Substrate Conformation Correlates with the Outcome of Hyoscyamine 6β-Hydroxylase Catalyzed Oxidation Reactions

Article abstract of DOI:10.1021/jacs.8b03729

Hyoscyamine 6β-hydroxylase (H6H) is an α-ketoglutarate dependent mononuclear nonheme iron enzyme that catalyzes C6-hydroxylation of hyoscyamine and oxidative cyclization of the resulting product to give the oxirane natural product scopolamine. Herein, the chemistry of H6H is investigated using hyoscyamine derivatives with modifications at the C6 or C7 position as well as substrate analogues possessing a 9-azabicyclo[3.3.1]nonane core. Results indicate that hydroxyl rebound is unlikely to take place during the cyclization reaction and that the hydroxylase versus oxidative cyclase activity of H6H is correlated with the presence of an exo-hydroxy group having syn-periplanar geometry with respect to the adjacent H atom to be abstracted.

Full text of DOI:10.1021/jacs.8b03729

Subscriber access provided by Kaohsiung Medical University
Communication
Substrate Conformation Correlates with the Outcome of
Hyoscyamine 6##-Hydroxylase Catalyzed Oxidation Reactions
Richiro Ushimaru, Mark Walter Ruszczycky, Wei-chen Chang, Feng Yan, Yung-nan Liu, and Hung-wen Liu
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b03729 • Publication Date (Web): 05 Jun 2018
Downloaded from http://pubs.acs.org on June 5, 2018
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 5
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
Substrate Conformation Correlates with the Outcome of Hyoscya-
mine 6-Hydroxylase Catalyzed Oxidation Reactions
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Richiro Ushimaru, Mark W. Ruszczycky, Wei-chen Chang, Feng Yan, Yung-nan Liu, and Hung-wen
Liu*
Department of Chemistry, and Division of Chemical Biology and Medicinal Chemistry, College of Pharmacy, University of
Texas at Austin, Austin, Texas 78712, United States
Supporting Information Placeholder
also dehydrogenations.9,13,17 The conversion of (S)-2-hy-
ABSTRACT: Hyoscyamine 6hydroxylase (H6H) is an α-
droxypropylphosphonate (HPP, 12) to fosfomycin (13) is
another example of this chemistry also catalyzed by a mon-
onuclear non-heme iron enzyme, HPP epoxidase (HppE),
which uses H2O2 rather than α-KG as the oxidant.18,19
ketoglutarate dependent mononuclear non-heme iron en-
zyme that catalyzes C6-hydroxylation of hyoscyamine and
oxidative cyclization of the resulting product to give the
oxirane natural product scopolamine. Herein, the chemistry
of H6H is investigated using hyoscyamine derivatives with
modifications at the C6 or C7 position as well as substrate
analogues possessing a 9-azabicyclo[3.3.1]-nonane core.
Results indicate that hydroxyl rebound is unlikely to take
place during the cyclization reaction and that the hydrox-
ylase versus oxidative cyclase activity of H6H is correlated
with the presence of an exo-hydroxy group having syn-peri-
planar geometry with respect to the adjacent H-atom to be
abstracted.
Scheme 1. Reactions catalyzed by (A) H6H, (B) CAS, (C)
LolO, and (D) HppE
The mononuclear non-heme iron dependent oxidases are
an important class of enzymes that catalyze a diverse array
of reaction types including hydroxylation, desaturation, epi-
merization, halogenation and epoxidation.1–4 Members of
this enzyme family typically require -ketoglutarate (α-KG)
as a co-substrate for catalysis and possess a highly conserved
His/His/Asp(Glu) facial triad that coordinates the catalytic
iron center.1–4 Despite significant progress in understanding
this class of enzymes, questions remain regarding how these
enzymes are able to catalyze specific transformations
thereby preventing alternative reaction outcomes.
