Molecular Basis of Bacillus subtilis ATCC 6633 Self-Resistance to the Phosphono-oligopeptide Antibiotic Rhizocticin

Article abstract of DOI:10.1021/acschembio.9b00030

Rhizocticins are phosphono-oligopeptide antibiotics that contain a toxic C-terminal (Z)-l-2-amino-5-phosphono-3-pentenoic acid (APPA) moiety. APPA is an irreversible inhibitor of threonine synthase (ThrC), a pyridoxal 5′-phosphate (PLP)-dependent enzyme that catalyzes the conversion of O-phospho-l-homoserine to l-threonine. ThrCs are essential for the viability of bacteria, plants, and fungi and are a target for antibiotic development, as de novo threonine biosynthetic pathway is not found in humans. Given the ability of APPA to interfere in threonine metabolism, it is unclear how the producing strain B. subtilis ATCC 6633 circumvents APPA toxicity. Notably, in addition to the housekeeping APPA-sensitive ThrC (BsThrC), B. subtilis encodes a second threonine synthase (RhiB) encoded within the rhizocticin biosynthetic gene cluster. Kinetic and spectroscopic analyses show that PLP-dependent RhiB is an authentic threonine synthase, converting O-phospho-l-homoserine to threonine with a catalytic efficiency comparable to BsThrC. To understand the structural basis of inhibition, we determined the crystal structure of APPA bound to the housekeeping BsThrC, revealing a covalent complex between the inhibitor and PLP. Structure-based sequence analyses reveal structural determinants within the RhiB active site that contribute to rendering this ThrC homologue resistant to APPA. Together, this work establishes the self-resistance mechanism utilized by B. subtilis ATCC 6633 against APPA exemplifying one of many ways by which bacteria can overcome phosphonate toxicity.

Full text of DOI:10.1021/acschembio.9b00030

Articles
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Molecular Basis of Bacillus subtilis ATCC 6633 Self-Resistance to the
Phosphono-oligopeptide Antibiotic Rhizocticin
Nektaria Petronikolou,†,⊥ Manuel A. Ortega,‡,⊥ Svetlana A. Borisova,‡ Satish K. Nair,*,†,‡,§
and William W. Metcalf*,‡,∥
†Department of Biochemistry, University of Illinois at Urbana−Champaign, Roger Adams Laboratory, 600 S. Mathews Ave., Urbana,
Illinois 61801, United States
‡Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana−Champaign, 1206 W. Gregory Drive, Urbana, Illinois
61801, United States
§Center for Biophysics and Computational Biology, University of Illinois at Urbana−Champaign, Roger Adams Laboratory, 600 S.
Mathews Ave., Urbana Illinois 61801, United States
∥Department of Microbiology, University of Illinois at Urbana−Champaign, Chemical and Life Sciences Laboratory, 601 S.
Goodwin Ave., Urbana, Illinois 61801, United States
S
* Supporting Information
ABSTRACT: Rhizocticins are phosphono-oligopeptide anti-
biotics that contain a toxic C-terminal (Z)-L-2-amino-5-
phosphono-3-pentenoic acid (APPA) moiety. APPA is an
irreversible inhibitor of threonine synthase (ThrC), a
pyridoxal 5′-phosphate (PLP)-dependent enzyme that cata-
lyzes the conversion of O-phospho-L-homoserine to L-
threonine. ThrCs are essential for the viability of bacteria,
plants, and fungi and are a target for antibiotic development,
as de novo threonine biosynthetic pathway is not found in
humans. Given the ability of APPA to interfere in threonine
metabolism, it is unclear how the producing strain B. subtilis
ATCC 6633 circumvents APPA toxicity. Notably, in addition
to the housekeeping APPA-sensitive ThrC (BsThrC), B.
subtilis encodes a second threonine synthase (RhiB) encoded within the rhizocticin biosynthetic gene cluster. Kinetic and
spectroscopic analyses show that PLP-dependent RhiB is an authentic threonine synthase, converting O-phospho-L-homoserine
to threonine with a catalytic efficiency comparable to BsThrC. To understand the structural basis of inhibition, we determined
the crystal structure of APPA bound to the housekeeping BsThrC, revealing a covalent complex between the inhibitor and PLP.
Structure-based sequence analyses reveal structural determinants within the RhiB active site that contribute to rendering this
ThrC homologue resistant to APPA. Together, this work establishes the self-resistance mechanism utilized by B. subtilis ATCC
6633 against APPA exemplifying one of many ways by which bacteria can overcome phosphonate toxicity.
