Structures and Mechanisms of the Non-Heme Fe(II)- and 2-Oxoglutarate-Dependent Ethylene-Forming Enzyme: Substrate Binding Creates a Twist

Article abstract of DOI:10.1021/jacs.7b06186

The ethylene-forming enzyme (EFE) from Pseudomonas syringae pv. phaseolicola PK2 is a member of the mononuclear nonheme Fe(II)- and 2-oxoglutarate (2OG)-dependent oxygenase superfamily. EFE converts 2OG into ethylene plus three CO₂ molecules while also catalyzing the C5 hydroxylation of l-arginine (l-Arg) driven by the oxidative decarboxylation of 2OG to form succinate and CO₂. Here we report 11 X-ray crystal structures of EFE that provide insight into the mechanisms of these two reactions. Binding of 2OG in the absence of l-Arg resulted in predominantly monodentate metal coordination, distinct from the typical bidentate metal-binding species observed in other family members. Subsequent addition of l-Arg resulted in compression of the active site, a conformational change of the carboxylate side chain metal ligand to allow for hydrogen bonding with the substrate, and creation of a twisted peptide bond involving this carboxylate and the following tyrosine residue. A reconfiguration of 2OG achieves bidentate metal coordination. The dioxygen binding site is located on the metal face opposite to that facing l-Arg, thus requiring reorientation of the generated ferryl species to catalyze l-Arg hydroxylation. Notably, a phenylalanyl side chain pointing toward the metal may hinder such a ferryl flip and promote ethylene formation. Extensive site-directed mutagenesis studies supported the importance of this phenylalanine and confirmed the essential residues used for substrate binding and catalysis. The structural and functional characterization described here suggests that conversion of 2OG to ethylene, atypical among Fe(II)/2OG oxygenases, is facilitated by the binding of l-Arg which leads to an altered positioning of the carboxylate metal ligand, a resulting twisted peptide bond, and the off-line geometry for dioxygen coordination.

Full text of DOI:10.1021/jacs.7b06186

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Article
Structures and Mechanisms of the Non-Heme Fe(II)- and 2-Oxoglutarate-
Dependent Ethylene-Forming Enzyme: Substrate Binding Creates a Twist
Salette Martinez, Matthias Fellner, Caitlyn Q Herr, Anastasia Ritchie, Jian Hu, and Robert P. Hausinger
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b06186 • Publication Date (Web): 05 Aug 2017
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Structures and Mechanisms of the Non-Heme Fe(II)- and 2-
Oxoglutarate-Dependent Ethylene-Forming Enzyme: Substrate
Binding Creates a Twist
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†,
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Salette Martinez, Matthias Fellner, Caitlyn Q Herr, Anastasia Ritchie, Jian Hu,‡,#,* and Rob-
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ert P. Hausinger†,‡,*
†Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan 48824
‡Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824
#Department of Chemistry, Michigan State University, East Lansing, Michigan 48824
ABSTRACT: The ethylene-forming enzyme (EFE) from Pseudomonas syringae pv. phaseolicola PK2 is a member of the
mononuclear non-heme Fe(II)- and 2-oxoglutarate (2OG)-dependent oxygenase superfamily. EFE converts 2OG into eth-
ylene plus three CO2 molecules while also catalyzing the C5 hydroxylation of L-arginine (L-Arg) driven by the oxidative
decarboxylation of 2OG to form succinate and CO2. Here we report eleven X-ray crystal structures of EFE that provide
insight into the mechanisms of these two reactions. Binding of 2OG in the absence of L-Arg resulted in predominantly
monodentate metal coordination, distinct from the typical bidentate metal-binding species observed in other family
members. Subsequent addition of L-Arg resulted in compression of the active site, a conformational change of the car-
boxylate side chain metal ligand to allow for hydrogen bonding with the substrate, and creation of a twisted peptide bond
involving this carboxylate and the following tyrosine residue. A reconfiguration of 2OG achieves bidentate metal coordi-
nation. The dioxygen binding site is located on the metal face opposite to that facing L-Arg, thus requiring reorientation
of the generated ferryl species to catalyze L-Arg hydroxylation. Notably, a phenylalanyl side chain pointing towards the
metal may hinder such a ferryl flip and promote ethylene formation. Extensive site-directed mutagenesis studies support-
ed the importance of this phenylalanine and confirmed the essential residues used for substrate binding and catalysis.
The structural and functional characterization described here suggests that conversion of 2OG to ethylene, atypical
among Fe(II)/2OG oxygenases, is facilitated by the binding of L-Arg which leads to an altered positioning of the carbox-
ylate metal ligand, a resulting twisted peptide bond, and the off-line geometry for dioxygen coordination.
INTRODUCTION
EFE is associated with selected plant pathovars such as
the bacteria Pseudomonas syringae and Ralstonia sola-
nacearum and the fungus Penicillium digitatum.17-18 The
archetype EFE, from P. syringae pv. phaseolicola PK2, cat-
alyzes two reactions in approximately a 1:2 ratio: (1) the
oxidative decarboxylation of 2-oxoglutarate (2OG) cou-
pled to C5 hydroxylation of L-arginine (L-Arg), with the
hydroxy-L-Arg intermediate decomposing to guanidine
and L-Δ-1-pyrroline-5-carboxylate (P5C), and (2) the de-
composition of 2OG to ethylene and three molecules of
CO2 (Scheme 1).19-20 The first reaction is typical of those
catalyzed by a large family of mononuclear non-heme
Fe(II)- and 2OG-dependent oxygenases21-24 and may be
the ancestral activity of this enzyme, whereas the second
(major) reaction is unique to EFE.