An atypical member of this enzyme family is hyoscya-
mine 6β-hydroxylase (H6H), which is involved in the bio-
synthesis of the anticholinergic alkaloid scopolamine (1) in
the Solanaceae family of plants.5–13 What makes H6H unu-
sual is its versatility, since it can catalyze hydroxylation (2
 3), dehydrogenation (3  1), and in vitro epoxidation (4
 1) reactions as shown in Scheme 1.7–10,11–13 Two other en-
zymes with similar catalytic properties are clavaminate syn-
thase (CAS)14 and LolO,15,16 which catalyze the conversion
of deoxyguanidino proclavaminate (5) to clavaminate (8)
and exo-1-acetamidopyrrolizidine (9) to N-acetylnorloline
(11), respectively (Scheme 1). Particularly notable are the
cyclization reactions (3  1, 6  7 & 10  11), which are
Similar to other α-KG-dependent non-heme iron en-
zymes,1–4 the hydroxylation reaction catalyzed by H6H is
predicted to involve H-atom abstraction from C6 of hyoscy-
amine (2) to give 15 by a high valent iron-oxo species (14)
that is generated via the reaction of the α-KG-Fe(II)-
substrate complex with O2 (see Scheme 2). Subsequent re-
bound of the metal-coordinated hydroxide then yields the
exo-6hydroxylated product (15  3). A similar iron-oxo
intermediary species could also play a key role in formation
of the oxirane ring (3  1). However, the reasons behind the
conversion of H6H to an oxidative cyclase rather than a hy-
droxylase when 3 is the substrate have yet to be established.
We report here the investigation of four substrate analogues
as mechanistic probes to delineate those features that deter-
mine the outcome of H6H-catalyzed reactions. The results
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 2 of 5
indicate that cyclization does not involve a diol intermediate
and that hydroxylation versus cyclization correlates with the
degree to which the abstracted exo-hydrogen eclipses the ad-
jacent hydroxy group in the unbound substrate.
suggested that one of the hydroxy groups in 20 had been ox-
+
idized to a carbonyl (calcd m/z for C17H22NO5 [M+H]+:
1
2
3
4
5
6
7
8
320.1492, obsd: 320.1500, see Figure S12). Isolation and
ESI-MS analysis of the reaction product following treatment
with Ac2O in pyridine (80 °C, 15 min) was consistent with
formation of a diacetylated species (e.g., 24, calcd m/z for
C21H26NO7+ [M+H]+: 404.1704, obsd: 404.1717, see Figure
S12). Furthermore, reduction of the reaction product with
sodium borohydride gave a compound that co-eluted with
(2′S)-20, but not with 6,7-dihydroxyhyoscyamine (25,
Figure S4). This implies that the keto functionality of the re-
action product is at C7 rather than C6, since reduction of the
keto group of 23 with NaBH4 should occur from the less hin-
dered exo face to regenerate 20.24 Based on these observa-
tions, the reaction product of 20 with H6H was assigned as
7-keto-6β-hydroxyhyoscyamine (23).
As shown in Scheme 3, the 7-keto product 23 could be
produced either by rebound of the hydroxyl group from
Fe(III)–OH to the radical intermediate (22  26), the direct
oxidation of 22 via electron transfer to the ferric iron (22 
23), or oxidative cyclization of 22 to 27 followed by ring
opening. When the reaction of 20 (as a 2′S/2′R mixture) was
conducted under 18O2, no 18O incorporation was found in 23
(Figure S5). Incorporation of 18O into 23 was observed when
the reaction was run in H218O; however, this could be fully
explained by hydration of the resulting ketone (Figure S5).
While these results do not rule out the possibility for stere-
oselective elimination of the exo-OH from 26, they do sug-
gest a mechanism involving either electron transfer (22 
23) or an oxirane intermediate (22  27  23). In either
case, neither 19 nor 20 appears to be an intermediate during
the cyclization of 3 to 1 arguing against the formation of a
1,2-diol intermediate and effectively ruling out routes C and
B-b (Scheme 2).