(Figure 1b).7 Notably, APPA is also found as the active
hosphonates are natural products characterized by the
Ppresence of an inert C−P bond in place of the more
warhead of plumbemycins, which are antibacterial tripeptides
common O−P bond found in phosphoric acids.1 As structural
analogs of numerous phosphate ester and carboxylic acid
intermediates found in metabolic processes, phosphonates act
as inhibitors of the corresponding enzymes. For instance, the
unusual nonproteinogenic amino acid (Z)-L-2-amino-5-phos-
phono-3-pentenoic acid (APPA) inhibits threonine synthases
(ThrC) irreversibly by mimicking the physiological substrate
O-phospho-L-homoserine (PHSer) (Figure 1a).2,3 ThrC is a
pyridoxal 5′-phosphate (PLP)-dependent enzyme that utilizes
PHSer as a substrate in the last step in threonine biosynthesis
(Figure 1a).4,5
produced by Streptomyces plumbeus.8,9 Although both phos-
phono-oligopeptides contain a C-terminal APPA residue, they
contain different amino acids at their N-terminus. This
difference is believed to be responsible for defining the
spectrum of target organisms affected by the two natural
products. Thus, due to differences in the specificities of
oligopeptide transporters, rhizocticin has antifungal but not
antibacterial activity, while plumbemycin has the opposite
spectrum. In either case, host peptidases cleave the peptide to
APPA is the pharmacophore present at the C-terminus of
the phosphono-oligopeptide antifungal rhizocticin,6 produced
by the Gram-positive bacterium Bacillus subtilis ATCC 6633
Received: January 9, 2019
Accepted: March 4, 2019
Published: March 4, 2019
© XXXX American Chemical Society
A
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Figure 1. Overview of rhizocticin biosynthetic gene cluster. (a) Reaction catalyzed by threonine synthases (BsThrC), which is inhibited by APPA.
(b) Structure of the phosphono-oligopeptides rhizocticin and plumbemycin. The nonproteinogenic phosphono-amino acid (Z)-L-2-amino-5-
phosphono-3-pentenoic acid is shown in red. (c) Rhizocticin biosynthetic gene cluster. Biosynthetic genes are shown in red and the putative gene
involved in self-resistance, RhiB, is shown in green. For comparison, the B. subtilis ATCC 6633 threonine biosynthetic operon is shown below
indicating the percent amino acid sequence identity between the predicted threonine synthase homologue RhiB and the BsThrC.
release the C-terminal APPA warhead after uptake, resulting in
growth inhibition.10
years since the identification of APPA as an inhibitor of ThrC,
the molecular details regarding the mechanism of inactivation
remain unclear.
Early in vivo antagonist experiments with various amino
acids suggested that APPA caused growth inhibition by
disrupting threonine-related metabolism.8,10 In vitro experi-
ments later revealed APPA to be an irreversible inhibitor of the
threonine biosynthetic enzyme ThrC, demonstrating satura-
tion kinetics with an apparent kinact of 1.50 min−1 and a Ki of
100 μM.2,3 Plausible mechanisms for inhibition include
covalent modification of the enzyme or modification of the
PLP cofactor, but the experimental data did not provide
support for either of these proposed mechanisms.2 In these
studies, APPA inhibited the threonine synthase from E. coli
(EcThrC) irreversibly at a molar ratio of 1:1 suggesting that
inhibition occurs through formation of a covalent adduct
between APPA and the enzyme.2,3 This mode of inhibition is
observed in other PLP-dependent enzymes such as inhibition
of 1-aminocyclopropane-1-carboxylate synthase by L-vinyl-
glycine.11 However, tryptic digestion of EcThrC and peptide
analysis did not reveal any modified peptides.2 This finding led
the authors to propose a mechanism of inhibition that involves
a conformational change of the enzyme leading to the
stabilization of an enzyme−inhibitor complex, a mechanism
similar to the one of D-amino acid aminotransferase inhibition
by the antibiotic D-cycloserine.2,12,13 Thus, despite almost 14
ThrC is an essential enzyme for growth of bacteria and fungi,
and this enzyme is not present in mammals. Given the
widespread occurrence of antibiotic resistance and the
impending need for the identification of novel antibiotics,
ThrC is a worthwhile microbial target, and APPA is an
attractive therapeutic candidate.14 As such, understanding the
mechanism of inhibition of ThrC by APPA as well as how the
producing organisms overcome APPA toxicity will inform on
the clinical potential of APPA.
Previous bioinformatic studies on the rhizocticin biosyn-
thetic gene cluster identified the rhiB gene, whose product
showed homology to threonine synthases (Figure 1c).15
Curiously, examination of the B. subtilis ATCC 6633 genome
also reveals the presence of a threonine biosynthetic operon
(thrACB), which is similar in genomic context and sequence to
the typical Bacillus threonine synthase involved in primary
metabolism (BsThrC) (Figure 1c).5,15,16 Sequence alignment
between RhiB and BsThrC revealed that they share greater
than 25% sequence identity further suggesting that RhiB is a
threonine synthase (Figure 1c). These observations prompted
us to consider that RhiB could function as an APPA-resistant
threonine synthase homologue, which could serve as a self-
B
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resistance mechanism to circumvent toxicity in the producing
organism.