Ethylene, H2C=CH2, is an important building block in
the production of plastics, fibers, foams, soaps, deter-
gents, antifreeze, etc.1 This hydrocarbon is produced
worldwide at levels that exceed all other synthetic organic
chemicals, with its production expected to increase at a
compound annual growth rate of ~11.26% from 2016-2025.2
Ethylene is primarily obtained from thermal cracking of
natural gas and petroleum,3 but this method of produc-
tion unfortunately releases into the atmosphere large
quantities of CO2 and other greenhouse gases with conse-
quent negative environmental impacts.4 Therefore, tech-
nologies are required that produce ethylene from renew-
able resources. Examples of these alternative or “green”
methods include production of ethylene from biomass5-6
and the use of genetically engineered microorganisms
including yeast,7-8 Escherichia coli,9-10 and cyanobacteria11-
To clarify how this unique member of the Fe(II)/2OG-
dependent oxygenases carries out its dual transfor-
mations, we crystallized EFE apoenzyme and ten enzyme
complexes with metals and substrates or substrate ana-
logues. We highlight the important differences between
our results and three just reported structures
(EFE•Mn•bis-Tris-propane (BTP), EFE•Mn•2OG, and
16
that express the efe gene encoding an ethylene-forming
enzyme (EFE). The use of transgenic organisms with this
gene offers a promising sustainable method for the com-
mercial production of ethylene.
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EFE•Fe•N-oxalylglycine (NOG)•L-Arg), especially those analyses provide new insights into the unique ethylene-
related to ethylene formation.25 In addition, we examine
the properties of a range of EFE variants and compare our
findings with results of prior mutagenesis efforts.25-27 To-
gether, our structural, mutagenesis, and biochemical
forming capability of EFE compared to other Fe(II)/2OG
oxygenases and they provide a blueprint for engineering
EFE to enhance 2OG transformation ethylene without
carrying out the wasteful hydroxylation of L-Arg.
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Scheme 1. Reactions catalyzed by EFE
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RESULTS
Compared to the structure of the apoprotein (Figure 1A),
Ni (and Mn) binding led to conformational changes of the
metal-chelating residues (Figure 1B). In particular, the
side chain of H268 flips up and participates in metal bind-
ing with the pre-positioned H189 and D191.
Overall Structure of EFE. We solved eleven crystal
structures of EFE: the EFE apoprotein, EFE•Ni, and
EFE•Mn with 2OG, 2OG•L-Arg, 2-oxoadipate (2OA)•L-
Arg,
2OG•Nγ-hydroxy-L-Arg
(HO-L-Arg),
2OG•L-
argininamide (L-Arg-amide), L-Arg, tartrate, malate, and
malate•L-Arg, with EFE•Mn•2OG•L-Arg-amide exhibiting
the best resolution at 1.14 Å (Figure 1, Tables S1-S3). Ten
structures contained a single EFE molecule per asymmet-
ric unit, consistent with the monomeric species observed
in our earlier size-exclusion chromatography/multi-angle
light scattering analysis,20 whereas EFE•Mn•2OG con-
tained two EFE molecules per unit cell.
In the EFE•Mn•2OG structure, the metal ion is hexa-
coordinated and electron density was observed near the
metal center corresponding to 2OG (Figure 1C). In con-
trast to the usual bidentate metal-binding mode of 2OG
found in other enzymes of this family,28-29 this substrate
was bound monodentate to Mn in EFE lacking L-Arg and
was observed in two inverted conformations (Figure 2A).
In the 2OGA conformation, the substrate bound Mn with
its C1 carboxyl group; in the 2OGB conformation, it bound
the metal using its C5 carboxyl group. Monodentate bind-
ing to Mn also was observed for the shorter dicarbox-
ylates, tartrate and malate, including for the
EFE•Mn•malate•L-Arg complex (Figure 1, D-E and J).
EFE contains a double-stranded β-helix (DSBH, also
known as the jellyroll or cupin fold)28 core typically found
in members of the Fe(II)/2OG-dependent oxygenases.
Nine β-strands, six from the major β-sheet and three from
the minor β-sheet, form the DSBH that is surrounded and
stabilized by 10 α-helices (Figure S1A). The eleven EFE
structures exhibited the same overall fold with only slight
differences observed (Figure S1B). In particular, residues
80-93 move significantly towards the active site when
metal and substrate are bound, acting like a “lid” that
covers or shields the active site from the solvent.
L-Arg Binding Leads to Bidentate Metal Coordina-
tion by 2OG. In the presence of L-Arg, 2OG shifts from
monodentate coordination of the Mn site to the typical
chelate binding mode (Figures 1G and 2B). Similarly, the
L-Arg analogues L-Arg-amide and HO-L-Arg also facili-
tate bidentate Mn chelation by 2OG (Figure 1, H and I).
2OA, an alternative 2-keto acid substrate greater in size
by one methylene unit compared to 2OG, also bound the
metallocenter in a chelate mode when L-Arg was present
(Figure 1F).
2OG Adopts Two Modes of Monodentate Metal
Binding in the Absence of L-Arg. The 2-histidine-1-
carboxylate metal-binding site of EFE is located in a cavity
formed by the major and minor β-sheets of the DSBH fold
with the H189, D191, and H268 ligands coming from β-8,
the loop connecting β-8 to β-9, and β-13 (Figure S1).
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Figure 1. Active site views of 11 EFE structures. Carbon atoms of the metal-chelating residues (H189, D191 and H268) are shown
in green; 2OG, 2OA, malate, or tartrate are depicted in yellow; L-Arg, HO-L-Arg, or L-Arg-amide are illustrated in magenta; Mn
and Ni atoms are shown as purple and green spheres. The 2FO - FC maps for metal atoms and ligands are shown as blue meshes at
1 σ. The anomalous maps for metal atoms are shown as magenta meshes at 5 σ. Metal chelation is indicated by dashed lines.
Bound water molecules are shown as red spheres. (A) EFE apoprotein. (B) EFE•Ni. (C) EFE•Mn•2OG. (D) EFE•Mn•tartrate. (E)
EFE•Mn•malate. (F) EFE•Mn•2OA•L-Arg. (G) EFE•Mn•2OG•L-Arg. (H) EFE•Mn•2OG•HO-L-Arg. (I) EFE•Mn•2OG•L-Arg-amide.
(J) EFE•Mn•malate•L-Arg. (K) EFE•Mn•L-Arg.