Scheme 2. Possible mechanisms for the epoxidation of 2.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Several possible mechanisms for the conversion of 3 to 1
are shown in Scheme 2. The 6-OH of 3 may coordinate the
iron center in the active site of H6H similar to substrate bind-
ing in the HppE-catalyzed epoxidation reaction (12 13,
Scheme 1).20–23 Such an interaction could facilitate H-atom
abstraction from the exo-C7 position in 3 by the Fe(IV)=O
complex (14) to generate the radical intermediate 1710 prior
to radical-mediated cyclization (route A). Ring formation
may also result from intramolecular nucleophilic addition of
the exo-C6-OH to the C7 carbocation in 18, which would be
produced via electron transfer from 17 to the Fe(III)OH
complex (route B-a). Alternatively, hydroxyl rebound to 17
could yield a 1,2-diol (19 or 20), which can then be con-
verted to 1 through nucleophilic displacement of the C7 hy-
droxy group (route C). The dihydroxylation could also occur
via a cation intermediate (18, route B-b).
To test whether a 1,2-diol intermediate is formed during
the catalytic cycle, 19 and 20 were synthesized (the latter as
a 3:2 mixture of 2′S and 2′R diastereomers) and incubated
with H6H from Hyoscyamus niger heterologously expressed
and purified using Escherichia coli (see Supporting Infor-
mation). No consumption of 19 (1 mM) was detected under
standard assay conditions (aerobic, 68 M H6H, 0.40 mM
FeSO4, 5.0 mM α-KG, 4.0 mM ascorbate, 50 mM tris(hy-
droxymethyl)aminomethane (Tris) buffer, pH 7.4). Replac-
ing α-KG with succinate, which is the by-product derived
from α-KG upon formation of the iron-oxo species,1–4 did
not change the outcome (Figure S2). These results indicated
that 19 is an unlikely intermediate in the conversion of 3 to
1 (Scheme 3).
Scheme 3. H6H-catalzyed oxidation of 20.
In contrast to 2, which is hydroxylated in the presence of
H6H, compounds 3 and 20 undergo dehydrogenation with
the former known to result in an oxirane. These observations
raised the question as to what determines hydroxylation ver-
sus dehydrogenation. One possibility is that the presence of
an exo-hydroxy functionality vicinal to the site of H-atom
abstraction plays a role in redirecting reaction flux to dehy-
drogenation. As a test of this hypothesis, 7β-hydroxyhyos-
cyamine (28)25,26 was prepared and incubated with H6H
On the contrary, consumption of (2′S)-20 was observed
under the standard assay conditions with α-KG, and a new
peak was detected by HPLC different from that of 1; how-
ever, no reaction was observed with succinate or (2′R)-20
(see Figure S3). ESI-MS analysis of the reaction product
ACS Paragon Plus Environment

Page 3 of 5
Journal of the American Chemical Society
(Scheme 4). In this experiment, formation of the C6-hydrox-
1
2
3
4
5
6
ylated product 19 from 28 was expected assuming the pres-
ence of the exo C7-OH leaves the H6H catalytic cycle essen-
tially unperturbed compared to that for 2. Instead, however,
28 was slowly cyclized to 1 in the presence of H6H under
standard conditions (Scheme 4), which was confirmed by
Scheme 5. (A) H6H-catalyzed oxidation of 30; (B) Corre-
lation of H6H-catalyzed reaction outcome with substrate
conformation.
+
ESI-MS spectroscopy (calcd m/z for C17H22NO4 [M+H]+:
304.1543, obsd: 304.1554) and co-elution with a standard
(Figures S6 & S12). No dihydroxylated product (e.g., 19)
could be detected. Hence, the presence of an exo-hydroxy
group at either C6 or C7 indeed influences the course of the
reaction. Consistent with previous reports,10 the observation
that 3, 20 and 28 are all substrates for H6H whereas 19 is not
implies that H-atom abstraction can occur from either the
exo-C6 or exo-C7 position but not from the corresponding
endo positions.
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 4. H6H-catalyzed oxidation of 28.
The observation that H6H catalyzes cyclization of hy-
droxy-azabicyclo[3.2.1]octanes (3 & 28) and hydroxylation
of hydroxy-azabicyclo[3.3.1]nonanes (31 & 34) suggests
that the orientation of the exo-C–H bond to be broken versus
the adjacent exo-C–O bond is correlated with the subsequent
reaction course. The X-ray crystal structure of 30 shows that
it adopts a staggered conformation with an H–C6–C7–H di-
hedral angle of 49° (Figure S8). Furthermore, gas phase
RB3LYP/6-31G* computations of models of 31a (with R =
CH3) implied that a similar HO–C6–C7–H dihedral angle is
retained in 31a (see Scheme 5B and Figure S42), which is
consistent with characterization by NMR (see Supporting In-
formation). Likewise, computational models of 34 (R =
CH3) show H–C6–C7–OH dihedral angles ranging from 35°
to 52° with respect to the exo-C6–H bond (see Figure S40).
