Modification of auxinic phenoxyalkanoic acid herbicides by the acyl acid amido synthetase GH3.15 from Arabidopsis

Article abstract of DOI:10.1074/jbc.RA118.004975

Herbicide-resistance traits are the most widely used agriculture biotechnology products. Yet, to maintain their effectiveness and to mitigate selection of herbicide-resistant weeds, the discovery of new resistance traits that use different chemical modes of action is essential. In plants, the Gretchen Hagen 3 (GH3) acyl acid amido synthetases catalyze the conjugation of amino acids to jasmonate and auxin phytohormones. This reaction chemistry has not been explored as a possible approach for herbicide modification and inactivation. Here, we examined a set of Arabidopsis GH3 proteins that use the auxins indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) as substrates along with the corresponding auxinic phenoxyalkanoic acid herbicides 2,4-dichlorophenoxylacetic acid (2,4-D) and 4-(2,4-dichlorophenoxy)butyric acid (2,4-DB). The IBA-specific AtGH3.15 protein displayed high catalytic activity with 2,4-DB, which was comparable to its activity with IBA. Screening of phenoxyalkanoic and phenylalkyl acids indicated that side-chain length of alkanoic and alkyl acids is a key feature of AtGH3.15’s substrate preference. The X-ray crystal structure of the AtGH3.15?2,4-DB complex revealed how the herbicide binds in the active site. In root elongation assays, Arabidopsis AtGH3.15-knockout and -overexpression lines grown in the presence of 2,4-DB exhibited hypersensitivity and tolerance, respectively, indicating that the AtGH3.15-cata-lyzed modification inactivates 2,4-DB. These findings suggest a potential use for AtGH3.15, and perhaps other GH3 proteins, as herbicide-modifying enzymes that employ a mode of action different from those of currently available herbicide-resistance traits.

Full text of DOI:10.1074/jbc.RA118.004975

JBC Papers in Press. Published on October 12, 2018 as Manuscript RA118.004975
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.RA118.004975
Modification of auxinic phenoxyalkanoic acid herbicides by the acyl acid
amido synthetase GH3.15 from Arabidopsis
*
Ashley M. Sherp, Soon Goo Lee, Evelyn Schraft, Joseph M. Jez
Department of Biology, Washington University, St. Louis, MO 63130, USA
*
Corresponding author; E-mail: jjez@wustl.edu
Running title: Herbicide modification by an acyl acid amido synthetase
Keywords: Arabidopsis thaliana, auxin, enzyme kinetics, herbicides, x-ray crystallography,
Gretchen Hagen 3, herbicide tolerance, acyl acid amido synthetase, 2,4-D, structure-activity
relationship
Herbicide-resistance traits are the
most widely used agriculture
hypersensitivity and tolerance,
respectively, indicating that the
biotechnology products. Yet, to maintain
their effectiveness and to mitigate
selection of herbicide-resistant weeds, the
discovery of new resistance traits that use
different chemical modes of action is
essential. In plants, the Gretchen Hagen
AtGH3.15-catalyzed modification
inactivates 2,4-DB. These findings
suggest a potential use for AtGH3.15, and
perhaps other GH3 proteins, as herbicide-
modifying enzymes that employ a mode of
action different from those of currently
available herbicide resistance traits.
3
(GH3) acyl acid amido synthetases
catalyze the conjugation of amino acids to
jasmonate and auxin phytohormones.
This reaction chemistry has not been
explored as a possible approach for
herbicide modification and inactivation.
Here, we examined a set of Arabidopsis
GH3 proteins that use the auxins indole-
Herbicide-resistance traits accounted for
47% of genetically-engineered soybean,
maize, canola, cotton, sugar beet, and alfalfa
plantings worldwide in 2017 (1). Since the
introduction of the first crops with a
glyphosate-resistant trait, the use of
3
-acetic acid (IAA) and indole-3-butyric
herbicides with distinct modes of action and
the discovery of new resistance traits have
become critical elements for increased
agricultural productivity and for effective
management of weed resistance (2-3). For
example, auxinic herbicides, based on
phenoxyalkanoic acid, benzoic acid,
pyridine carboxylic acid, and quinoline
carboxylic acid chemical scaffolds mimic
the plant hormone auxin (indole-3-acetic
acid; IAA) and are used extensively in
agronomic and non-crop applications for
broadleaf weed control (Fig. 1) (4-5). These
molecules elicit the same type of growth and
developmental responses as IAA, but due to
higher stability in the plant result in longer-
lasting and stronger effects such as plant
overgrowth (4-5).
acid (IBA) as substrates along with the
corresponding auxinic phenoxyalkanoic
acid herbicides 2,4-
dichlorophenoxylacetic acid (2,4-D) and
4
-(2,4-dichlorophenoxy)butyric acid (2,4-
DB). The IBA-specific AtGH3.15 protein
displayed high catalytic activity with 2,4-
DB, which was comparable to its activity
with IBA. Screening of phenoxyalkanoic
and phenylalkyl acids indicated that side-
chain length of alkanoic and alkyl acids is
a key feature of AtGH3.15's substrate
preference. The X-ray crystal structure
of the AtGH3.15•2,4-DB complex
revealed how the herbicide binds in the
active site. In root elongation assays,
Arabidopsis AtGH3.15-knockout and -
overexpression lines grown in the
Of the auxinic herbicides, 2,4-
presence of 2,4-DB exhibited
dichlorophenoxyacetic acid (2,4-D; Fig. 1)

