Transition State Structure and Inhibition of Rv0091, a 5′-Deoxyadenosine/5′-methylthioadenosine Nucleosidase from Mycobacterium tuberculosis

Article abstract of DOI:10.1021/acschembio.6b00144

5′-Methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) is a bacterial enzyme that catalyzes the hydrolysis of the N-ribosidic bond in 5′-methylthioadenosine (MTA) and S-adenosylhomocysteine (SAH). MTAN activity has been linked to quorum sensing pathways, polyamine biosynthesis, and adenine salvage. Previously, the coding sequence of Rv0091 was annotated as a putative MTAN in Mycobacterium tuberculosis. Rv0091 was expressed in Escherichia coli, purified to homogeneity, and shown to be a homodimer, consistent with MTANs from other microorganisms. Substrate specificity for Rv0091 gave a preference for 5′-deoxyadenosine relative to MTA or SAH. Intrinsic kinetic isotope effects (KIEs) for the hydrolysis of [1′-³H], [1′-¹⁴C], [5′-³H₂], [9-¹⁵N], and [7-¹⁵N]MTA were determined to be 1.207, 1.038, 0.998, 1.021, and 0.998, respectively. A model for the transition state structure of Rv0091 was determined by matching KIE values predicted via quantum chemical calculations to the intrinsic KIEs. The transition state shows a substantial loss of C1′-N9 bond order, well-developed oxocarbenium character of the ribosyl ring, and weak participation of the water nucleophile. Electrostatic potential surface maps for the Rv0091 transition state structure show similarity to DADMe-immucillin transition state analogues. DADMe-immucillin transition state analogues showed strong inhibition of Rv0091, with the most potent inhibitor (5′-hexylthio-DADMe-immucillinA) displaying a K_i value of 87 pM.

Full text of DOI:10.1021/acschembio.6b00144

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Article
“
Transition state structure and inhibition of Rv0091, a 5#-deoxyadenosine /
5#-methylthioadenosine nucleosidase from Mycobacterium tuberculosis.”
Hilda A. Namanja-Magliano, Christopher F. Stratton, and Vern L. Schramm
ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.6b00144 • Publication Date (Web): 28 Mar 2016
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Transition state structure and inhibition of Rv0091,
a 5′ꢀdeoxyadenosine / 5′ꢀmethylthioadenosine nucleosidase from
Mycobacterium tuberculosis
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†
†
Hilda A. NamanjaꢀMagliano, Christopher F. Stratton, & Vern L. Schramm*
Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue,
Bronx, New York 10461, United States
†These authors contributed equally to this work.
ABSTRACT
5
′ꢀMethylthioadenosine/Sꢀadenosylhomocysteine nucleosidase (MTAN) is a bacterial enzyme
that catalyzes the hydrolysis of the Nꢀribosidic bond in 5′ꢀmethylthioadenosine (MTA) and Sꢀ
adenosylhomocysteine (SAH). MTAN activity has been linked to quorum sensing pathways,
polyamine biosynthesis, and adenine salvage. Previously, the coding sequence of Rv0091 was
annotated as a putative MTAN in Mycobacterium tuberculosis. Rv0091 was expressed in
Escherichia coli, purified to homogeneity, and shown to be a homodimer, consistent with
MTANs from other microorganisms. Substrate specificity for Rv0091 gave a preference for 5′ꢀ
deoxyadenosine relative to MTA or SAH. Intrinsic kinetic isotope effects (KIEs) for the
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3
15
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hydrolysis of [1′ꢀ H], [1′ꢀ C], [5′ꢀ H ], [9ꢀ N], and [7ꢀ N]MTA were determined to be 1.207,
2
1
.038, 0.998, 1.021, and 0.998, respectively. A model for the transition state structure of Rv0091
was determined by matching KIE values predicted via quantum chemical calculations to the
intrinsic KIEs. The transition state shows a substantial loss of C1′–N9 bond order, wellꢀ
developed oxocarbenium character of the ribosyl ring, and weak participation of the water
nucleophile. Electrostatic potential surface maps for the Rv0091 transition state structure show
similarity to DADMeꢀimmucillin transition state analogues. DADMeꢀimmucillin transition state
analogues showed strong inhibition of Rv0091, with the most potent inhibitor (5′ꢀhexylthioꢀ
DADMeꢀimmucillinA) displaying a K value of 87 pM.
i
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Mycobacterium tuberculosis is the causative agent of tuberculosis (TB), a leading cause of
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death among infectious diseases. In 2014, 9.6 million cases of TB were reported, and 1.5 million
people died of TBꢀrelated infections. Accordingly, the continued rise of multidrugꢀresistant,
extensively drugꢀresistant, and totally drugꢀresistant strains of M. tuberculosis has become a
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major public health concern. These challenges highlight the pressing need for new antiꢀ
tubercular therapies with novel targets and mechanism of action.