To assess this hypothesis, we reconstituted the activity of
both the housekeeping BsThrC and the putative resistant RhiB
in vitro. We demonstrate that RhiB is a PLP-dependent
threonine synthase capable of generating threonine from
PHSer. Kinetic assays revealed the enzyme to be 7-fold less
efficient at generating threonine as compared to the BsThrC.
However, while BsThrC is sensitive to APPA, RhiB was not
subject to inhibition by this compound. We also determined
the 2.0-Å resolution cocrystal structure of BsThrC in complex
with APPA demonstrating that inhibition occurs via the
formation of a covalent complex between APPA and the bound
PLP cofactor. Structure-based analysis of the sensitive and
resistant threonine synthases provide insights into the
molecular basis for the observed APPA resistance in RhiB.
This work uncovers the self-resistance mechanism utilized by
B. subtilis ATCC 6633 against APPA, exemplifying one of
many ways by which antibiotic producing bacteria can ensure
self-resistance during the biosynthesis of a toxic metabolite.
Figure 2. In vitro reconstitution of His6-RhiB activity. 1H NMR
spectra of (a, b) reaction assays following incubation of PHSer with
either (a) His6-RhiB or (b) His6-BsThrC, (c) threonine standard
solution, and (d) reaction assay with no enzyme present. Only the
1
RESULTS AND DISCUSSION
upfield portion of the H NMR spectrum is shown for clarity.
■
RhiB Is a Bona Fide Threonine Synthase Insensitive
to APPA. For biochemical studies, recombinant RhiB was
expressed and purified from an E. coli heterologous expression
system, and its enzymatic activity was tested in vitro. Soluble
His6-RhiB required coexpression with the E. coli chaperones
GroEL/GroES. Significantly, throughout the purification
process, His6-RhiB exhibited a yellow color characteristic of
either a bound flavin or PLP cofactor. Threonine synthases
characterized to date employ the use of PLP as a cofactor to
convert O-phospho-L-homoserine into threonine.5,17,18 UV−
vis spectroscopy of purified His6-RhiB revealed the presence of
an absorption maximum at 420 nm characteristic of PLP-
containing enzymes similar to other characterized threonine
synthases (Supplementary Figure S1). We next sought to
determine the ability of this enzyme to convert PHSer into
threonine. Upon incubation of His6-RhiB with PHSer, a new
displayed an apparent kcat of 0.60 s−1 and an apparent KM of
824 μM.
The presence of two bona fide threonine synthases in B.
subtilis ATCC 6633 suggests that RhiB might be an insensitive
variant, which could serve as a self-resistance mechanism to
avert APPA toxicity. To test this hypothesis, APPA was
purified as previously described,15 and the ability of RhiB and
BsThrC to catalyze threonine formation was assessed in the
presence of APPA by measuring PHSer consumption via 31P
NMR. Regardless of the APPA concentration used (either 5×
or 20× molar excess), upon incubation of APPA with RhiB, no
change in activity was observed compared to enzyme assays
performed in the absence of APPA (Figure 4). Interestingly,
incubation of BsThrC with APPA resulted in the formation of
a new spectral feature at ∼519 nm (Supplementary Figure S2).
This chromophore is consistent with the formation of a
quinonoid species that may arise upon the enzymatic Cα
proton abstraction from the APPA−PLP adduct.2,3 This initial
step is consistent with the enzymatic mechanism employed by
threonine synthases, which commences with the abstraction of
the alpha proton present in the PLP−PHSer adduct.4 Activity
assays performed with BsThrC in the presence of 5× molar
excess APPA resulted in decreased enzymatic activity when
compared to assays performed in the absence of APPA (Figure
4). These results suggest that APPA is capable of inhibiting
BsThrC but not RhiB, proving that the latter is an APPA-
insensitive threonine synthase.
Structural Characterization of BsThrC in Complex
with APPA Reveals Mode of Inhibition. To establish the
molecular basis by which APPA inhibits threonine synthase
activity, we determined the 2.0-Å resolution X-ray crystal
structures of BsThrC in its PLP and PLP−APPA bound states
(Table 1). As expected, the overall structure of BsThrC is
highly similar to other threonine synthases sharing the
canonical fold-type II of PLP-dependent enzymes.23,24 The
overall fold of BsThrC recapitulates the architecture of other
enzymes of the tryptophan synthase family.23,24 The structure
consists of an N-terminal α/β domain (domain 1; helices α1,
α2, and α7−α11 and strands β1, β2, and β7−β10), followed by
a second α/β domain (domain 2; helices α3−α6 and strands
1
product appeared as observed by one-dimensional H NMR
with a doublet signal at 1.34 ppm and a J coupling constant of
6.4 Hz (Figure 2a). This diagnostic signal is characteristic of
the Hγ present in threonine suggesting RhiB to be a threonine
synthase.