was the first to be commercialized in 1945
and is the most widely used
the endogenous auxin indole-3-butyric acid
(IBA), to regulate plant growth, seed
development, light signaling, and pathogen
responses (25-33). GH3 proteins catalyze
the adenylation of the carboxylate on these
molecules to form an acyl-AMP
phenoxyalkanoic acid herbicide with ~46
million pounds applied in the US per year,
predominantly in the Midwest, Great Plains,
and the Northwestern United States (6-7).
Related to 2,4-D, 4-(2,4-
intermediate, which undergoes nucleophilic
attack by an amino acid to yield the
dichlorophenoxy)butyric acid (2,4-DB; Fig.
1
) has also been used to control annual and
conjugated product (28, 30). Biochemical
and structural studies of GH3 proteins from
Arabidopsis thaliana have identified
perennial broadleaf weeds since 1958 (8-9).
After foliar application, 2,4-DB is taken up
by the leaves and roots and converted
through peroxisomal β-oxidation to the
active herbicide 2,4-D (8-10). At the
molecular level, 2,4-D binds to the auxin
receptor F-box protein TIR1, which
facilitates interaction between the receptor
and co-repressor Aux/IAA proteins (11-12).
This leads to ubiquitination and degradation
of the Aux/IAA proteins to modulate
downstream interactions with auxin
response factors that control transcription of
auxin responsive genes (13-14). Although
both IAA and 2,4-D target the auxin
receptor, 2,4-D is metabolized more slowly
than IAA, which enhances herbicidal effects
through elevated expression of auxin
responsive genes leading to plant death (6,
jasmonate-, IAA-, and IBA-specific
members of the family (25-27, 29, 32-33).
Given the chemical similarity between
phenoxyalkanoic acids (i.e., 2,4-D and 2,4-
DB) and auxins (IAA and IBA) (Fig. 1), we
examined the potential of selected
Arabidopsis GH3 proteins to modify either
2,4-D or 2,4-DB. The IBA-specific
Arabidopsis GH3.15 protein (AtGH3.15)
displayed high catalytic activity with 2,4-
DB, which was comparable to that of IBA,
and the X-ray crystal structure of the
enzyme in complex with the herbicide
shows how the molecule binds in the active
site. When grown on 2,4-DB, A. thaliana T-
DNA insertions in AtGH3.15 and
35S:FLAG-AtGH3.15 overexpression lines
show hypersensitivity and tolerance,
1
5-16). For agricultural biotechnology
applications, herbicide tolerance traits have
relied on the identification of enzymes that
either chemically inactivate the herbicide or
prevent inhibition of a target by the
herbicide (17-24). For example, isolation of
a microbial aryloxyalkanoate dixoygnease
that cleaves 2,4-D provides tolerance to this
auxinic herbicide and is the basis for 2,4-D
resistant crops currently entering the market
respectively, in root elongation assays.
These findings suggest a potential use for
AtGH3.15, and perhaps other GH3 proteins,
as herbicide-modifying enzymes that
employ a mode of action that differs from
available auxinic herbicide resistance traits.
RESULTS
Screen of Arabidopsis GH3 proteins
with 2,4-D and 2,4-DB and comparison to
auxin substrates. The auxinic herbicides
2,4-D and 2,4-DB minic the biological
activity of the endogenous auxins IAA and
IBA, respectively. Previous biochemical
studies identified IAA- and IBA-specific
GH3 acyl acid amido synthetases from
Arabidopsis (26, 32-33). Using purified
(
23). Access to tolerance traits with distinct
modes of action is critical for reducing the
emergence of herbicide resistant weeds (2-
6
).
In plants, the Gretchen Hagen 3 (GH3)
acyl acid amido synthetases conjugate
amino acids to carboxylic acid-containing
hormones, such as jasmonic acid, IAA, and
2