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In all cases of bound 2OG, the carboxylate distal to the
metal was stabilized by a salt bridge with R277 and the
carboxylate coordinating the metal was stabilized by hy-
drogen bonds with R171 (Figure 2). Efforts to produce the
R277A variant led to the formation of inclusion bodies
and we did not study this protein further. Furthermore,
while the R171A variant enzyme was soluble, it produced
no detectable ethylene and only a trace of P5C under the
standard assay conditions (Figure 3A and Table S4). These
results indicate the critical nature of these two arginine
residues for interacting with 2OG.
to the near elimination of ethylene production (Figure 3B,
Table S4), but several of these variant proteins continued
to produce significant levels of P5C. Single substitutions
of V196 and A281 to arginine resulted in enzymes produc-
ing negligible levels of products. By contrast, the A281V
variant produced low levels of products in comparison to
the WT enzyme. The V196F variant was insoluble and
found in inclusion bodies. Notably, the A198V variant
produced large amounts of P5C but very little ethylene.
These results highlight the importance of maintaining a
hydrophobic environment surrounding the 2OG-binding
pocket, with strict limitations on the size of the hydro-
phobic residue. These residues may function in 2OG sta-
bilization, binding of dioxygen, or release of the small
nonpolar product ethylene.
In contrast to its carboxylate-binding interactions, 2OG
binds to a pocket lined by hydrophobic residues (L173,
F175, L206, V270, A279, and F283 (Figure S2A); and the
nearby V196, A198, and A281 (Figure 3B)). Replacing F283
by tryptophan, tyrosine, arginine, alanine, and valine led
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Figure 2. Active site comparisons of EFE•Mn•2OG and EFE•Mn•2OG•L-Arg. Carbon atoms of the metal-chelating residues are
shown in green; 2OG and stabilizing residues are depicted in yellow; L-Arg and its stabilizing residues are illustrated in magenta
(these residues are cyan when L-Arg is absent); and water and Mn are shown as red and purple spheres, respectively. The 2FO - FC
maps for metal atoms and ligands are shown as blue meshes at 1 σ. The anomalous maps for metal atoms are shown as magenta
meshes at 5 σ. Metal chelation is indicated by dashed lines colored the same as the metal, hydrogen bonds are indicated by red
dashed lines. (A) EFE•Mn•2OG (1.85 Å). (B) EFE•Mn•2OG•L-Arg (1.43 Å).
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L-Arg-Binding Introduces Significant Conforma-
tional Changes in the Active Site. L-Arg was observed
to bind near, but did not coordinate, the metal center in
EFE•Mn•2OG•L-Arg (Figure 1G) as well as in
the newly created ferryl species would need to “flip” to a
very different position, a notion that has been proposed
for other off-line members of this enzyme family.30-31 The
repositioning of F283 upon binding of L-Arg (Figure 4B)
may have implications regarding this ferryl flip. This resi-
due adopts a position pointing at the metal center (~4.6 Å
away) and may hinder the flipping of the ferryl species
(Figure 4A). Substitution by the larger residues tyrosine,
tryptophan, and arginine essentially abolished ethylene-
forming activities (Figure 3B, Table S4), probably due to
diminished dioxygen binding, while greatly decreasing
P5C formation. Conversely, substitution with the smaller
residues valine and alanine allowed for significant levels
of L-Arg hydroxylation in the absence of ethylene for-
mation.
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EFE•Mn•malate•L-Arg,
EFE•Mn•L-Arg-amide,
EFE•Mn•HO-L-Arg, EFE•Mn•2OA•L-Arg, and EFE•Mn•L-
Arg (Figure 1, F, H-K). In all structures with bound L-Arg
and HO-L-Arg a single conformation was observed, with
only the L-Arg-amide ligand showed two conformations
differing slightly in the position of the C5 and NE (Figure
1I). The binding of L-Arg led to several notable structural
changes at or near the active site: (i) 2OG shifts to biden-
tate coordination of the metal and defines a dioxygen
binding site (Figures 1G, 2B, and 4A), (ii) D191 alters its
orientation to engage in hydrogen bonding interactions
with the NH1 of L-Arg (Figures 2B and 4B), (iii) significant
positional changes were noted for several residues, lead-
ing to a twisted peptide bond between D191 and Y192
(Figure 4, B and C), and (iv) helix α-8 is brought closer to
the metal center making the active site more compact
(Figure S1B).
L-Arg binding induces a repositioning of the metal lig-
and D191, allowing it to form a hydrogen bond with the
newly introduced substrate (Figures 1G, 2B, and 4B). Ac-
companying the shift in D191 conformation, Y192 also un-
dergoes a large change in position to hydrogen bond with
L-Arg (Figure 4B). These changes create a twisted peptide
bond between D191 and Y192 at the enzyme active site
(Figure 4C). A peptide is annotated as twisted when one
of its dihedral angles deviate over 30° from its ideal value
(in this case from 180°).32 For the six L-Arg and L-Arg ana-
log bound structures, the ω torsion angle (D191-Cα, D191-
C, Y192-N, Y192-C) is 146.8° on average compared to the 5
unbound structures with an angle of 168.1° (Table S5). We
substituted Y192 with arginine, phenylalanine, or trypto-
phan, leading to reduced product formation in the latter
two cases (Figure 3A, Table S4). These results suggest
Y192 is important, but not essential, to catalysis and it
most likely both helps position L-Arg in the correct orien-
tation for the hydroxylation step and directs the 2OG
conversion to ethylene.