In addition to RhiB, B. subtilis ATCC 6633 encodes for an
endogenous ThrC (BsThrC) present within the threonine
biosynthetic operon (Figure 1c). Recombinant BsThrC was
purified as an N-terminal His6 tagged construct, and the
recombinant enzyme exhibited a UV−vis absorption maximum
of 420 nm indicating this protein also copurified with PLP
(Supplementary Figure S1). As with RhiB, enzyme activity
assays with BsThrC incubated in the presence of PHSer led to
1
the formation of threonine as observed by H NMR (Figure
2b). Threonine formation was not detected in the absence of
enzyme (Figure 2c,d).
To gain more insights into the efficiency of the enzymes in
catalyzing the formation of threonine, steady state kinetic
parameters for both enzymes were determined (Figure 3).
Upon formation of threonine catalyzed by threonine synthases,
inorganic phosphate is released as a product of the
reaction.19−21 Phosphate release was thus measured using a
previously reported UV−vis continuous coupled spectropho-
tometric assay.22 Recombinant BsThrC exhibited an apparent
k
cat of 1.74 s−1 and an apparent KM of 329 μM, while His6-RhiB
C
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Figure 3. Kinetic characterization of His6-RhiB and His6-BsThrC. (a) His6-BsThrC and (b) His6-RhiB activity was measured by determining Pi
release from O-phosphohomoserine. Enzymatic rates were plotted as a function of O-phosphohomoserine concentration, and the data were fit to
the Michaelis−Menten equation. Results are means standard error of mean (SEM) of triplicate experiments.
Table 1. Data Collection and Refinement Statistics
BsThrC−PLP/
BsThrC−APPA
BsThrC−Ala (6CGQ)
(6NMX)
Data Collection
space group
C2221
P21
a, b, c (Å), β (deg)
49.8, 127.9, 237.1
49.5, 104.2, 125.6,
99.6
resolution (Å)
Rsym (%)
I/σ(I)
completeness (%)
redundancy
total reflns
118.5−2.0
12.7 (102.7)
14.6 (2.2)
100 (97.6)
8.2 (8.4)
411421
123.8−1.97
12.9 (85.2)
10.7 (2.1)
91.3 (95.7)
4.7 (4.7)
379109
a,b
unique reflns
Refinement
50330
80926
resolution (Å)
49.7−2.0
50265
0.18/0.23
123.8−1.97
80926
0.16/0.21
no. reflns used
c
Rwork/Rfree
Number of Atoms
Protein
PO4
5121
5
10437
Figure 4. BsThrC and RhiB activity in the presence of APPA. End
point assays were performed by measuring the amount of PHSer
consumed at the end of the reaction. Data are plotted as the means
standard error of the mean (SEM) of triplicate experiments. 5× APPA
is 2.5 μM when incubated with BsThrC and 15 μM when incubated
with RhiB. 20× APPA is 60 μM.
PLP
PLP-Ala
PLP−APPA
water
B-factors
protein
PO4
PLP
PLP-Ala
15
21
108
849
370
28.23
25.91
32.52
21.08
22.93
22.44
β3−β6) and a C-terminal tail (helices α12 and α13) that
extends from one subunit to the other (Figure 5a). The
enzyme active site forms at the interface of the two α/β
domains of the same subunit while the C-terminal helix α13
interacts with domain 2 of the other subunit (Figure 5a,b).
Solution studies were consistent with a homodimeric
assembly for BsThrC (Figure S1), and the two molecules in
the crystallographic asymmetric unit of the enzyme recapitu-
lated this functional homodimer (Figure 5a). In this
homodimer, one subunit is in an open conformation with
the PLP bound to the active site Lys59 through an aldimine
linkage while the other subunit is in the closed conformation
(Figure 5b−d). In the closed conformation, a ligand is bound
to the cofactor even though no substrate was added during the
purification or crystal screening process. However, the
crystallization condition contained a mixture of amino acids
(0.2 M sodium L-Glu, 0.2 M DL-Ala, 0.2 M Gly, 0.2 M DL-Lys
HCl, 0.2 M DL-Ser). Modeling and refinement efforts support
the judicious placement of Ala as the ligand adjacent to the
PLP cofactor (Supplementary Figure S3). Consistent with this
PLP−APPA
water
18.61
29.55
34.41
rms deviations
bond lengths (Å)
bond angles (deg)
MOLPROBITY statistics
clash score
0.007
0.873
0.007
0.916
2.71
0.29/1.18/98.53
4.13
0.29/1.08/98.63
Ramachandran outliers/
allowed/favored (%)
a
b
Highest-resolution shell is shown in parentheses. Rsym = ∑|Ii −
⟨I⟩|/∑Ii, where Ii = intensity of the ith reflection and ⟨I⟩ = average
intensity of symmetry-related observations of a unique reflection. R-
c
factor = ∑|Fobs − Fcalc|/∑|Fobs| and R-free is the R value for a test set
of reflections consisting of a random 5% of the diffraction data not
used in refinement.