-
recombinant protein, we examined the
ability of the IAA-modifying AtGH3.1,
AtGH3.2, AtGH3.5, and AtGH3.17 (32) and
the IBA-modifying AtGH3.15 (33) to use
either 2,4-D or 2,4-DB as substrates (Table
(deprotonated molecular ion (M-H) m/z =
376.2; Fig. S1). Assays in the absence of
protein or any one substrate did not yield a
peak corresponding to the conjugated
product. To confirm that the amino acid
preference of AtGH3.15 was the same with
2,4-DB as with IBA, the substrate profile
was examined using 2,4-DB and each amino
acid (Fig. S2). The amino acid profile was
the same for AtGH3.15 with 2,4-DB as with
IBA (33) with cysteine, histidine,
1
; Fig. 2). None of the IAA-specific
Arabidopsis GH3 proteins tested used 2,4-D
as a substrate; however, AtGH3.15 exhibited
a low activity with this herbicide. Except
for AtGH3.1, the GH3 proteins accepted
2
,4-DB as a substrate to varying degrees.
The most efficient enzyme was the IBA-
specific AtGH3.15 with a catalytic
efficiency (kcat/K ) 3- to 5-fold higher than
m
AtGH3.2 and AtGH3.5, respectively.
AtGH3.17 had the lowest activity, roughly
methionine, glutamine, and tyrosine having
the highest activity. Steady-state kinetics
with cysteine, histidine, methionine,
glutamine, and tyrosine were determined
and confirm that, as with IBA, glutamine is
the preferred amino acid for AtGH3.15 with
2,4-DB (Table 2).
To determine if AtGH3.15 was active
with other auxinic herbicides (Fig. S3), the
benzoic acid dicamba (3,6-dichloro-2-
methoxybenzoic acid), the pyridine
5
0-fold that of AtGH3.15, with 2,4-DB as a
substrate. For comparison, previously
reported data on these GH3 proteins (32-33)
with the auxin substrates IAA and IBA are
summarized (Table 1; Fig. 2). Although
2
,4-D mimics the biological effect of IAA, it
is not used as a substrate for the four IAA-
modifying GH3 proteins examined here and
is a poor substrate for the IBA-specific
AtGH3.15. In contrast to the 2,4-D/IAA
pairing, the catalytic efficiencies of
carboxylic acids clopyralid (3,6-
dichloropyridine-2-carboxylic acid),
picloram (4-amino-3,5,6-trichloro-2-
pyridine carboxylic acid), and triclopyr
([[3,5,6-trichloro-2-pyridinyl]oxy]acetic
acid), and the phenoxyalkanoic acids
dichlorprop (2-(2,4-
dichlorophenoxy)propanoic acid), mecoprop
(2-(4-chloro-2-methylphenoxy)propionic
acid), 4-chloro-2-methylphenoxy acetic acid
(MCPA), 2,4,5-trichlorophenoxyacetic acid
(2,4,5-T), and 4-(4-chloro-2-
methylphenoxy)butanoic acid (MCPB) were
tested as substrates. AtGH3.15 displayed no
detectable activity with dicamba, the
pyridine carboxylic acids, dichlorprop,
mecoprop, and MCPA. The catalytic
efficiency of AtGH3.15 with 2,4,5-T was
comparable to that with 2,4-D (Table 3).
Similarly, AtGH3.15 accepted MCPB as a
AtGH3.2, AtGH3.5, AtGH3.15, and
AtGH3.17 for the 2,4-DB/IBA pair were
generally comparable with AtGH3.15 as the
most robust enzyme for these molecules.
Biochemical analysis of AtGH3.15. As
the most active GH3 protein tested with 2,4-
DB, AtGH3.15 was further characterized for
its amino acid substrate profile and with
other phenoxyalkanoic and phenylalkyl
acids. As noted above, biochemical analysis
of AtGH3.15 yielded steady-state kinetic
parameters for 2,4-DB that were comparable
to those obtained for IBA with glutamine
(
Table 1). QTRAP mass spectrometry
analysis confirmed formation of the 2,4-DB-
glutamine conjugate in vitro. Incubation of
AtGH3.15 with 2,4-DB, ATP, and
m
substrate with a kcat/K 1.8-fold lower than
either 2,4-DB or IBA (Table 3).
Chemically, MCPB is identical to 2,4-DB
except for a methyl group substituted for a
glutamine lead to formation of the conjugate
3

chlorine at C2 (Fig. S3). There results are
consistent with a preference for longer-chain
phenoxyalkanoic acids.
To further probe the structure-activity
relationship of AtGH3.15 assays were
performed with phenoxybutanoic and
phenylalkyl acids (Fig. S3; Table 3).
Kinetic analysis with 4-(2-
chlorophenoxy)butanoic acid and 4-(2,6-
dimethylphenoxy)butanoic acid suggests
that the removal of the chlorine from the C4
position does not reduce catalytic efficiency;
however, extension of this position reduces
catalytic activity, as observed with 4-(4-
methoxyphenoxy)butanoic acid.
points the reactive carboxylate group toward
the phosphate group that undergoes the
adenylation reaction (Fig. 3C). This 2,4-DB
molecule stacks with Phe166, forms a
hydrogen bond contact with Ser122 (which
was modeled in two alternate side-chain
conformations), and is situated in a space
bordered by Met162, Val163, Phe325, and
Phe332 (Fig. 3D). The second 2,4-DB
molecule positions its carboxylate group
away from the nucleotide binding site and is
situated deeper in the acyl acid binding site
(Fig. 3C). This ligand forms a charge-
charge interaction between its carboxylate
and the side-chain of Arg214 (Fig. 3D).
Ser299 contributes a hydrogen bond
Comparison of the catalytic efficiencies of
4
-phenoxybutyric acid, 4-phenylbutryic
interaction to the carboxylate. The
acid, 5-phenylvaleric acid, and 5-(4-
fluorophenyl)valeric acid also indicate that
compounds longer in length from
carboxylate to the substituted phenyl group
are superior substrates.
substituted phenyl ring is positioned to form
van der Waals contacts with Ile143, Leu181,
and Phe219. It is not clear if the binding of
two 2,4-DB molecules in the acyl acid site
of AtGH3.15 is biochemically relevant or is
an artifact of crystallization. Because of the
large size of the site, it is possible that
binding of one ligand deeper in the pocket
positions the second for efficient catalysis.
Three-dimensional structure of
AtGH3.15 in complex with 2,4-DB. To
provide insight on how 2,4-DB interacts
with AtGH3.15, the protein was crystallized
in the presence of the ligand. The 2.15 Å
resolution structure of the AtGH3.15•2,4-
DB complex was solved by molecular
replacement (Table 4; Fig. 3A). The
overall fold of the resulting structure was
Effect of AtGH3.15 knockout and
overexpression in Arabidopsis on 2,4-DB
and 2,4-D treatment. 2,4-DB, like IBA,
inhibits primary root elongation (34-36). To
determine if the in vitro activity of
2
similar (1.4 Å root mean square deviation
AtGH3.15 with 2,4-DB had in planta
effects, previously generated and
for 550 C -atoms) to that of the previously
α
reported AtGH3.15•AMP complex (33) with
the conformationally mobile C-terminal
domain adopting the 'open' active site
conformation. Examination of the electron
density maps in the active site revealed two
large patterns of density in the acyl acid
binding site, which were subsequently
modeled and refined as two molecules of
characterized knockout and overexpression
lines of AtGH3.15 (33) were used in
primary root elongation assays with 2,4-DB.
Seedlings of wild-type, two T-DNA
insertion lines (SALK_108265 and
SALK_079153), and three overexpression
lines (35S:FLAG-GH3.15 1-5, 35S:FLAG-
GH3.15 2-7, and 35S:FLAG-GH3.15 8-2)
were grown on 1 µM 2,4-DB for 10 days to
determine if altered expression of AtGH3.15
altered plant responses to 2,4-DB treatment
(Fig. 4). Mock-treated seedlings were
2
,4-DB (Fig. 3B). Comparison with the
position of AMP in the AtGH3.15•AMP
complex indicates that only one 2,4-DB
molecule is positioned in an orientation that
4