For the structures containing both 2OG (or 2OA) and
L-Arg (or L-Arg-amide or HO-L-Arg), the C1 carboxyl and
C2 keto groups of the 2-oxo acid coordinate the metal in a
bidentate manner and are nearly in the same plane as
H268 and D191 (the distal histidine and carboxylate lig-
ands). This configuration defines the open coordination
site for dioxygen binding opposite H189 as shown for
EFE•Mn•2OG•L-Arg (Figure 4A). Indeed, the highest reso-
lution structures containing HO-L-Arg (1.17 Å) and L-Arg-
amide (1.14 Å) revealed a water molecule bound at this
site. This configuration has been termed “off-line”30 be-
cause a ferryl species formed with this same orientation
would not point towards the oxygenase substrate; i.e., the
C5 of L-Arg which becomes hydroxylated by EFE. Rather,
to abstract the pro-S-hydrogen from this carbon atom of
the substrate, located only ~4.2 Å from the metal center,
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Figure 3. Activity comparisons of WT and selected EFE variants. Ethylene and P5C production by variants of (A) selected hydro-
philic residues near the active site along with their location (orange) in the EFE•Mn•2OG•Arg structure, (B) selected hydropho-
bic residues and their locations (dark blue), and (C) additional EFE variants and their locations (purple). Carbon atoms of metal
ligands are shown in white, 2OG in yellow, and L-Arg in magenta. Mn atoms are purple spheres. Assay conditions: 2 mL reaction
consisting of 0.5 mM 2OG, 0.5 mM L-Arg, 0.2 mM Fe(II), 0.4 mM L-ascorbic acid, and 1 µM EFE in 25 mM HEPES, pH 7.5, incu-
bated at 25 °C for 80 min and terminated with HCl. WT enzyme generates ~2 ethylene per P5C, and comparisons are shown as
percentage of the WT values. Error bars represent the standard errors of at least two independent enzyme preparations. Variants
marked with * were found in inclusion bodies.
In addition to the hydrogen bonding with D191 and
Y192, L-Arg binds in a hydrophilic pocket with stabiliza-
tion achieved by interactions with E84, V85, T86, R316,
and Van der Waals interactions with C317 (Figures 3A,
S2B, and S2C). E84 forms hydrogen bonds with both the
NE and amide nitrogens of L-Arg. Substitution of this
residue with alanine leads to near elimination of ethylene
and P5C production (Figure 3A, Table S4). Both T86 and
the carbonyl oxygen of V85 form hydrogen bonds to the
amide nitrogen. Hydrogen bonding with R316 stabilizes
the substrate carboxylate, and this residue undergoes a
reorientation when L-Arg binds (Figure S2C). We substi-
tuted the neighboring residue, C317, with serine or aspar-
agine leading to relatively minor changes in activity (Fig-
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ure 3A, Table S4). We conclude that this residue is not matic network with F314 and Y318 (all located in the α-8
critical to catalysis.
1
helix) along with Y192 and F283. Substitution of Y306 with
alanine or phenylalanine decreased the level of all prod-
ucts. Similarly, elimination of aromatic interactions by
substituting F310 with arginine proved detrimental for
EFE activity; however, substitution with the aromatic res-
idue tryptophan resulted in retention of substantial activ-
ities. The large reduction in catalysis by EFE when these
residues were changed to non-aromatic side chains is
likely the result of destabilization throughout the helix
that prevents it from moving closer to the active site
when L-Arg binds, as needed to make it ready for dioxy-
gen binding. No activity changes were expected for the
L73K, L73R, R184A, and E213A variants with substitutions
positioned distant to the active site, and only modest ac-
tivity losses were observed (Figure 3C, Table S4). Surpris-
ingly, the E215A and E213A/E215A variants exhibited sub-
stantial reductions in EFE activities despite the distance
of E215 from the active site (Figure 3C, Table S4); we have
no explanation for this sensitivity. In contrast, the Δ338-
350 truncation variant was highly active, consistent with
the non-essential role of the carboxyl terminus of EFE
from P. syringae pv. phaseolicola PK2 which is extended
compared to a related sequence for a known ethylene-
forming enzyme that is missing this region (Figure S3).
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Figure 4. Identification of the O2 binding site and a twisted
peptide bond in EFE•Mn•2OG•L-Arg. (A) The C1-carboxylate
oxygen of 2OG (yellow carbons) binds approximately trans to
the distal H189 and the C2-keto oxygen binds opposite D191,
thus defining a dioxygen-binding site in the “off-line” config-
uration that points away from the C5 position of the L-Arg
substrate (magenta carbons). The 2-histidine-1-carboxylate
metal-binding motif is shown with green carbons, and the
nearby F283 residue is shown in blue. The dioxygen-binding
site is illustrated by a water molecule (red sphere) seen in
our EFE•Mn•2OG•HO-L-Arg structure. (B) Conformational
changes occurring upon L-Arg binding. Comparison of the
active sites of EFE•Mn•2OG in cyan and EFE•Mn•2OG•L-Arg
in magenta, based on an active site alignment. Metal chela-
tion is shown by dashed lines, a key hydrogen bond between
NH1 of L-Arg and OD1 of D191 is indicated by a red dashed
line. Alteration of the D191 orientation leads to a switch in
metal chelation from OD1 to OD2. (C) Ball and stick represen-
tations of the D191-Y192 peptide bond before and after upon
L-Arg binding, showing the dihedral peptide angle ω (Cα, C,
N+1, Cα+1). The twisted peptide is highlighted in yellow.
DISCUSSION
EFE exhibits the typical DSBH fold and 2-histidine-1-
carboxylate metal-binding motif found in other members
of the Fe(II)/2OG oxygenase family, yet it uniquely cata-
lyzes the transformation of 2OG into ethylene. Below, we
compare our findings derived from 11 EFE structures to
those associated with three just-published EFE struc-
tures,25 especially in terms of understanding how the en-
zyme catalyzes its two reactions. In addition, we place the
previously published mutagenesis studies into a structural
context.