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′ꢀMethylthioadenosine/Sꢀadenosylhomocysteine nucleosidases (MTANs) are involved in
Sꢀadenosylmethionine (SAM)ꢀrelated pathways, including methylation, polyamine biosynthesis,
adenine salvage, the biosynthesis of quorum sensing autoinducers and radical reactions involving
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SAM. MTANs catalyze the hydrolysis of the Nꢀribosidic bond in 5′ꢀmethylthioadenosine
MTA), Sꢀadenosylhomocysteine (SAH), and 5′ꢀdeoxyadenosine (5′ꢀdAdo; Figure 1). The
(
absence of MTANs in human metabolism makes this protein class an appealing target for
antibacterials that function through blocking the quorumꢀsensing pathway or SAM recycling.
Figure 1. The MTAN reaction. MTANs catalyze the hydrolysis of the Nꢀribosidic bond of methylthioꢀ
adenosine (MTA), Sꢀadenosylhomocysteine (SAH), and 5′ꢀdeoxyadenosine (5′ꢀdAdo) to produce
methylthioribose (MTR), Sꢀribosylhomocysteine (SRH), and 5′ꢀdeoxyribose (5′ꢀdR), respectively. MTAN;
methylthioadenosine nucleosidase.
Previously, we reported models for the transition state (TS) structures of MTANs from
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Escherichia coli, Streptococcus pneumonia, and Neisseria meningitidis using a combination of
experimental kinetic isotope effect (KIE) measurements and computational methods. Intrinsic
KIEs report on bond vibrational changes between the ground state (GS) and TS of chemical
reactions, and can be used as boundary constraints on quantum chemical calculations to model
the structure of reactants at the TS. Such analyses provide information that can be leveraged in
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the design of chemically stable analogues that mimic features of the TS structure.
TS
analogues designed for the MTAN reaction function as extremely potent inhibitors with Kd
1
1ꢀ13
values in the picomolar (pM) and femtomolar (fM) range.
Importantly, these inhibitors have
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been shown to disrupt quorum sensing pathways in E.coli and Vibrio cholerae, and inhibit
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growth in Helicobacter pylori by blocking menaquinone biosynthesis.
The Rv0091 protein from M. tuberculosis was annotated previously as a putative biꢀ
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functional MTA/SAH nucleosidase. Herein, we report the expression, purification, and kinetic
characterization of Rv0091. We demonstrate the preferred substrate for Rv0091 is 5′ꢀdAdo and
the protein exhibits less efficient nucleosidase activity with MTA and SAH substrates. A model
of the Rv0091 TS structure for MTA hydrolysis was produced by matching KIE values predicted
via quantum chemical calculations to a family of intrinsic KIEs. This model reveals the Rv0091
TS structure and DADMeꢀimmucillin TS analogues share similar electrostatic distributions. The
potential of DADMeꢀimmucillin TS analogues to modulate Rv0091 activity was investigated via
in vitro inhibition assays.
RESULTS AND DISCUSSION
Oligomeric State and Substrate Specificity for Rv0091
A synthetic gene was designed for Rv0091 (NCBI GenBank: CCP42816.1) and purchased
from DNA2.0 Inc. in a pJexpress414 expression vector. Rv0091 was heterologously expressed in
2+
E. coli with an Nꢀterminal His6 tag and purified to homogeneity via Ni ꢀaffinity
chromatography. The purified protein was analyzed via SDSꢀPAGE to reveal a protein band of a
molecular weight consistent with the predicted monomer size for Rv0091 (29.4 kDa; Figure 2).
To determine the oligomeric state of Rv0091, the purified protein was treated with
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glutaraldehyde to enable intersubunit crosslinking. SDSꢀPAGE analysis of the crosslinked
protein revealed a band consistent with a dimeric oligomerization state (Figure 2). These data
are consistent with previous reports for MTANs from other organisms, which have been
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characterized as homodimers in solution.