comparable between the various lines (Fig.
A, upper panels). Seedlings treated with 1
µM 2,4-DB (Fig. 4A, lower panels) showed
statistically significant differences between
wild-type Col-0, SALK_108265,
AtGH3.2, AtGH3.5, and AtGH3.17) did not
have activity with 2,4-D (Table 1; Fig 2).
This suggests that although 2,4-D is a potent
IAA analog targeting the TIR1 auxin
receptor, it does not serve as an IAA mimic
for the GH3 proteins. This difference may
contribute to potentiation of the herbicidal
effect of 2,4-D.
4
SALK_079153, 35S:FLAG-GH3.15 1-5,
3
5S:FLAG-GH3.15 2-7, and 35S:FLAG-
GH3.15 8-2 (Fig. 4B). The T-DNA
knockout lines (SALK_108265 and
In contrast, AtGH3.15 is highly active
with both IBA and its auxinic herbicide
counterpart 2,4-DB (Table 1; Fig 2).
Additional biochemical analysis with a
range of substrates (Fig. S3) shows that
AtGH3.15 does not accept benzoic acid
(dicamba), pyridine carboxylic acid
(clopyralid, picloram, and triclopyr), and
short side-chain phenoxyalkonaoic acid
(dichlorprop, mecoprop, MCPA) auxin
herbicides as substrates. AtGH3.15 did use
of 2,4-D and 2,4,5-T, which differs from
2,4-D by one additional chlorine, but with
catalytic efficiencies 50- to 100-fold lower
than that observed with either IBA or 2,4-
DB (Tables 1 & 3; Fig 2). The kinetic
analysis with MCPB, an analog of 2,4-DB,
and other longer side-chain
phenoxyalkonaoic acid and phenylalkyl acid
substrates indicates that substitutions of the
phenyl group are not as important as side-
chain length for activity; however, changes
to phenyl group substituents that lengthen
the moieties, such as the methoxy group of
4-(4-methoxyphenoxy)butanoic acid, reduce
catalytic efficiency (Table 3).
The x-ray crystal structure of AtGH3.15
in complex with 2,4-DB (Fig. 3) provides
insight on how this molecule is recognized
largely through apolar surface contacts,
although some hydrogen bond interactions
contribute. The orientation of one 2,4-DB
molecule in the active site with its reactive
carboxylate toward the location of the
ATP/AMP binding site suggests how a
productive first-half reaction leading to the
adenylated reaction intermediate occurs. As
noted in the results, the binding of two 2,4-
SALK_079153) showed hypersensitivity to
treatment with 2,4-DB compared to wild-
type Col-0 (Fig. 4). The overexpression
lines (35S:FLAG-GH3.15 1-5, 35S:FLAG-
GH3.15 2-7, and 35S:FLAG-GH3.15 8-2)
were resistant to treatment with 2,4-DB
compared to wild-type Col-0, as they
maintained active root elongation in the
presence of the herbicide (Fig. 4). As
AtGH3.15 uses IBA and 2,4-DB, but does
not prefer either IAA or 2,4-D as a substrate
in vitro, wild-type Col-0, knockout, and
overexpression lines were also screened on
2
,4-D in the root elongation assay. There
were no statistically significant differences
between wild-type, T-DNA insertion, and
overexpression lines grown on 20 nM, 40
nM, or 80 nM 2,4-D for 10 days (Fig. S4),
which is consistent with AtGH3.15 either
not having a role in 2,4-D metabolism or
downstream herbicide effects.
DISCUSSION
Multiple studies highlight the diverse
roles for GH3 acyl acid amido synthetases
as modulators of jasmonate and auxin
phytohormones (25-33), but the potential
function of these proteins as modifiers of
herbicides in plants has not been fully
examined. Motivated by the possibility that
the phenoxyalkanoic acid auxinic herbicides
2
,4-D and 2,4-DB mimic IAA and IBA (Fig.
1
), respectively, in triggering auxin-linked
responses, we examined if GH3 proteins that
conjugate amino acids to these hormones
can modify 2,4-D and 2,4-DB. Surprisingly,
the IAA-modifying GH3 proteins (AtGH3.1,
5