conclusion, prior experiments with E. coli threonine synthase
show Ala as the substrate for half transamination reactions.2 In
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Figure 5. Crystal structure of BsThrC. (a) Ribbon diagram of the functional homodimer of BsThrC as generated by the space group symmetry
operations. In blue is domain 1, in yellow is domain 2, and the C-terminal tail is shown in red. The helix α13 of one monomer interacts with the
domain 2 of the other monomer. (b) Surface representation of the functional dimer. In blue is the BsThrC-PLP subunit in the open conformation
and in pink is the BsThrC-Ala subunit in the closed conformation. (c, d) Zoomed in view of the surface in the open and closed conformations,
respectively. Upon binding of the substrate, domain 2 (shown in gray) moves and closes the active site. Gray, domain 2, blue, domain 1 in the open
state, pink, domain 1 in the closed state. (e) Conformational changes of domain 2 upon binding of the substrate. Only domain 2 is shown. Blue,
open conformation, pink, closed conformation, dashed line, residues missing from crystal structure. (f, g) Stick representation of residues that
interact with the bound ligands (alanine and phosphate, respectively) showing their position before (blue) and after (pink) binding. Dashed lines
represent hydrophobic interactions and solid lines represent hydrogen bonding interactions (distances shown are in Å). Residues that interact with
the cofactor are not shown.
addition, a phosphate group was also modeled in the active site
of the BsThrC−Ala monomer (Supplementary Figure S3),
which was presumably carried over during purification of the
enzyme.
The structure of the BsThrC−APPA complex reveals a
closed conformation, and a superposition with BsThrC bound
to Ala shows that the active sites are nearly identical (Figure
6b). The carboxylate group of APPA is engaged by hydrogen
bonding interactions with the hydroxyl group of the highly
conserved Ser82 and the main chain amines of the highly
conserved Thr83 and Thr86 (Figure 5f and Supplementary
Figure S5). The inhibitor is further anchored by interactions
between the phosphonate oxygens and the side chains of the
highly conserved (among endogenous threonine synthases)
Thr86, Asn152, Ser153, Arg158, and Asn186, as well as Lys59,
which is the lysine that forms an aldimine linkage with PLP
when the enzyme is in the resting state (Supplementary Figure
S5). Finally, Phe132 and Ile240 line the enzyme pocket near
the β, γ, and δ carbons of APPA further supporting the
inhibitor (Supplementary Figure S5). Hence, APPA inhibits
threonine synthase by mimicking the physiological substrate
and trapping the enzyme in a closed conformation, and upon
formation of the APPA−PLP adduct, ThrC abstracts the Cα
proton from this species. This mechanism for inhibition is
supported by the formation of a stable quinonoid intermediate
(Supplementary Figure S2), and given the structural
resemblance of APPA to PHSer, it is expected for the enzyme
to be able to catalyze this initial step. When ThrC acts on the
PLP derivative of PHSer, further proton abstractions on the
adduct take place leading to the nonhydrolytic elimination of
the phosphate in PHSer.4,20,21,27 However, due to the presence
of a stable C−P bond in APPA, the required nonhydrolytic
elimination needed to yield threonine, hence resetting the
catalytic cycle, is hampered preventing the enzyme from
As observed in prior structures of threonine synthases, the
enzyme undergoes a conformational change upon binding of
the substrate (Ala) to control proton transfers and solvent
accessibility during the reaction.24−26 The superposition of the
open and closed conformations reveals that major changes take
place in domain 2 upon substrate binding (Figure 5d).
Specifically, the loop that connects strand β4 with helix α5
(residues Gly108−Gly113) is disordered in the absence of
substrate but becomes structured in the presence of substrate,
with residues Phe112 and Gly113 adopting an α-helical
conformation that extends helix α5. This change is
accompanied by a significant movement of strands β3, β4,
and β5 and helix α6 resulting in a closure of the active site.
Finally, when the substrate binds, residues Asn152 and Ser153
are now within hydrogen bonding distance to the phosphate
group of the substrate.
Crystals of BsThrC in complex with the inhibitor APPA
showed an orange color, in agreement with the observed shift
in the absorption spectrum of the enzyme after binding of the
inhibitor (Supplementary Figure S2a,b and S4).2 The complex
crystallized as a dimer of functional dimers, and electron
density corresponding to the inhibitor was present in all 4
molecules in the asymmetric unit (Figure 6a). Notably, the
structure reveals that APPA binds to BsThrC by forming a
covalent complex with the PLP cofactor and not with any of
the residues in the active site (Figure 6a).