This continues a general trend of staggered substrate confor-
mations observed among other α-KG-dependent nonheme
iron enzymes catalyzing the -hydroxylation of alcohols to
produce vicinal diols (e.g., OrfP,27 PolL,28 GA 2β oxidase,29
KdoO,30 BcmG,31,32 and RbtG33). In contrast, gas phase
models of 3 (R = CH3) exhibit dihedral angles less than 4°
versus the exo-C7–H bond (see Scheme 5B & Figure S38).
One possible explanation for this correlation is that a sim-
ilar geometric arrangement is maintained between the exo-
C–OH bond and the adjacent partially filled p-orbital in the
radical intermediate resulting from H-atom abstraction. This
would then facilitate cyclization of radicals derived from 3
and 28 (i.e., 17 & 29) as opposed to oxygen rebound for rad-
icals derived from 31 and 34. However, while gas phase
UB3LYP/6-31G* computations indicated that the HO–C6–
C7–p angle increases to no more than 22° in the modeled
radicals derived from 3 (R = CH3, see Figure S39), angles
less than 15° (i.e., periplanar) could also be found among
optimized conformers of radicals derived from 31 and 34
(see Figures S41 & S43). Therefore, interactions between
the enzyme and bound substrate may be important to main-
tain the initial substrate geometry in order to explain the ob-
To explore the relationship between substrate structure
and reaction course, an analogue with a 9-azabicy-
clo[3.3.1]nonane core (30) was synthesized (Scheme 5A).
Two new products in an approximately 3:2 ratio were de-
tected by HPLC when 30 was incubated with H6H for 10
min under standard conditions (Figure S9). These were as-
signed as the mono-hydroxylated species 31a (or its isomer
31b) and 34 based on NMR and ESI-MS (calcd m/z for
+
C18H26NO4 [M+H]+: 320.1856, obsd: 320.1848 and
320.1849 for 31 and 34, respectively, see Supporting Infor-
mation). The identity of the major product was confirmed to
be 34 by co-elution with a synthetic standard (Figure S10).
Upon further incubation, these products are consumed with
concomitant formation of two new species in a roughly 3:1
ratio both having masses consistent with dihydroxylated de-
+
rivatives of 30 (calcd m/z for C18H26NO5 [M+H]+:
336.1805, obsd: 336.1799 and 336.1795, see Figures S9 &
S12). The major dihydroxylated species was identified as the
6,7-dihydroxylated compound 33a (or its isomer 33b) by
HPLC co-elution with a mixed synthetic standard of 33a &
33b (Figure S11). Purification of the mono-hydroxylated
products (31 & 34) followed by reincubation with H6H
showed that both could be converted to 33 (Figure S10).
While the structure of the minor dihydroxylated species
could not be determined, it does not coelute with the mixed
standard of 33a & 33b by HPLC. The only evidence for cy-
clization was an LCMS signal consistent with 32 or a C6/C8-
+
keto derivative of 30 (calcd m/z for C18H24NO4 [M+H]+:
318.1700, obsd: 318.1695, see Figure S12) when purified 30
was incubated with H6H. However, the MS signal intensity
of this species was less than 5% relative to that of the major
dihydroxylation product (33). Therefore, if cyclization does
take place, then it is only a very minor side reaction.
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 4 of 5
(6) Fodor, G.; Romeike, A.; Janzeó, G.; Koczor, I. Tetrahedron Lett.
1959, 7, 19–23.
served correlation. Alternatively, a discrete radical interme-
1
2
3
4
5
6
7
8
diate susceptible to rebound may only form from substrates
with staggered (31 & 34) rather than eclipsed alignments (3
& 28). In the latter case, cyclization may proceed in concert
with H-atom abstraction resulting in a one-step-two-electron
reduction of the Fe(IV)=O complex. While enzymatic
Fe(IV)=O complexes are typically modeled as stepwise one-
electron acceptors, Fe(IV)=O complexes have been impli-
cated in two-electron processes,34,35 and a similar chemistry
may be at work in the catalytic cycle of H6H.