DB molecules in the acyl acid site of
AtGH3.15 maybe biochemically relevant or
an artifact of crystallization, but is not
unprecedented. For example, a set of
stacked alrestatin molecules in aldose
reductase was proposed to contribute to
ligand specificity (34). It is possible that the
large size of the AtGH3.15 acyl acid binding
site and binding of two substrates in
different orientations contributes to efficient
catalysis. This detail requires additional
detailed biochemical analyses. Overall, the
AtGH3.15•2,4-DB complex is the first of a
GH3 protein with a herbicide bound and
shows how binding in the site is largely
dictated by surface contacts.
correspond to GH3 protein expression
changes (25-26, 30, 32-33). These
differences highlight the need for further
investigations into the metabolism of these
molecules, which may alter local
concentrations within different tissues and
cell types of the plant and the fluxes that
control plant growth and development.
Overall, the biochemical and in planta
analysis of AtGH3.15 suggests a possible
model for how altered expression affects
plant growth (Fig. 5). Loss of AtGH3.15 in
the T-DNA insertion lines would remove
background conjugation to 2,4-DB, allowing
more of the herbicide to be metabolized in
the peroxisome (35-37). This results in the
shortened root phenotype compared to wild-
type Arabidopsis seedlings. In contrast,
overexpression of AtGH3.15 would increase
2,4-DB conjugate formation, which results
in the observed tolerance to herbicide
treatment and the longer root phenotype.
This parallels the effect of treating
The in planta effect of AtGH3.15
knockout or overexpression indicates that
changes in expression alter sensitivity to
2
,4-DB (Fig. 4). Previous work
characterized these plant lines (33). In the
root elongation assays with Arabidopsis
seedlings (Fig. 4), knockout lines of
AtGH3.15 showed hypersensitivity to
treatment with 2,4-DB, whereas,
Arabidopsis AtGH3.15 knockout and
overexpression lines with the auxin IBA
(33). Overall, the biochemical, structure,
and in planta experiments suggest the use of
AtGH3.15 as a possible resistance trait for
2,4-DB.
overexpression lines of AtGH3.15 displayed
clear tolerance to 2,4-DB. As with other
plants, metabolism of 2,4-DB to 2,4-D by β-
oxidation in the peroxisome, a process
similar to conversion of IBA to IAA, leads
to auxinic herbicide effects in Arabidopsis
While monocots and leguminous plants
are inherently tolerant to 2,4-DB
(
35-37). Interestingly, this experiment with
application, they are not completely resistant
and dicots remain susceptible (4-9).
AtGH3.15 and 2,4-DB, along with other
reported studies of various GH3 proteins and
their responses to different phytohormones
such as IAA, IBA, and jasmonates (25-26,
Overexpression of AtGH3.15 in planta
could potentially enhance the tolerance of
plants to 2,4-DB application. There are
several possible advantages to exploring
AtGH3.15 as a possible 2,4-DB resistance
trait. In comparison to overexpression of
IAA-specific GH3 proteins, which results in
severe growth phenotypes, such as dwarfing
3
0, 32-33), highlight differences between in
vitro steady-state kinetics and in planta
responses. The K values reported for
m
various GH3 proteins with their cognate
plant hormone substrates are typically in the
(29), overexpression of AtGH3.15 in
3
00-800 µM range; however,
Arabidopsis yielded no detrimental growth
changes (33). Moreover, the distinct amino
acid substrate profile of AtGH3.15 versus
the IAA-specific proteins (Table 2; Fig S2),
overexpression and knockout plant lines of
the different GH3-encoding genes exhibit
growth phenotypes with phytohormone
treatments in the range of 1-10 µM that
6