E
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Figure 6. Crystal structure of BsThrC−APPA. (a) Difference Fourier map (Fo − Fc) calculated with the bound ligands removed prior to one round
of refinement and map calculation and shown at a contour of 2.0σ (blue). It is evident from the electron density that the APPA is covalently bound
to PLP and not to any active site residues. (b) Superposition of the BsThrC−APPA (teal) and BsThrC−Ala (pink) active sites. The two active sites
are identical further supporting the hypothesis that APPA inhibits threonine synthase by “trapping” the enzyme in the closed conformation. The
PLP−APPA adduct is shown in orange and the PLP−Ala adduct in yellow. (c) Superposition of BsThrC−APPA (teal) and RhiB (yellow) active
sites. The RhiB model was generated by SWISS-MODEL44 using the BsThrC−APPA structure as a template. The labels correspond to the RhiB
residues. (d) Phe132 in BsThrC is ∼3.8 Å away from the Cδ of APPA. In RhiB, the corresponding Phe has been substituted by Tyr. (e) Sequence
alignment of BsThrC and RhiB. Star, lysine that forms the internal aldimine; circles, residues that interact with APPA; squares, residues that interact
with the cofactor.
completing a turnover thus trapping it in its “closed” state
(Supplementary Figure S6). Due to the numerous proton
abstractions and protonations occurring during the ThrC
catalytic cycle, further spectroscopic studies of the PLP−APPA
adduct following incubation with ThrC are warranted. Despite
this limitation, our studies provide the first direct evidence for
this previously proposed mechanism of ThrC inhibition by
APPA.2
Structural Comparison of BsThrC and RhiB Active
Sites Reveals Insights into the Structural Basis of RhiB
Resistance to APPA. As no active site residues in BsThrC are
involved in covalent interactions with the inhibitor, this raises
the question as to why APPA is unable to inhibit RhiB.
Presumably, APPA could bind to the PLP cofactor in RhiB as
it does to BsThrC. To address this question, we generated a
homology model for RhiB using the BsThrC−APPA structure
as a template and compared their active sites. The most
notable difference was the substitution of Phe132 in BsThrC
with Tyr188 in RhiB (Figure 6c). In BsThrC, Phe132 is ∼3.8 Å
away from the δ carbon of the phosphonic group of APPA,
which is the position of the oxygen of the phosphate group in
PHSer (Figure 6d). This led us to the hypothesis that
introduction of a hydroxyl group in that position may affect
binding of the inhibitor.
activity assays in the absence and presence of APPA. The
substitution of a Phe in Y188F_RhiB resulted in decreased
enzymatic activity in the presence of 5× molar excess of APPA
when compared to assays performed in the absence of inhibitor
(Figure 4). Similarly, the substitution of a Tyr in
F132Y_BsThrC resulted in a variant that was less sensitive
to the inhibitor compared to the wild-type enzyme (Figure 4).
These results suggest that the introduction of a hydroxyl group
near the δ carbon of APPA affects its binding to PLP and the
substitution of Phe132 in BsThrC with a Tyr188 in RhiB
contributes to resistance to APPA in the latter enzyme.
Interestingly, the activity levels seen in the Y188F_RhiB
mutant suggest that while Tyr188 is important for rendering
RhiB resistant to APPA additional structural features within
RhiB must contribute as well for the observed APPA
resistance.
A closer examination of the two active sites shows that the
highly conserved Asn186 in BsThrC is substituted by Ala243 in
RhiB (Figures 5g and 6b,c,e). As Asn186 is involved in
engaging the phosphate group of the substrate PHSer (Figure
5g), the substitution of Asn186 and Phe132 in BsThrC by
Ala243 and Tyr188 in RhiB may result in different binding
orientation of the substrate−PLP adduct in RhiB, which may
account for the 7-fold difference in the catalytic efficiencies
(kcat/KM) of the two enzymes. As there is a complex network
of interactions between the enzyme active site residues and the
To investigate this hypothesis in detail, we generated the
F132Y_BsThrC and Y188F_RhiB variants and performed
F
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Crystallization of BsThrC. Initial crystallization conditions were
determined by the sparse matrix sampling method using commercial
screens. Crystals of BsThrC-APPA were grown using the hanging
drop vapor diffusion method. Briefly, 1 μL of protein at 4 mg mL−1
concentration was incubated with 15 mol equiv of APPA (1.5 mM)
for 1.5 h at ambient temperature, mixed with 1 μL of precipitant
solution (0.03 M MgCl2 hexahydrate, 0.03 M CaCl2 dihydrate, 18%
v/v PEG 550 MME, 9% w/v PEG 20000, 0.1 M MES/imidazole, pH
6.5), and equilibrated over a well containing the same precipitant
solution at 9 °C. The crystals were sequentially soaked in precipitant
solution supplemented with increasing concentrations of 20% (v/v)
and 30% (v/v) ethylene glycol prior to vitrification by direct
immersion in liquid nitrogen.
