α-KG, Fe2+), 1 hr;
iii) full reaction, 2 hr; iv) full reaction, 3 hr; v) reaction in the absence of Fe2+, vi)
reaction in the absence of α-KG. D) HPLC analysis of enzymatic reactions
catalysed by i) AdeV and ii) AdeV(H251F).
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Figure 3. A) 11 substrates used for AdeV enzyme activity test in this study. B)
HPLC profiles of enzymatic reactions catalysed by AdeV. i) 2’-dIMP; authentic
sample. ii) 2’-dIMP; full reaction (AdeV, 2’-dIMP, α-KG, Fe2+), 1 hr. iii) 2’-ddAMP;
authentic sample. iv) 2’-ddAMP; full reaction (AdeV, 2’-ddAMP, α-KG, Fe2+), 1
hr. C) HPLC analysis of enzymatic reaction catalysed by AdeV. i) Equimolar 2’-
dIMP and 2’-dAMP. Authentic sample. ii) Equimolar 2’-dIMP and 2’-dAMP; full
reaction (AdeV, mixed substrates, α-KG, Fe2+), 1 hr. iii) Equimolar 2’-ddAMP
and 2’-dAMP; authentic sample. iv) Equimolar 2’-ddAMP and 2’-dAMP; full
reaction (AdeV, mixed substrates, α-KG, Fe2+), 1 hr.
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Figure 4. Phylogenetic tree between function-known α-KG-dependent oxygenases and halogenases. Cur, BarB2, CmaB, SyrB2 and CytC3 are CP-tethered
halogenases, which are highlighted in dark green. WelO5/AmbO5 (alkaloids highlighted in yellow), BesD and AdeV are CP-independent halogenases, while EcdG，
GloF, KtzP, AsnO, VioC, MppO, VldW, Fr9P, LipL, KanJ, HygX and TmpA are oxygenases.
Table 1. Cross-comparison of AdeV with previously characterized iron-α-ketoglutarate-dependent halogenases SyrB2, WelO5 and BesD.
[23]
SyrB2 [12] [13]
Fe-α-KG
WelO5 [15] [16]
Fe-α-KG
BesD [17] [18] AdeV
Halogenase
type
Fe-α-KG
Fe-α-KG
ACP(PCP)-
free?
No
Yes
Yes
Yes
Substrates
Amino acid-
CP
No
Alkaloid
No
Amino acid Nucleoside
Substrate
phosphate-
tethered?
Chlorination
timing
No
Yes
After CP-
tethered
No
Late-stage
modification
No
Early stage
Yes
Early stage
No
Cryptic
enzyme?
dA, showing that the nucleotide form containing a phosphate
group at 5’ position is crucial for AdeV substrate reactivity. To the
best of our knowledge, AdeV represents the first halogenase to
selectively act upon nucleotide 5’-phosphates. A phylogenetic
tree of all characterized α-KG-dependent oxygenases and
halogenases was generated and we could speculate that, using
the AdeV sequence as a probe, we could find more halogenated
nucleotide natural products through genome mining. The
Conclusion
Iron(II)-α-KG-dependent halogenases act through free radical
mechanisms and in all examples, except WelO5 and BesD, act
upon substrates tethered with ACP or PCP
The chlorinase enzyme identified in our report, AdeV, selectively
acts upon 2’-dAMP as its native substrate. We also identified that
AdeV can accept 2’-ddAMP and 2’-dIMP as substrates but not 2’-
[15-16]
(Scheme 3A).
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nucleoside halogenase may also aid us in creating new
nucleoside analogues through biological, chemical or biochemical
routes [26] [27] [28] [29] for clinical use.
[9]
M. C. Andorfer, J. C. Lewis, Annu Rev
Biochem 2018, 87, 159-185.
[10] D. S. Gkotsi, H. Ludewig, S. V. Sharma, J.
A. Connolly, J. Dhaliwal, Y. Wang, W. P.
Unsworth, R. J. K. Taylor, M. M. W.
McLachlan, S. Shanahan, J. H. Naismith,
R. J. M. Goss, Nat Chem 2019.
[11] Z. D. Miles, S. Diethelm, H. P. Pepper, D.
M. Huang, J. H. George, B. S. Moore, Nat
Chem 2017, 9, 1235-1242.
[12] F. H. Vaillancourt, E. Yeh, D. A. Vosburg,
S. E. O'Connor, C. T. Walsh, Nature 2005,
436, 1191-1194.
[13] L. C. Blasiak, F. H. Vaillancourt, C. T.
Walsh, C. L. Drennan, Nature 2006, 440,
368-371.
Acknowledgements
We acknowledge Prof. Yuhui Sun, Prof. Liu Tiangang at Wuhan
University, Dr. Jason Chan at HongKong University of Science and
Technology and Prof. Lin Shuangjun at Shanghai Jiaotong University,
China, Prof. Tang Gongli and Dr Yuanhua at the Shanghai Institute of
Organic Chemistry, Dr Gu Lianghu at Huazhong University of Science
and Technology for encouragement and aiding in experiments. We
acknowledge Dr Deng Hai at Aberdeen University, UK for critical
reading of the manuscript. This work was financially supported by the
Program for Changjiang Scholars of the Ministry of Education of the
People’s Republic of China (Grant T2016088), the National Natural
Science Foundation for Distinguished Young Scholars (Grant
81725021), and the Innovative Research Groups of the National
Natural Science Foundation of China (Grant 81721005).