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Figure 2. SDS-PAGE analysis of purified monomeric and crosslinked Rv0091. Rv0091 was purified
to homogeneity and SDSꢀPAGE analysis revealed a band consistent with the predicted mass (29.4 kD) of
the monomeric protein. The pure protein was treated with 0.1% to 2% glutaraldehyde to allow for interꢀ
subunit crosslinking. A band consistent with a dimeric oligomerization state was observed for the
crosslinked protein. Lane 1, protein molecular weight standards; Lane 2, Rv0091 + 0.1% glutaraldehyde;
Lane 3, Rv0091 + 0.5% glutaraldehyde; Lane 4, Rv0091 + 2% glutaraldehyde; Lane 5, Rv0091 before
crosslinking.
Recently,
a
5′ꢀmethylthioadenosine phosphorylase (MTAP) was identified in
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M. tuberculosis and M. smegmatis. MTAPs catalyze the phosphorolysis of MTA to adenine
and 5′ꢀmethylthioriboseꢀ1′ꢀphosphate. Prior to these reports, MTAPs were believed to be absent
from bacteria, and the biological reason for why M. tuberculosis and M. smegmatis express both
MTAP and MTAN is currently unknown. We explored the substrate specificity of Rv0091 by
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determining the kinetic constants with MTA, SAH, and 5′ꢀdAdo. These experiments indicate
the preferred substrate for Rv0091 is 5′ꢀdAdo, which displayed a specificity constant (k / K )
cat
M
7ꢀfold greater than MTA and 700ꢀfold greater than SAH (Table 1). MTA was reported as the
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preferred substrate for M. tuberculosis MTAP, and addition of phosphate to the Rv0091
reaction mixture did not enhance the rate of MTA hydrolysis. These data indicate Rv0091 does
not exhibit MTAP activity and suggests Rv0091 primarily functions as a 5′ꢀdAdo nucleosidase.
a
Table 1. Kinetic parameters for Rv0091 with 5′-dAdo, MTA, and SAH.
b
k
cat
ꢀ1
K
(µM)
M
kcat/ K
M
(M sec )
Substrate
ꢀ1
ꢀ1
(sec )
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′ꢀdAdo
MTA
SAH
0.47 ± 0.02
0.023 ± 0.001
0.004 ± 0.001 63.1 ± 7.0
10.9 ± 1.5
3.7 ± 0.4
43.4 × 103
6.3 × 10
3
0.06 × 10
a
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Parameters obtained using a luciferaseꢀbased assay at 25 ºC.
b
Adenosine and 2′ꢀdeoxyadenosine showed less substrate activity
than MTA and were not further characterized. 5′ꢀdAdo,
′ꢀdeoxyadenosine; MTA, 5′ꢀmethylthioadenosine; SAH,
Sꢀadenosylhomocysteine.
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The catalytic efficiencies (k / K ) for Rv0091 with 5′ꢀdAdo and MTA (Table 1) are lower
cat
M
6
–1
–1
than those of E. coli MTAN (kcatꢀMTA / KMꢀMTA = 2.3 x 10 M sec ; k
/ KMꢀ5´dAdo
=
catꢀ5´dAdo
6
–1
–1 23
0
.8 x 10 M sec ). The low catalytic efficiency of enzymes from M. tuberculosis, such as
Rv0091, has been attributed to the slow doubling time of M. tuberculosis (24 hrs) as compared to
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E. coli (20 min).
Determination of Intrinsic KIEs for MTA Hydrolysis
KIEs for the hydrolysis of MTA by Rv0091 were measured via the competitive radiolabel
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approach
using MTA substrates with isotopic labels incorporated at sensitive or remote
positions (Table 2). Isotope effects on enzymatic reactions measured via internal competition
provide V / K KIEs, which report on all steps from substrate binding, up to and including the first
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irreversible chemical step. The most valuable information for interrogating TS structure is
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derived from intrinsic KIEs, which reflect the chemical step alone. For a given isotope x, the
x
x
intrinsic KIE on an enzymatic reaction ( k) can be extracted from the V / K KIE using
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Northrop’s equation (eq 1), when forward commitment (C ), reverse commitment (C ), and the
f
r
x
equilibrium isotope effect ( K ) are known.
eq
(1)
As the Rv0091 reaction was irreversible under the conditions used to measure KIEs, C is
r
x
x
assumed to be zero. As such, eq 1 is reduced to eq 2 whereby k can be extracted from the V / K
KIE using C alone:
f
(2)
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The V / K and intrinsic KIEs for the hydrolysis of MTA by Rv0091 are reported in Table 2.
Experimental KIEs were corrected for remote effects to provide the V / K KIEs, and intrinsic
values were determined using eq 2 where C = 0.079. The value of C for Rv0091 was measured
f
f
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experimentally using the substrate trapping method developed by Rose, as previously described
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(Figure S1).
Table 2. Kinetic isotope effects for the hydrolysis of MTA by Rv0091.