which primarily use aspartate and glutamate,
may help maintain inactive forms of 2,4-DB
and contribute to tolerance (25-33). Amino
acid conjugated forms of IAA have varied
roles with the IAA-aspartate and IAA-
glutamate conjugates leading to hormone
degradation and IAA-alanine and IAA-
leucine conjugates providing storage forms
of the auxin (31, 38-39). The best-studied
IAA and 2,4-D conjugates are those of
aspartate and glutamate, which suggests that
these molecules can be hydrolyzed back into
free acid forms (39). With 2,4-D conjugates
this contributes to maintaining the effect of
the herbicide (39). Currently, there is a lack
of information on the metabolic fates of IBA
and auxinic herbicides conjugated to other
amino acids. Potential glutamine, cysteine,
histidine, methionine, and tyrosine
To date, the molecular basis for enzyme-
based herbicide protection strategies rely on
a limited number of mechanisms - the use of
modified enolpyruvylshikimate-3-phosphate
synthase to prevent inhibition by glyphosate
and glufosinate (21); acetylation of
herbicide (18-19); degradation of 2,4-D by
aryloxyalkanoate dixoygneases (23);
conversion of dicamba by mono-oxygenases
(22); modification of acetolactate synthase
to prevent inhibition by sulfonylurea
herbicides; degradation of oxynil herbicide
by a nitrilase (24); and use of p-
hydroxyphenylpyruvate dioxygenases for
mesotrione and isoxaflutole tolerance (20).
Amino acid conjugation of herbicides may
provide an additional resistance mechanism.
The benefits of herbicide-tolerant crops
and the selection of resistant weeds,
conjugates of IBA and 2,4-DB formed by
AtGH3.15 need to be more fully explored
with regard to biological fate and herbicide
action.
In addition to the activity of AtGH3.15
with 2,4-DB, the structure of this enzyme in
complex with the herbicide serves as a
starting point to engineer variants that
modify 2,4-D with amino acids that are
neither aspartate nor glutamate, as a means
of exploiting potential differences in
herbicide metabolism. As 2,4-D is a widely
used herbicide, the ability to engineer
activity of AtGH3.15 with 2,4-D would
result in a greater agricultural impact than
with 2,4-DB. Before the discovery and
commercialization of auxin herbicides, like
highlights the importance of discovery and
development of new modes-of-action for
herbicide tolerance. Stacked traits, the
ability to tolerate different herbicidal
applications, are also important for the
future of herbicide tolerance in genetically
modified crops to help combat the selection
of herbicide resistant weeds in the future
(41). The development of 2,4-DB tolerant
crops via expression of AtGH3.15, or the
use of an engineered variant that efficiently
conjugates 2,4-D, would help to broaden the
tool kit of herbicidal tolerance modes of
action.
EXPERIMENTAL PROCEDURES
Protein preparation, enzymatic
2
,4-D, perennial weeds were particularly
analyses, and site-directed mutagenesis.
Recombinant AtGH3.1, AtGH3.2, AtGH3.5,
AtGH3.15, and AtGH3.17 were expressed in
E. coli and purified by nickel-affinity and
size-exclusion chromatographies (32-33).
Enzymatic activity of various GH3 proteins
was monitored using a coupled-enzyme
assay system (28, 32-33). Standard
difficult to control (40); however, to reduce
the development of weeds with herbicide
resistance multiple different modes of action
for tolerance traits are needed. For example,
extensive reliance on glyphosate in early
agricultural biotech crops led to selection of
weed populations with inherent tolerance to
the herbicide and spurred the development
of new herbicide resistance traits.
conditions were 50 mM Tris (pH 8.0), 3 mM
MgCl , 1 mM ATP, 1 mM
2
7

phosphoenolpyruvate, 0.2 mM NADH, 2
units myokinase, 4 units pyruvate kinase, 4
units lactate dehydrogenase and 10 µg
protein in a 200 µL reaction volume.
Concentrations used for various assays are
noted in table legends. Reactions were
performed in a 96-well plate format with
initial velocity data acquired on a Tecan
Infinite 200 with data fit to the Michaelis-
Menten equation using SigmaPlot.
refinement were performed with COOT (44)
and PHENIX, respectively. Data collection
and refinement statistics are summarized in
Table 4. Coordinates and structure factors
were deposited in the PDB (PDB: 6E1Q).
Arabidopsis knockout and
overexpression lines and root elongation
assays. Confirmation and characterization
of the two homozygous T-DNA insertion
lines (SALK_108265C and SALK_071953)
in the At5g13370 gene that codes for
Mass spectrometry. Reactions were
AtGH3.15 was previously described (33).
Generation and characterization of the three
independent A. thaliana Col-0 lines
performed in the presence and absence of
~20 µg AtGH3.15 with 50 mM Tris (pH 8),
mM MgCl , 2 mM ATP, 2 mM 2,4-DB,
3
2
overexpressing N-terminally FLAG-tagged
AtGH3.15 under control of the cauliflower
mosaic virus 35S promoter was also
previously reported (33). Root elongation
assays to examine the effect of AtGH3.15
expression changes on resistance to 2,4-D
and 2,4-DB, used seeds that were surface
sterilized with 70% (v/v) ethanol for 5 min,
and 2 mM glutamine in a 200 µl volume.
Reactions were allowed to react for 10
minutes at room temperature and then
placed at -20 °C. The reactions were
directly infused into the mass spectrometer.
The MS1 (Q1) scan was acquired with the
6
500-QTRAP (Sciex) in low mass (LM)
electrospray ionization in negative ion mode
at a capillary voltage of -4500 and a mass
range of 50-600 m/z.
9
0% (v/v) ethanol for 1 min, and
resuspended in 0.1% (w/v) sterile agar.
Sterilized seeds were stratified at 4 °C for 2-
4
days and plated on Murashige and Skoog
Protein crystallography. Crystals of
AtGH3.15 in complex with 2,4-DB were
grown by vapor diffusion in hanging drops
of a 1:1 mixture of protein (13 mg mL ) and
crystallization buffer (1.2 M potassium
phosphate (dibasic)/0.8 M sodium phosphate
(MS) plates with 0.6% (w/v) agar and
supplemented with 0.5% (v/v) sucrose.
Treatments were performed at 1 µM 2,4-DB
and 20, 40 and 80 nM 2,4-D with mock-
treated plates receiving equivalent amounts
of 70% (v/v) ethanol (2,4-DB and 2,4-D
were dissolved in 70% ethanol). Plates were
sealed with 3M micropore tape and
-
1
(
monobasic) and 0.1 M sodium
acetate/acetic acid, pH 4.5) with 2.5 mM
,4-DB at 4 °C. Crystals were frozen in
2
incubated at 22 °C under continuous white
light for 10 days. Seedlings were excised
from media and measured using a ruler.
Percent root length versus mock-treated was
calculated as: (root length of treated
seedlings)/(average root length of mock
treated seedlings)*100.
liquid nitrogen with mother liquor
supplemented with 15% (v/v) glycerol as a
cryoprotectant. Diffraction data was
collected at the SBC-19ID beamline of the
Argonne National Laboratory Advanced
Photon Source (APS) with indexing and
scaling performed using HKL3000 (42).
Molecular replacement was performed using
PHENIX (43) with the three-dimensional
structure of AtGH3.15 (PDB: 6AVH; 33) as
a search model. Model building and
ACKNOWLEDGEMENTS
This work was supported by the NSF
(MCB-1614539 to J.M.J.). A.M.S was an
8