Comparisons to Prior EFE Structures. Zhang et al.
reported a model for an EFE•Mn•2OG structure (PDB ID
5MOF, 1.45 Å)25 that exhibits substantial differences from
our model (PDB ID 5V2X, 1.85 Å). In 5MOF, 2OG has bi-
dentate binding to Mn at 70% occupancy with the sixth
coordination site being water at 100% occupancy. The C5
carboxyl group weakly interacts with R277, with only O4
forming a hydrogen bond. At the same position, a chlo-
ride ion (modeled at 30% occupancy) strongly interacts
with R277. In addition, they model several water mole-
cules at 30% occupancy around 2OG and at the positions
of the metal-chelating O2 and O5 atoms (Figure S4B). In
5V2X, we interpreted the map with 2OG binding as a
monodentate ligand to Mn, with two water molecules
coordinated at 100% occupancy. We chose two confor-
mations (~50%+~30%) for 2OG, either with the C1 or C5
carboxyl group facing R277, in each case with both car-
boxylate oxygen atoms involved in a salt bridge (Figure
2A and S4A). Both interpretations are consistent that
2OG does not bind tightly to the active site in the absence
of L-Arg or an L-Arg analog. We further argue that the
Non-Active Site Regions. We examined the effects on
EFE activities of a series of additional variants that in-
volved residues more distant from the active site (Figure
3C and Table S4). S81 is located in a region that undergoes
major rearrangement upon binding of the metal ion and
substrates (Figure S1B). Both the S81R and S81Y variants
exhibited substantial reductions in product formation,
likely due to increased instability of this region. Because
E285 hydrogen bonds with Y306 in helix α-8, we investi-
gated the E285Q and E285A variants. Both species lost
nearly all ethylene-forming activity, while retaining some
P5C production. Residues Y306 and F310 form an aro-
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There are at least two possible explanations for why
monodentate binding is more plausible for the structure
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lacking L-Arg because the electron density for the biden-
tate linkage is not strong in our case and forcing a biden-
tate model would lead to an overbuilt situation. Further-
more, our observed strong interaction of 2OG with R277
appears to be a better interpretation than including a
chloride ion at this position. In both EFE•Mn•2OG struc-
tures, the D191 coordination is not switched to introduce
the twisted peptide bond, consistent with our other
monodentate-bound structures that lack L-Arg,
EFE•Mn•tartrate (Figure 1D) and EFE•Mn•malic acid (Fig-
ure 1E), which also contain water molecules at the fifth
and sixth coordination sites. Also, our structural interpre-
tation of predominantly monodentate coordination is
compatible with our previously reported anaerobic UV-
visible difference spectra of the Fe(II)-containing en-
zyme.20 In those studies, a low intensity (~28 M-1 cm-1)
metal-to-ligand charge-transfer (MLCT) transition at 515
nm was observed for the EFE•Fe(II)•2OG sample, but the
addition of L-Arg led to intensification of the MLCT tran-
sition at 510 nm (~79 M-1 cm-1) to a value that is more typ-
ical of related enzymes. The MLCT transition is known to
be associated with chelate binding of the metal in other
members of this enzyme family,20 so the sample free of L-
Arg exhibits a perturbed binding mode for 2OG. Our
model of the EFE•Mn•2OG complex reveals the nature of
this perturbation as a mixed ligand state with primarily
monodentate coordination.
Zhang et al. observed the second L-Arg conformation in
5LUN.25 First, the metal might need to be Fe for the se-
cond conformation to appear; however, this hypothesis is
unlikely because L-Arg does not directly interact with the
metal. Furthermore, all other observations seen in our
Mn structures appear to match those of 5LUN, including
the two conformations of D191. More likely, the substitu-
tion of 2OG by NOG in 5LUN could influence the binding
of L-Arg and make this second conformation possible.
Our finding of clear coupling between 2OG binding and
L-Arg binding adds support to this notion.
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Another point of discrepancy between the Zhang et al.
studies25 and our findings centers on the intrinsic con-
formation of NOG/2OG. They emphasize the atypical
situation found in their 5LUN structure in which the
plane formed by the C5/O3/O4 carboxylate atoms differs
significantly from the plane formed by the C3(or N in
NOG)/C4/C5 carbon atoms (Figure S5B).25 In contrast,
these groups of atoms are coplanar in our
EFE•Mn•2OG•L-Arg structure (Figure 4A and S5A), as well
as
those
for
EFE•Mn•2OG•L-Arg-amide
and
EFE•Mn•2OG•HO-L-Arg (Figure 1, H and I). The crystal
structures of several other enzyme family members have
NOG bound in nearly coplanar orientations (e.g., PDB IDs
3O2G, 3HQR, 4NM6),33-34 so the identity of the ligand
does not account for these observed differences. One pos-
sible explanation is that the polar amide group in NOG is
not compatible with the unusual hydrophobic environ-
ment of the 2OG binding site in EFE, so that NOG has to
adopt a distorted and distinct conformation.
Zhang et al. additionally presented the EFE•Fe•NOG•L-
Arg structure (NOG is an inhibitor of the enzyme; PDB ID
5LUN, 1.08 Å)25 that matched many features of our
EFE•Mn•2OG•L-Arg (and several other) structures. At this
high resolution, the authors reported two conformations
(A:B in a 50:50 ratio, Figure S5) with two models of D191
coordination and their effects on the active site. Interest-
ingly, although the authors did not mention it in their
report, a similar twisted peptide bond between D191 and
Y192 is identified in their conformation A, with ω being
146.6°, while conformation B had an angle of 152.5° (aver-
age of four chains in the unit cell Table S5). In their con-
formation A, the side chain of D191 forms a hydrogen
bond with L-Arg via OD1 and chelates the metal via OD2
(Figure S5A). NOGA forms a salt bridge with R277 while
also binding bidentate to iron. This conformation has C5
of L-Arg pointed toward the metal and matches very well
with our EFE•Mn•2OG•L-Arg structure. In contrast, their
conformation B has D191 chelating iron similar to our
structures that lack L-Arg or its analogs (Figure S5B). Fur-
thermore, in conformation B the L-Arg shifts towards the
metal (but with the C5 carbon pointed away) and NOG,
still with bidentate coordination, now interacts weakly
with R277 using a single hydrogen bond. The authors
concluded that conformation B facilitates ethylene pro-
duction. Significantly, we have not seen this second con-
formation of L-Arg (or L-Arg analog) in any structure we
solved so far. For EFE•Mn•2OG•L-Arg-amide we modeled
two slightly different L-Arg-amide conformations (Figure
1I); however, neither matches the conformation of 5LUN
and we observed no effects on the rest of the active site.