(7) Hashimoto, T.; Yamada, Y. Plant Physiol. 1986, 81, 619−625.
(8) Hashimoto, T.; Yamada, Y. Eur. J. Biochem. 1987, 164, 277–285.
(9) Hashimoto, T.; Kohno, J.; Yamada, Y. Plant Physiol. 1987, 84,
144–147.
(10) Hashimoto, T.; Kohno, J.; Yamada, Y. Phytochemistry 1989, 28,
1077–1082.
(11) Matsuda, J.; Okabe, S.; Hashimoto, T.; Yamada, Y. J. Biol. Chem.
1991, 266, 9460−9464.
(12) Hashimoto, T.; Matsuda, J.; Yamada, Y. FEBS Lett. 1993, 329,
35–39.
9
H6H represents an excellent system for studying the par-
titioning of radical-mediated catalytic cycles among differ-
ent reaction paths. Herein, evidence is provided that the ox-
idative cyclization catalyzed by H6H does not involve re-
bound of the hydroxyl group following H-atom abstraction.
Furthermore, cyclization versus rebound appears to require
that an exo-OH not only be adjacent to the site of H-atom
abstraction but also have the correct syn-periplanar configu-
ration. Future investigation of these properties and the chem-
istry underlying them will provide new insights into the me-
chanics of nonheme iron biocatalysts.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(13) Li, J.; van Belkum, M. J.; Vederas, J. C. Bioorg. Med. Chem. 2012,
20, 4356−4363.
(14) Elson, S. W.; Baggaley, K. H; Gillett, J.; Holland, S.; Nicholson,
N. H.; Sime, J. T.; Woroniecki, S. R. J. Chem. Soc., Chem. Commun.
1987, 1736–1738.
(15) Pan, J.; Bhardwaj, M.; Faulkner, J. R.; Nagabhyru, P.; Charlton,
N. D.; Higashi, R. M.; Miller, A.-F.; Young, C. A.; Grossman, R. B.;
Schardl, C. L. Phytochemistry 2014, 98, 60−68.
(16) Pan, J.; Bhardwaj, M.; Zhang, B.; Chang, W.-c.; Schardl, C. L.;
Krebs, C.; Grossman, R. B.; Bollinger, J. M., Jr. Biochemistry 2018,
57, 2074–2083.
(17) Krol, W. J.; Basak, A.; Salowe, S. P.; Townsend, C. A. J. Am.
Chem. Soc. 1989, 111, 7625–7627.
(18) Liu, P.; Murakami, K.; Seki, T.; He, X.; Yeung, S.- M.; Kuzuyama,
T.; Seto, H.; Liu, H.-w. J. Am. Chem. Soc. 2001, 123, 4619–4620.
(19) Wang, C.; Chang, W.-c.; Guo, Y.; Huang, H.; Peck, S. C.; Pande-
lia, M. E.; Lin, G.-m.; Liu, H.-w.; Krebs, C.; Bollinger, J. M., Jr. Sci-
ence 2013, 342, 991−995.
ASSOCIATED CONTENT
The Supporting Information is available free of charge on
the ACS Publications website at DOI: XXX. Details regard-
ing H6H assays along with HPLC protocols, chemical syn-
thesis of compounds and computational methods and results.
(20) Higgins, L. J.; Yan, F.; Liu, P; Liu, H.-w.; Drennan, C. L. Nature
2005, 437, 838−844.
(21) Chang, W.-c.; Dey, M.; Liu, P.; Mansoorabadi, S. O.; Moon, S.-
J.; Zhao, B. K.; Drennan, C. L.; Liu, H.-w. Nature 2013, 496, 114−118.
(22) Yun, D.; Dey, M.; Higgins, L. J.; Yan, F.; Liu, H.-w.; Drennan, C.