NSF Graduate Research Fellow (DGE-
143954). The mass spectrometry
1
measurement was performed at the
Proteomics & Mass Spectrometry Facility at
the Donald Danforth Plant Science Center
based upon work supported by grants from
the National Science Foundation (DBI-
1
427621) for acquisition of the 6500
QTRAP LC-MS/MS. Portions of this
research were carried out at the Argonne
National Laboratory Structural Biology
Center of the Advanced Photon Source, a
national use facility operated by the
University of Chicago for the Department of
Energy Office of Biological and
Environmental Research under Grant DE-
AC02-06CH11357.
CONFLICT OF INTEREST
STATEMENT
The authors have no competing financial
interests.
AUTHOR CONTRIBUTIONS
A.M.S. and J.M.J. designed the research;
A.M.S., S.G.L., and E.S. performed the
research; A.M.S., S.G.L., and J.M.J.
analyzed data; A.M.S. and J.M.J. wrote the
paper with all authors providing editorial
input.
9

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3

TABLES
Table 1. Kinetic comparison of Arabidopsis GH3 proteins with auxinic herbicides (2,4-D and
2
,4-DB) and auxins (IAA and IBA).
substrate
parameter
cat (min )
AtGH3.1
AtGH3.2
--
AtGH3.5
--
AtGH3.15
1.3 ± 0.1
3,790 ± 420
6
11 ± 0.7
590 ± 100 4,830 ± 790
315
0.8 ± 0.1
560 ± 160
23
9.9 ± 0.2
530 ± 43
313
AtGH3.17
-
1
2
,4-D
k
K
k
k
K
k
k
K
k
k
K
--
--
--
--
--
--
--
--
(µM) -
--
--
--
--
m
1
-1
cat/K
m
(M s )
-
1
2
,4-DB
cat (min )
11 ± 1.7
2,330 ± 260
78
17 ± 1.6
510 ± 105
556
3.4 ± 0.4
1,160 ± 190
48
14.5 ± 1.4
770 ± 110
314
1.9 ± 0.5
m
(µM) -
1
-1
cat/K
m
(M s )
--
6
2.7 ± 0.1
68 ± 6
662
1.2 ± 0.1
1,600 ± 160
13
-
1
IAA
IBA
cat (min )
5.7 ± 0.6
530 ± 150
179
m
(µM) -
1
-1
cat/K
m
(M s )
-
1
cat (min )
17 ± 20
17 ± 1.8
72 ± 40
(µM) -
17,000 ± 12,500 2,190 ± 390 16,500 ± 9,630
17 129 73
m
1
-1
k
m
cat/K (M s )
Steady-state kinetic parameters for the auxinic herbicides were determined using varied
concentrations of either 2,4-D or 2,4-DB at fixed concentrations of amino acid (10 mM) and
ATP (1 mM). The amino acid substrate used for each GH3 protein was follows: AtGH3.1, Asn;
AtGH3.2 and AtGH3.5, Asp; AtGH3.15, Gln; and AtGH3.17, Glu. Kinetic parameters for IAA
with AtGH3.1, AtGH3.2, AtGH3.5, and AtGH3.17 were previously published (32). Kinetic
parameters for IAA and IBA with AtGH3.15 and for IBA with the other proteins were previously
published (33). Average values ± SD (n=3) are shown.
4

Table 2. Steady-state kinetic analysis of AtGH3.15.
-
1
-1 -1
substrate
2
k
cat (min )
K
m
(µM)
k
cat/K
m
(M s )
315
887
119
78
,4-DB 11.0 ± 0.7
590 ± 100
970 ± 100
Gln
Cys
His
Met
Tyr
52.3 ± 1.5
52.5 ± 9.7 7,240 ± 2,100
43.1 ± 1.2 9,290 ± 620
27.5 ± 0.6 10,600 ± 590
12.0 ± 1.8 18,400 ± 4,100
42
11
Steady-state kinetic parameters were determined using varied concentrations of 2,4-DB at fixed
concentrations of Gln (10 mM) and ATP (1 mM) or with varied concentrations of the indicated
amino acid at fixed concentrations of 2,4-DB (10 mM) and ATP (1 mM). Average values ± SD
(n=3) are shown. Kinetic parameters for 2,4-DB from Table 1 are shown for comparison.
5