Mechanism of Ethylene formation. A notable struc-
tural aspect of EFE centers on the apparent dioxygen
binding site in the EFE•Mn•2OG•L-Arg, EFE•Mn•2OA•L-
Arg, EFE•Mn•2OG•L-Arg-amide, and EFE•Mn•2OG•HO-L-
Arg crystal structures (Figures 4, A and B, and S6B). In
these complexes, C5 of L-Arg is located close to the metal
as needed for performing the typical steps of substrate
hydroxylation. On the other hand, the open sixth coordi-
nation site for dioxygen binding is on the opposite face of
the metal from this substrate, indicated by a water mole-
cule
in
the
EFE•Mn•2OG•L-Arg-amide
and
EFE•Mn•2OG•HO-L-Arg structures (Figures 4 and S6B).
Several Fe(II)/2OG oxygenases exhibit a similar off-line
configuration30 (Figure S6A), and the reaction mecha-
nisms for those enzymes have been suggested to involve a
ferryl flip in which the initially generated ferryl group
(with the oxygen atom pointed away from substrate) un-
dergoes a conformational change that allows the ferryl
oxygen to approach the substrate.31 The extent of shift
required for EFE to hydroxylate L-Arg (~130° based on the
positions of the waters in the L-Arg-amide and HO-L-Arg
structures) appears to exceed that of most other repre-
sentatives (Figure S6A). Furthermore, L-Arg binding
causes the F283 side chain to shift position to approach
the metal (Figure 4B); thus, F283 may partially hinder the
flipping of the ferryl group. In addition, the distinctly
altered position of D191 compared to other off-line en-
zymes (Figure S6C) may reduce the tendency of the ferryl
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group to flip by reducing the space available on the oppo- supported their hypothesis by using quantum calculations
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site side of ferryl group. The generation of a ferryl group
that is poorly positioned to react with substrate L-Arg
could reasonably facilitate the aberrant degradation of
2OG, thus leading to ethylene formation.
of carboxylate model compounds with good leaving
groups at the C3 position.25 Such fragmentation of succin-
ate would release the formate dianion, a very poor leaving
group; however, simultaneous electron transfer from the
formate dianion to the ferryl group would form carbon
dioxide and Fe(II)-OH. In contrast, we had proposed ear-
lier that a cyclic peroxide-Fe(IV) intermediate formed
during the EFE reaction would decarboxylate and frag-
ment to release ethylene while generating an oxalate-
bound ferryl species.20 The latter species could decom-
pose further by several mechanisms including (A) decar-
boxylation with formation of a formyl radical anion
bound to Fe(III)-OH and subsequent release of a second
CO2, (B) decarboxylation with formation of bicarbonate
bound to Fe(II), or (C) formation of bicarbonate while
generating a formyl radical anion bound to Fe(III) with
subsequent release of CO2 (Scheme 2). Notably, these
reactions are reminiscent of steps proposed in the de-
composition of oxalate by oxalate decarboxylase and oxa-
late oxidase.37-38 Because of the multiple pathways availa-
ble for oxalate metabolism and the precedent for this type
of chemistry, we continue to favor an ethylene-forming
mechanism that includes formation of transiently-bound
oxalate at the metallocenter.
The occurrence of the twisted peptide bond at the ac-
tive site of EFE appears to be unique, not only within re-
lated enzymes but also in general. Deviations greater than
20° from planarity are considered very rare features.
Whereas twisted peptide bonds are not generally associ-
ated with active sites and catalysis,35 the fact that the
D191-Y192 peptide bond is severely twisted only upon L-
Arg binding suggests it may play a significant role in the
reaction. We postulate that such a strong structural con-
straint imposed by L-Arg (or L-Arg analog) binding could
alter the energy state of the enzyme-substrate complex,
facilitating ethylene formation through an unknown
mechanism, as the other non-ethylene producing mem-
bers within the same enzyme family lack this feature.
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In addition to the extreme off-line orientation of the
dioxygen binding site for L-Arg hydroxylation and the
unusual twisted peptide bond at the active site, the highly
hydrophobic environment of 2OG could stimulate the
decarboxylative fragmentation of this substrate into eth-
ylene. Zhang et al. propose that an EFE intermediate with
succinate bound to a ferryl group decomposes to produce
ethylene by a Grob-type fragmentation.36 The authors
Scheme 2. Potential mechanisms for decomposing a putative oxalate intermediate of EFE
Structural Context for Previously Described EFE
variants. Our site-directed mutagenesis efforts con-
firmed the importance of many residues that bind sub-
strates and affect catalysis, with our product measure-
ments including not just ethylene, but also P5C. An
alignment of EFE sequences from eight strains known to
produce ethylene (Figure S3)17, 27 reveals extensive conser-
vation of these residues, with identity noted in many cas-
es. Zhang et al. also reported the loss or diminishment of
ethylene formation in several variants affecting substrate-
binding residues.25 Two mutational studies of P. syringae
pv. phaseolicola PK2 efe were reported prior to elucida-
tion of the structure. Nagahama et al. substituted gluta-
mine for each of the ten histidine residues of EFE and
studied the ethylene-producing activities of recombinant
E. coli cells producing these proteins.26 In addition, Jo-
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has been described for similar substitutions in other fami-
hansson et al. created recombinant yeast cells producing
an assortment of EFE variants and studied their ethylene
production activities.27 We depict the locations of these
prior substitutions in the EFE structure in Figure S7 and
summarize their ethylene-forming activities in Table S6.
Substitution of the universally-conserved metal-binding
ligands (H189, D191, and H268) by alanine eliminates ac-
tivity,27 as does the substitution of H189 by glutamine.26 In
contrast, the H168Q variant retains a trace of activity, as
ly members.39 Curiously, among the residues distant from
the active site, the H233A variant was fully active27 while
the H233Q protein was inactive,26 and substantial activity
losses were reported for the H116Q, H168Q, H169Q,
H284Q, and H309Q variants. The reasons for these great
diminishments in activity are unclear.