The BsThrC−Ala/BsThrC−PLP crystals were pooled from the
initial screening trays. Briefly, 0.2 μL of protein at 6 mg mL−1 was
mixed with 0.2 μL of MORPHEUS crystallization screen28 condition
H1 (0.2 M sodium L-glutamate, 0.2 M DL-alanine, 0.2 M glycine, 0.2
M DL-lysine HCl, 0.2 M DL-serine, 10% w/v PEG 20000, 20% v/v
PEG MME 550, 0.1 M MES/imidazole, pH 6.5) using the sitting drop
vapor diffusion method and equilibrated over a well containing the
same precipitant solution at 9 °C. The crystals were sequentially
soaked in precipitant solution supplemented with increasing
concentrations of 20% (v/v) and 30% (v/v) ethylene glycol prior
to vitrification by direct immersion in liquid nitrogen.
Data Collection, Phasing, and Structure Determination. X-
ray diffraction data were collected at Life Sciences Collaborative
Access Team (LS-CAT), Sector 21, Argonne National Laboratory. All
data were indexed, integrated, and scaled using AutoProc.29 Both
structures were determined by molecular replacement as implemented
in the Phenix program suite;30 for the BsThrC−PLP/BsThrC−Ala
structure, the coordinates of threonine synthase from Aquifex aeolicus
Vf5 (59% sequence identity, 77% sequence similarity, 97% cover,
PDB ID 2ZSJ) were used as a search model, while for the BsThrC−
APPA complex the refined coordinates of BsThrC−PLP/BsThrC−Ala
were used. In both cases, the resultant solutions were subsequently
used as starting models for several rounds of automated model
building using Phenix Autobuild31 and Buccaneer,32−34 followed by
rounds of manual rebuilding using Coot,35 and refinement using
either Phenix Refine36 or REFMAC5.34 Ligands were built in Coot,35
and the resultant models were further refined and manually inspected.
In the final stages of refinement, water molecules were added with
Phenix Refine and confirmed by manual inspection. In all cases, the
quality of the in-progress model was routinely monitored using both
the free R factor37 and MolProbity38 for quality assurance. The final
models contain four Ramachandran outliers (Thr233 in chains A, B,
and C and Thr337 in Chain D). These outliers are located on loops
and were built to the best of our ability.
different moieties of the cofactor−substrate/inhibitor adduct,
it is very likely that in the RhiB active site the adduct is
engaged in a shifted orientation. This presumed difference in
binding orientation of the adduct may affect the binding
kinetics or affinity of RhiB for APPA and is to be further
investigated. Nonetheless, our mutagenesis data clearly show
that the F132Y substitution in BsThrC is sufficient to render
BsThrC less sensitive to the inhibitor.
CONCLUDING REMARKS
■
We show here that Bacillus subtillis ATCC 6633 possesses two
threonine synthases, one that is sensitive (BsThrC) and one
that is resistant (RhiB) to APPA, the active component of the
rhizocticin natural product produced by this organism. In
addition, our structural-sequence analyses between RhiB and
the 2.0-Å resolution crystal structure of BsThrC in complex
with APPA suggested that a single amino acid substitution in
the RhiB active site (Tyr188) contributes to rendering this
ThrC homologue resistant to APPA. In support of this finding,
we generated a BsThrC variant tolerant to APPA by
introducing a single site mutation within its active site
(F132Y_BsThrC variant). Together, this work uncovers the
self-resistance mechanism utilized by B. subtilis ATCC 6633
against APPA; the rhizocticin producing organism evades
APPA toxicity by employing a second threonine synthase
(RhiB) that is insensitive to APPA.
MATERIALS AND METHODS
■
BsThrC and RhiB Activity Assays. The following conditions
were used to determine the ability of BsThrC and RhiB to catalyze
threonine formation: 20 mM HEPES-NaOH, pH 7.5, 1 mM PHSer,
50 μM enzyme (either BsThrC or RhiB), and 5 μM PLP in a final
volume of 500 μL, and samples were incubated for 2 h at 37 °C.
Following the incubation period, proteins were removed using 3 kDa
MWCO Amicon centrifugal filters (14 000g, 4 °C); the filtrate was
collected and lyophilized to dryness. Dried solids were then dissolved
1
in 500 μL of D2O and analyzed via H NMR.