Table 3. Kinetic analysis of AtGH3.15 with phenoxyalkanoic and phenylalkyl acids.
-
1
-1 -1
substrate
IBA
2
k
cat (min )
K
m
(µM)
k
cat/K
m
(M s )
313
6
315
3
228
113
506
28
126
42
445
597
9.9 ± 0.2
1.3 ± 0.1
11.0 ± 0.7
530 ± 40
3,790 ± 420
590 ± 100
,4-D
,4-DB
,4,5-trichlorophenoxyacetic acid (2,4,5-T)
-(4-chloro-2-methylphenoxy)butanoic acid (MCPB) 15.6 ± 0.5
2
2
4.3 ± 0.2 23,200 ± 2,300
4
1,130 ± 100
3,550 ± 520
180 ± 70
4
-(2-chlorophenoxy)butanoic acid
-(2,6-dimethylphenoxy)butanoic acid
-(4-methoxyphenoxy)butanoic acid
-phenoxybutyric acid
-phenylbutryic acid
-phenylvaleric acid
-(4-fluorophenyl)valeric acid
24 ± 1.4
5.4 ± 0.3
29.8 ± 2.9 14,800 ± 2,470
16.2 ± 3.8
15.0 ± 0.5
26.6 ± 0.3
24.0 ± 0.4
4
4
4
4
5
2,140 ± 260
6,000 ± 420
960 ± 50
5
680 ± 50
Steady-state kinetic parameters were determined using varied concentrations of each substrate
with fixed concentrations of Gln (10 mM) and ATP (1 mM). Average values ± SD (n=3) are
shown. Kinetic parameters for IBA, 2,4-D, and 2,4-DB from Table 1 are shown for comparison.
6

Table 4. Summary of crystallographic statistics for the AtGH3.15•2,4-DB complex.
Data Collection
Space group
C222
1
Cell dimensions
a = 153.8 Å, b = 154.8 Å,
c = 73.4 Å
Wavelength (Å)
0.979
Resolution (Å) (highest shell)
Reflections (total/unique)
Completeness (highest shell)
38.7 - 2.15 (2.19- 2.15)
319,808 / 47,900
99.5% (98.1%)
18.5 (2.0)
<
I/σ> (highest shell)
sym (highest shell)
Refinement
R
4.7% (55.7%)
R
cryst / Rfree
0.182 / 0.213
4,513
No. of protein atoms
No. of waters
290
No. of ligand atoms
R.m.s.d., bond lengths (Å)
R.m.s.d., bond angles (°)
Avg. B-factor (Å ): protein, water, ligand
Stereochemistry: most favored, allowed, disallowed
26
0.009
0.919
2
44.9, 87.0, 48.5
98.4, 1.4, 0.2 %
7

FIGURE LEGENDS
Figure 1. Structures of phenoxyalkanoic acid auxinic herbicides and endogenous auxins.
8

Figure 2. Comparison of Arabidopsis GH3 proteins auxinic herbicide (2,4-D and 2,4-DB) and
auxin (IAA and IBA) substrate profiles. Catalytic efficiencies (kcat/K ) for AtGH3.1, AtGH3.2,
m
AtGH3.5, AtGH3.15, and AtGH3.17 with 2,4-D (black), IAA (grey), 2,4-DB (dark grey), and
IBA (white) are summarized based on steady-state data from Table S1.
9

Figure 3. X-ray crystal structure of AtGH3.15 in complex with 2,4-DB. (A) Overall three-
dimensional structure of the AtGH3.15•2,4-DB complex. The ribbon diagram shows the N- and
C-terminal domains with α-helices (rose) and β-strands (blue). The bound 2,4-DB molecules are
shown as space-filling models. (B) Electron density for 2,4-DB molecules in the active site is
o c
shown as a 2F -F omit map (1.0 σ). (C) Surface view of 2,4-DB binding in the AtGH3.15
active site. The position of AMP from the previously reported AtGH3.15•AMP complex (33) is
also shown. (D) AtGH3.15 acyl acid binding site. The position of AMP from the previously
reported AtGH3.15•AMP complex (33) is also shown.
1
0

Figure 4. AtGH3.15 knockout and overexpression results in hypersensitivity and resistance to
,4-DB in root elongation assays. Wild-type Col-0 (Col); Overexpression lines (35S: FLAG-
2
GH3.15) are indicated as GH3.15 1-5, 2-7, and 8-2. (A) Seedlings were grown under continuous
white light for 10 days at 22 °C on medium supplemented with ethanol (mock) or 1 µM 2,4-DB.
Scale bar = 1 cm. (B) Percent root length versus mock-treated was calculated via the equation
(root length of treated seedlings)/(average root length of mock treated seedlings)*100. Error bars
represent ± SE of the means (n = 20). *P < 0.05, **P < 0.001, ***P < 0.0001 versus wild-type.
1
1

Figure 5. Conversion of 2,4-DB to 2,4-D and potential role of AtGH3.15 as a 2,4-DB tolerance
mechanism. Metabolism of 2,4-DB to 2,4-D by β-oxidation in the peroxisome leads to auxinic
herbicide effects. Overexpression of AtGH3.15 likely inactivates 2,4-DB by conjugate
formation.
1
2

Modification of auxinic phenoxyalkanoic acid herbicides by the acyl acid amido
synthetase GH3.15 from Arabidopsis
Ashley M. Sherp, Soon Goo Lee, Evelyn Schraft and Joseph M. Jez
J. Biol. Chem. published online October 12, 2018
Access the most updated version of this article at doi: 10.1074/jbc.RA118.004975
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