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Scheme 3. Two-pathway reaction mechanism of EFE with insights provided by the enzyme structuresa
aEFE•Fe(II) binds 2OG predominantly in two monodentate conformations, with a small amount of chelate coordination to the
metal. The nearby binding of L-Arg leads to a change in orientation of D191 (shown in cyan) as it forms a hydrogen bond with
this substrate and the 2OG switches to bidentate coordination. Dioxygen binds to the metallocenter opposite of L-Arg to form
the Fe(III)-superoxo species that transforms to a cyclic peroxide-Fe(IV) intermediate. The mechanism diverges at this intermedi-
ate, with the predominant pathway invoking a decarboxylative fragmentation to release ethylene with oxalic acid transiently
bound to the ferryl species. Subsequent reactivity of this intermediate yields CO2 and bicarbonate or two molecules of CO2
(Scheme 2) and the recycled EFE•Fe(II). The second pathway depicts oxidative decarboxylation of 2OG to yield succinate and a
ferryl species pointing away from the L-Arg substrate. A ferryl flip repositions the ferryl group to point toward C5 of L-Arg, with
F283 (and the repositioned D191) partly hindering this process. Hydrogen atom abstraction, hydroxyl radical rebound, elimina-
tion
of
guanidine
from
5-hydroxy
L-Arg,
and
ring
formation
complete
this
cycle.
CONCLUSIONS
zyme. Based on our earlier biochemical and spectroscopic
studies and new insights from the present structural and
mutagenesis investigations, we propose an updated ver-
Our structural and mutational studies of EFE provide
keen insights into the catalytic mechanisms of this en-
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Jian Hu 0000-0001-6657-9826
sion of the dual-pathway EFE reaction mechanism
(Scheme 3). In this revised model, the EFE-catalyzed me-
tabolism of L-Arg (blue pathway) appears to take place
via the prototypical hydroxylation mechanism; i.e., a fer-
ryl intermediate is generated by the oxidative decarboxy-
lation of 2OG, the ferryl flips its orientation in order to
abstract a hydrogen atom from the nearby C5 position,
and hydroxyl radical rebound leads to an unstable hy-
droxylated intermediate that decomposes to guanidine
and P5C. Three novel aspects of this mechanism are (1)
the predominant monodentate coordination of 2OG to
the metal in the absence of L-Arg, (2) a shift in the orien-
tation of the D191 metal-binding ligand upon binding of
L-Arg accompanied by formation of a twisted peptide
bond at the active site, and (3) the drastically off-line di-
rection of the ferryl group with F283 and D191 hindering
the flip needed for reaction with the L-Arg substrate. The-
se features along with the hydrophobic environment of
2OG may be important for catalysis of the atypical eth-
ylene-forming reaction carried out by this enzyme (red
pathway).
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Author Contributions
ǁS.M. and M.F. contributed equally to this work.
ACKNOWLEDGMENTS
This work was initiated with support by the National Insti-
tutes of Health (Grant GM063584 to R.P.H.). We thank
Nicholas Henning for assistance with some assays and crea-
tion of some variants, and Tuo Zhang for help with collection
of crystallographic data. We thank the beamline scientists at
LS-CAT at APS for assistance during data collections.
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REFERENCES
(1) Chenier, P. J. Survey of Industrial Chemistry, Springer US:
Boston, MA, 2002; pp 143-162.
(2) Global Ethylene Market By End Use, By Region,
Competition Forecast and Opportunities, 2011
Newswire 2017.
(3) Chenier, P. J. Survey of Industrial Chemistry, Springer US:
Boston, MA, 2002; pp 117-141.
(4) Ghanta, M.; Fahey, D.; Subramaniam, B. Appl Petrochem
Res 2014, 4, 167-179.
- 2025. PR
We previously showed that the two reactions can be
separated by the use of substrate analogues.20 For exam-
ple, 2OA allows for hydroxylation of L-Arg, but it cannot
generate ethylene. Similarly, HO-L-Arg can stimulate
2OG-dependent formation of ethylene, but it does not
undergo hydroxylation. Many variants involving residues
that interact with 2OG or L-Arg lead to substantial reduc-
tion or loss of activity, but thus far no substitution has led
to the exclusive production of ethylene. Of interest, how-
ever, several variants (most notably A198V) were highly
capable of L-Arg hydroxylation yet had severely repressed
ethylene-forming ability. The findings presented here
provide new insights into the properties of this unique
enzyme and offer the potential for creating a version of
EFE that exhibits high-level synthesis of ethylene in the
absence of the wasteful L-Arg hydroxylation reaction.
(5) Lundgren, A.; Hjertberg, T. Surfactants from Renewable
Resources, John Wiley & Sons, Ltd: Chichester, U.K., 2010; pp
109-126.
(6) Dijkmans, T.; Pyl, S. P.; Reyniers, M.-F.; Abhari, R.; Van
Geem, K. M.; Marin, G. B. Green Chem. 2013, 15, 3064-3076.
(7) Johansson, N.; Persson, K. O.; Quehl, P.; Norbeck, J.;
Larsson, C. FEMS Yeast Res. 2014, 14, 1110-1118.
(8) Johansson, N.; Quehl, P.; Norbeck, J.; Larsson, C. Microb.
Cell Fact. 2013, 12, 1-7.
(9 ) Yan, D.; Lenz, P.; Hwa, T. Appl. Environ. Microbiol. 2011,
77, 6763-6771.
(10) Lynch, S.; Eckert, C.; Yu, J.; Gill, R.; Maness, P.-C.
Biotechnol Biofuels 2016, 9, 1-10.
(11) Zhu, T.; Xie, X.; Li, Z.; Tan, X.; Lu, X. Green Chem. 2015, 17,
421-434.
(12) Markham, J. N.; Tao, L.; Davis, R.; Voulis, N.; Angenent, L.
T.; Ungerer, J.; Yu, J. Green Chem. 2016, 18, 6266-6281.
(13) Guerrero, F.; Carbonell, V.; Cossu, M.; Correddu, D.;
Jones, P. R. PLoS ONE 2012, 7, e50470.
(14) Xiong, W.; Morgan, J. A.; Ungerer, J.; Wang, B.; Maness,
P.-C.; Yu, J. Nature Plants 2015, 1, 15053.
(15) Zavřel, T.; Knoop, H.; Steuer, R.; Jones, P. R.; Červený, J.;
Trtílek, M. Bioresource Technol. 2016, 202, 142-151.