Kinetic Characterization of BsThrC and RhiB. Michaelis−
Menten kinetics were performed utilizing a coupled assay for the
continuous monitoring of inorganic phosphate (Pi) release by the
enzyme.22 Briefly, in the presence of inorganic phosphate, 2-amino-6-
mercapto-7-methylpurine riboside (MESG) is converted enzymati-
cally by purine nucleoside phosphorylase (PNP) to ribose 1-
phosphate and 2-amino-6-mercapto-7-methylpurine. This conversion
of MESG results in a shift in maximum absorbance from 330 nm for
the substrate to 360 nm for the product (ε360 = 11 000 M−1 cm−1).
This shift allows for continuous monitoring of Pi production by
measuring the increase in absorbance at 360 nm. The following
conditions were used: 50 mM HEPES-NaOH, pH 7.5, 100 μM PLP,
200 μM MESG, 0.00375 U μL−1 PNP, either 0.25 μM RhiB or 0.1
μM BsThrC, and various concentrations of O-phospho-L-homoserine
(PHSer). Measurements were performed in triplicate.
Purification of Rhizocticin and APPA from B. subtilis 6633.
Purification of rhizocticin was performed as previously described.15
Following purification, acid hydrolysis with 6 M HCl was performed
to obtain APPA following previously published procedures.6,10
Binding of APPA. Binding of the inhibitor to the enzyme was
monitored spectrophotometrically by monitoring the formation of a
chromophore at ∼500 nm.2 BsThrC and RhiB were incubated with
APPA (488 μM) in a 2:1 ratio for 15 min. Following the incubation
period, both proteins were washed 5 times using 10 kDa MWCO
Amicon centrifugal filters (500 μL, 14 000g, 4 °C, 20 min) with buffer
(20 mM HEPES-NaOH, pH 7.5, 300 mM NaCl and 10% (v/v)
glycerol), and the absorption spectra were recorded on a Cary 4000
UV−visible spectrophotometer at ambient temperature.
Inhibition Assays: 31P NMR. Enzyme inhibition assays were
carried out at 30 °C in 50 mM HEPES-NaOH (pH 7.5). In a final
volume of 400 μL, 0.5 μM holo-BsThrC wild-type or mutant was
preincubated with 2.5 μM APPA for 1 h at 30 °C, and then 1.6 mM
PHSer (∼5 × KM) was added. For RhiB wild-type and mutant, 3 μM
holoenzyme was preincubated with 15 μM or 60 μM APPA for 1 h at
30 °C, and then 4 mM PHSer (∼5 × KM) was added. After 30 min
incubation of the enzymes with PHSer, the samples were passed
through 10 kDa MWCO Amicon centrifugal filters, and 300 μL of the
filtrate was removed and mixed with 300 μL of D2O. For
quantification during 31P NMR, 0.5 mM dimethylphosphinic acid
was added as internal standard in all samples, and PHSer
consumption was measured for the calculation of enzyme activity.
The activity of the enzymes in the presence of APPA was expressed as
“percentage of PHSer consumed” with respect to the corresponding
wild-type reaction that contained no APPA (considered as 100%). All
measurements were performed in triplicate.
Estimation of PLP Content: Holoenzyme Concentration.
Protein concentrations were determined by measuring the absorbance
at 280 nm using the molecular weights and extinction coefficients
calculated by the ProtParam tool (ExPASY server) (Table S3).39 In
order to calculate the concentration of holo-RhiB and holo-BsThrC in
the protein preparations, the absorbance at 388 nm before (free PLP)
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and after (total PLP) the addition of 0.2 M NaOH was measured, as
previously described.40−43 The difference between the two values
corresponds to the PLP concentration that is bound to the enzyme
(holoenzyme concentration). Free PLP has a maximum absorbance at
388 nm with an extinction coefficient of 6600 M−1 cm−1 in 0.1 M
NaOH.43 The ε388 of free PLP in 0.2 M NaOH was measured by
using 40 μM and 80 μM PLP and was found to be ∼6525 M−1 cm−1.
All measurements were performed in triplicate.
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acschem-
bio.9b00030.
■
S
Additional materials and methods, supporting figures,
and supporting tables (PDF)
Accession Codes
The structures presented in this article have been deposited in
the PDB under accession codes (6CGQ) and (6NMX).
AUTHOR INFORMATION
Corresponding Authors
*W.W.M. E-mail: metcalf@illinois.edu.
*S.K.N. E-mail: snair@illinois.edu.
■
ORCID
Satish K. Nair: 0000-0003-1790-1334
William W. Metcalf: 0000-0002-0182-0671
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Author Contributions
⊥N.P. and M.A.O. contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
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■
We thank the staff at Life Sciences Collaborative Access Team
(LS-CAT, Argonne National Laboratory, Argonne, IL) for
facilitating data collection. We also thank K.S. Ju, M.N.
̃
Goettge, E. Parkinson, M.M. Lopez-Munoz, and K.K. Wang for
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(to S.K.N. and W.W.M.) and R01GM127659 (to W.W.M.).
́
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