[14] L. Gu, B. Wang, A. Kulkarni, T. W.
Geders, R. V. Grindberg, L. Gerwick, K.
Hakansson, P. Wipf, J. L. Smith, W. H.
Gerwick, D. H. Sherman, Nature 2009,
459, 731-735.
Keywords: biological halogenation, adechlorin biosynthesis,
nucleotide antibiotic, Fe2+-α-ketoglutarate dependent halogenase,
C-H functionalization
[15] M. L. Hillwig, X. Liu, Nat Chem Biol
2014, 10, 921-923.
References
[16] A. J. Mitchell, Q. Zhu, A. O. Maggiolo, N.
R. Ananth, M. L. Hillwig, X. Liu, A. K.
Boal, Nat Chem Biol 2016, 12, 636-640.
[17] J. A. Marchand, M. E. Neugebauer, M. C.
Ing, C. I. Lin, J. G. Pelton, M. C. Y.
Chang, Nature 2019, 567, 420-424.
[18] M. E. Neugebauer, K. H. Sumida, J. G.
Pelton, J. L. McMurry, J. A. Marchand, M.
C. Y. Chang, Nat Chem Biol 2019, 15,
1009-1016.
[19] X. Feng, N. Al Maharik, A. Bartholome, J.
E. Janso, U. Reilly, D. O'Hagan, Org
Biomol Chem 2017, 15, 8006-8008.
[20] C. Zhao, J. Qi, W. Tao, L. He, W. Xu, J.
Chan, Z. Deng, PLoS One 2014, 9,
e114722.
[1]
F. H. Vaillancourt, E. Yeh, D. A. Vosburg,
S. Garneau-Tsodikova, C. T. Walsh, Chem
Rev 2006, 106, 3364-3378.
A. Butler, M. Sandy, Nature 2009, 460,
848-854.
J. Latham, E. Brandenburger, S. A.
Shepherd, B. R. K. Menon, J. Micklefield,
Chem Rev 2018, 118, 232-269.
C. Dong, F. Huang, H. Deng, C.
Schaffrath, J. B. Spencer, D. O'Hagan, J.
H. Naismith, Nature 2004, 427, 561-565.
D. O'Hagan, C. Schaffrath, S. L. Cobb, J.
T. Hamilton, C. D. Murphy, Nature 2002,
416, 279.
A. S. Eustaquio, F. Pojer, J. P. Noel, B. S.
Moore, Nat Chem Biol 2008, 4, 69-74.
J. W. Schmidberger, A. B. James, R.
Edwards, J. H. Naismith, D. O'Hagan,
Angew Chem Int Ed Engl 2010, 49, 3646-
3648.
C. Dong, S. Flecks, S. Unversucht, C.
Haupt, K. H. van Pee, J. H. Naismith,
Science 2005, 309, 2216-2219.
[2]
[3]
[4]
[5]
[6]
[7]
[21] J. B. Tunac, M. Underhill, J Antibiot
(Tokyo) 1985, 38, 1344-1349.
[22] aY. Gao, G. Xu, P. Wu, J. Liu, Y. S. Cai,
Z. Deng, W. Chen, Appl Environ
Microbiol 2017, 83; bP. Wu, D. Wan, G.
Xu, G. Wang, H. Ma, T. Wang, Y. Gao, J.
Qi, X. Chen, J. Zhu, Y. Q. Li, Z. Deng, W.
Chen, Cell Chem Biol 2017, 24, 171-181.
[8]
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RESEARCH ARTICLE
[23] M. L. Hillwig, Q. Zhu, K. Ittiamornkul, X.
Liu, Angew Chem Int Ed Engl 2016, 55,
5780-5784.
[24] W. S. Glenn, E. Nims, S. E. O'Connor, J
Am Chem Soc 2011, 133, 19346-19349.
[25] D. Khare, B. Wang, L. Gu, J. Razelun, D.
H. Sherman, W. H. Gerwick, K.
Hakansson, J. L. Smith, Proc Natl Acad
Sci U S A 2010, 107, 14099-14104.
[26] D. S. Gkotsi, J. Dhaliwal, M. M.
McLachlan, K. R. Mulholand, R. J. Goss,
Curr Opin Chem Biol 2018, 43, 119-126.
[27] A. Deb Roy, S. Gruschow, N. Cairns, R. J.
Goss, J Am Chem Soc 2010, 132, 12243-
12245.
[28] J. T. Payne, P. H. Butkovich, Y. Gu, K. N.
Kunze, H. J. Park, D. S. Wang, J. C.
Lewis, J Am Chem Soc 2018, 140, 546-
549.
[29] T. Hayashi, M. Ligibel, E. Sager, M. Voss,
J. Hunziker, K. Schroer, R. Snajdrova, R.
Buller, Angew Chem Int Ed Engl 2019.
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Nucleotide
halogenase:
Zhao Chunhua, Yan Shan, Li Qin, Zhu
Hucheng, Zhong Zhiyu, Ye Ying, Zixin
Deng, Zhang Yonghui*
Nucleoside antibiotic adechlorin
halogenation requires an iron-α-
ketoglutarate-dependent
halogenase
AdeV,
which
zhangyh@mails.tjmu.edu.cn
regioselectively halogenates the C-
2’ position of dAMP, ddAMP or
dIMP.
Page No. – Page No.
Title
An iron(II)- and α-
ketoglutarate-dependent
halogenase acts on nucleotide
substrates
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Text for Table of Contents, 450 characters with spaces.
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