(16) Veetil, V. P.; Angermayr, S. A.; Hellingwerf, K. J. Microb.
Cell Fact. 2017, 16, 34.
(17) Weingart, H.; Völksch, B.; Ullrich, M. S. Phytopath. 1999,
89, 360-365.
(18) Fukuda, H.; Kitajima, H.; Fujii, T.; Tazaki, M.; Ogawa, T.
FEMS Microbiol. Lett. 1989, 59, 1-5.
(19) Fukuda, H.; Ogawa, T.; Tazaki, M.; Nagahama, K.; Fujiil,
T.; Tanase, S.; Morino, Y. Biochem. Biophys. Res. Commun. 1992,
188, 483-489.
(20) Martinez, S.; Hausinger, R. P. Biochemistry 2016, 55,
5989-5999.
(21) Martinez, S.; Hausinger, R. P. J. Biol. Chem. 2015, 290,
20702-20711.
(22) Hausinger, R. P. 2-Oxoglutarate-Dependent Oxygenases,
Schofield, C. J.; Hausinger, R. P., Eds. The Royal Society of
Chemistry: Cambridge, UK, 2015; pp 1-58.
(23) Kal, S.; Que, L. J. Biol. Inorg. Chem. 2017, 22, 339-365.
(24) Wu, L.-F.; Meng, S.; Tang, G.-L. Biochim. Biophys. Acta
2016, 1864, 453-470.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge via the
Internet at http://pubs.acs.org.
Experimental procedures, eight supplementary fig-
ures, and eight supplementary tables (PDF) along with X-ray
crystallographic data for eleven EFE structures (CIF). The
reported coordinates and structure factors have been depos-
ited in the PDB as apoprotein (PDB ID 5V2U); EFE•Ni, PDB
ID 5V2V); Mn•2OG (PDB ID 5V2X); Mn•2OG•L-Arg (PDB ID
5V2Y); Mn•2OA•L-Arg (PDB ID 5V2Z); Mn•2OG•HO-L-Arg
(PDB ID 5VKA); Mn•2OG•L-Arg-amide (PDB ID 5VKB);
Mn•malate•Arg (PDB ID 5V34); Mn•Arg (PDB ID 5V31);
Mn•tartrate (PDB ID 5V2T) and Mn•malate (PDB ID 5V32)
AUTHOR INFORMATION
Corresponding Authors
* hausinge@msu.edu
* hujian1@msu.edu
ORCID
Robert P. Hausinger 0000-0002-3643-2054
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(25) Zhang, Z.; Smart, T. J.; Choi, H.; Hardy, F.; Lohans, C. T.;
(32) Chen, V. B.; Arendall, B., III; Headd, J. J.; Keedy, D. A.;
1
2
3
4
5
6
7
8
Abboud, M. I.; Richardson, M. S. W.; Paton, R. S.; McDonough,
M. A.; Schofield, C. J. Proc. Natl. Acad. Sci. U.S.A. 2017, 114, 4667-
4672.
(26) Nagahama, K.; Yoshino, K.; Matsuoka, M.; Tanase, S.;
Ogawa, T.; Fukuda, H. J. Ferment. Bioeng. 1998, 85, 255-258.
(27) Johansson, N.; Persson, K. O.; Larsson, C.; Norbeck, J.
BMC Biochem. 2014, 15, 1-8.
(28) Aik, W. S.; Chowdhury, R.; Clifton, I. J.; Hopkinson, R. J.;
Leissing, T.; McDonough, M. A.; Nowak, R.; Schofield, C. J.;
Walport, L. J. 2-Oxoglutarate-Dependent Oxygenases, Schofield,
C. J.; Hausinger, R. P., Eds. The Royal Society of Chemistry:
Cambridge, U.K., 2015; pp 59-94.
Immormino, R. M.; Kapral, G. J.; Murray, L. W.; Richardson, J. S.;
Richardson, D. C. Acta Crystallogr. 2010, D66, 12-21.
(33) Chowdhury, R.; McDonough, M. A.; Mecinovíc, J.;
Loenarz, C.; Flashman, E.; Hewitson, K. S.; Domene, C.;
Schofield, C. J. Structure 2009, 17, 981-989.
(34) Hu, L.; Li, Z.; Cheng, J.; Rao, Q.; Gong, W.; Liu, M.; Shi, Y.
G.; Zhu, J.; Wang, P.; Xu, Y. Cell 2013, 155, 1545-1555.
(35) Berkholz, D. S.; Driggers, C. M.; Shapovatov, M. V.;
Dunbrack, R. L., Jr.; Karplus, P. A. Proc. Natl. Acad. Sci. U.S.A.
2012, 109, 449-453.
9
(36) Grob, C. A.; Baumann, W. Helv. Chim. Acta 1955, 38, 594-
610.
10
11
12
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14
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21
22
23
24
25
26
27
28
29
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34
35
36
37
38
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(29) Clifton, I. J.; McDonough, M. A.; Ehrismann, D.; Kershaw,
N. J.; Granatino, N.; Schofield, C. J. J. Inorg. Biochem. 2006, 100,
644-669.
(30) Hausinger, R. P. Crit. Rev. Biochem. Molec. Biol. 2004, 39,
21-68.
(37) Molt, R. W., Jr.; Lecher, A. M.; Clark, T.; Bartlett, R. J.;
Richards, N. G. J. J. Am. Chem. Soc. 2015, 137, 3248-3252.
(38) Zhu, W.; Easthon, L. M.; Reinhardt, L. A.; Tu, C.; Cohen,
S. E.; Silverman, D. N.; Allen, K. N.; Richards, N. G. Biochemistry
2016, 55, 163-173.
(31) Zhang, Z.; Ren, J.-s.; Harlos, K.; McKinnon, C. H.; Clifton,
I. J.; Schofield, C. J. FEBS Lett. 2002, 517, 7-12.
(39) Grzyska, P. K.; Müller, T. A.; Campbell, M. G.; Hausinger,
R. P. J. Inorg. Biochem. 2007, 101, 797-808.
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ACS Paragon Plus Environment