L. J. Am. Chem. Soc. 2011, 133, 11262–11269.
(23) Yan, F.; Moon, S.-J.; Liu, P.; Zhao, Z.; Lipscomb, J. D.; Liu, A.;
Liu, H.-w. Biochemistry 2007, 46, 12628–12638.
(24) Grundt, P.; Kopajtic, T. A.; Katz, J. L.; Newman, A. H. Bioorg.
Med. Chem. Lett. 2004, 14, 3295–3298.
(25) Ishimaru, K.; Shimomura, K. Phytochemistry 1989, 28, 3507–
3509.
(26) Muñoz, M. A.; Muñoz, O.; Joseph-Nathan, P. J. Nat. Prod. 2006,
69, 1335–1340.
(27) Chang, C.-Y., Lyu, S.-Y., Liu, Y.-C., Hsu, N.-S., Wu, C.-C., Tang,
C.-F., Lin, K.-H., Ho, J.-Y., Wu, C.-J., Tsai, M.-D., and Li, T.-L. An-
gew. Chem., Int. Ed. 2014, 53, 1943−1948.
(28) Qi, J.; Wan, D.; Ma, H.; Liu, Y.; Gong, R.; Qu, X.; Sun, Y.; Deng,
Z.; Chen, W. Cell. Chem. Biol. 2016, 23, 935–944.
(29) Thomas, S. G.; Phillips, A. L.; Hedden, P. Proc. Natl. Acad. Sci.
U.S.A. 1999, 96, 4698–4703.
(30) Chung, H. S.; Raetz, C. R. H. Proc. Natl. Acad. Sci. U.S.A. 2010,
108, 510–515.
(31) Patteson, J. B.; Cai, W.; Johnson, R. A.; Santa Maria, K. C.; Li, B.
Biochemistry 2018, 57, 61–65.
AUTHOR INFORMATION
Corresponding Author
*h.w.liu@mail.utexas.edu
ORCID
Hung-wen Liu: 0000-0001-8953-4794
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
We thank Dr. Takashi Hashimoto at Nara Institute of Science
and Technology for generously providing the clone of h6h.
This work was supported by grants from the National Insti-
tutes of Health (GM113106 and GM040541) and the Welch
Foundation (F-1511).
REFERENCES
(1) Hausinger, R. P. Biochemical diversity of 2-oxoglutarate dependent
oxygenases. In 2-Oxoglutarate-dependent oxygenases (Schofield, C. J.,
and Hausinger, R. P., Eds.) The Royal Society of Chemistry, Cam-
bridge, U.K. 2015, 1−58,
(32) Meng, S.; Han, W.; Zhao, J.; Jian, X.-H.; Pan, H.-X.; Tang, G.-L.
Angew. Chem., Int. Ed. 2018, 57, 719–723.
(33) Bai, J.; Yan, D.; Zhang, T.; Guo, Y.; Liu, Y.; Zou, Y.; Tang, M.;
Liu, B.; Wu, Q.; Yu, S.; Tang, Y.; Hu, Y. Angew. Chem., Int. Ed. 2017,
56, 4782–478.ꢀ
(34) Pestovsky, O.; Bakac, A. J. Am. Chem. Soc. 2004, 126, 13757–
13764.
(35) Oh, N. Y.; Suh, Y.; Park, M. J.; Seo, M. S.; Kim, J.; Nam, W.
Angew. Chem. 2005, 117, 4307–4311.
(2) Kovaleva, E. G.; Lipscomb, J. D. Nat. Chem. Biol. 2008, 4, 186–
193.
(3) Costas, M.; Mehn, M. P.; Jensen, M. P.; Que, L., Jr. Chem. Rev.
2004, 104, 939–986.
(4) Krebs, C.; Fujimori, D. G.; Walsh, C. T.; Bollinger, M. J., Jr. Acc.
Chem. Res. 2007, 40, 484–492.
(5) Klinkenberg, I.; Blokland, A. Neurosci. Biobehav. Rev. 2010, 34,
1307−1350.
ACS Paragon Plus Environment

Page 5 of 5
Journal of the American Chemical Society
1
2
3
4
H6H
α-ketoglutarate
O2
5
6
7
8
9
H6H
α-ketoglutarate
O2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
5
ACS Paragon Plus Environment