Heavy
MTA
Light
MTA
Type of
KIE
primary
-secondary
primary
-secondary
remote
V / K
KIEs
1.035 ± 0.004
1.194 ± 0.009b
1.019 ± 0.006b
0.998 ± 0.004
0.998 ± 0.008
Intrinsic
KIEs
Calculated
c
d
KIEs
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b
[
1′ꢀ C]
[5′ꢀ H ]
1.038 ± 0.005
1.207 ± 0.010
1.021 ± 0.007
0.998 ± 0.005
0.998 ± 0.009
1.039
1.209
1.023
0.985
2
3
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a
[
1′ꢀ H]
[5′ꢀ C]
α
1
5
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3
[9ꢀ N, 5′ꢀ C]
[5′ꢀ H ]
2
1
5
14
3
[7ꢀ N, 5′ꢀ C]
[5′ꢀ H ]
β
2
3
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[5′ꢀ H ]
[5′ꢀ C]
0.990
2
a
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b
3
The KIE on the remote [5′ꢀ C] label is assumed to be unity. Experimental values were corrected for the remote [5′ꢀ H ]
2
3
c
KIE using the expression KIE = KIE × [5′ꢀ H ] KIE. Intrinsic KIEs were determined by correcting V / K values using eq
obs
2
d
2
assuming C = 0.079. Calculated values (Gaussian 09, RB3LYP/6ꢀ31g(d) theory) corresponds to those for the final TS
f
model shown in Figure 4.
The primary C1′ KIE for N-ribosyltransferases is sensitive to atomic motion along the
reaction coordinate, and provides information for differentiating S 1 and S 2 reactions. For a
N
N
fully dissociative S 1 reaction, with complete loss of bond order to the leaving group and no
N
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participation of the incoming nucleophile at the TS, a primary 1′ꢀ C KIE near unity would be
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predicted. For S 1 mechanisms in which partial bond order to either the leaving group or the
N
1
4
nucleophile is retained at the TS, 1′ꢀ C KIEs would range from 1.01–1.03. By contrast, primary
1
4
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1′ꢀ C KIEs for associative S 2 mechanisms range from 1.08 to 1.13. The 1′ꢀ C KIE measured
N
for MTA hydrolysis by Rv0091 was 1.038 ± 0.005 (Table 2), which suggests either the N9 of
the adenine leaving group and/or the oxygen of the nucleophilic water (Ow) retain partial bond
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order with the C1′ position at the TS. The primary 9ꢀ N KIE reports on loss of C1′–N9 bond
order and rehybridization of the N9 position. A value of 1.021 ± 0.007 was obtained for the
1
5
primary 9ꢀ N KIE on the Rv0091 reaction (Table 2), which indicates the C1′–N9 bond
undergoes significant, but not full, loss of bond order at the TS.
The αꢀsecondary 1′ꢀH KIE for Nꢀribosyltransferases reports directly on changes in
hybridization at the C1′ position, and reflects the degree of oxocarbenium character retained by
2
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the ribose ring in the TS structure. A highly dissociative TS, with considerable oxocarbenium
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character, will typically display 1′ꢀ H KIEs in the range of 1.06 to 1.22. By contrast, an
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associative TS with less oxocarbenium character will display 1′ꢀ H KIE values ranging from
3
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.00 to 1.03. The 1′ꢀ H KIE measured for hydrolysis of MTA by Rv0091 was 1.207 ± 0.010
(
Table 2), which suggests a TS structure with significant oxocarbenium character. Finally, the
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βꢀsecondary 7ꢀ N KIE, which reports on the protonation state at the N7 position of adenine, was
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measured as 0.998 ± 0.005. Previous studies suggest intrinsic 7ꢀ N KIEs near unity for the
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MTAN reaction indicate protonation of the N7 position at the TS.
Computational Models of the Rv0091 TS Structure
To investigate the mechanism of MTA hydrolysis by Rv0091, we developed a model of the
Rv0091 TS structure using intrinsic KIEs as experimental boundaries for density functional
theory (DFT) calculations. As there is no structural information available for the conformation of
MTA bound to Rv0091, the input geometry for the TS structure search was taken from the
2
0
coordinates for 5′ꢀmethylthiotubercidin bound to E. coli MTAN (PDB: 1NC1). A family of
energyꢀminimized TS structures was generated by constraining the C1′–N9 bond distance along
the reaction coordinate as detailed below. Predicted KIEs were calculated (Gaussian 09,
2
9
RB3LYP/ 6ꢀ31g(d) theory) from the scaled vibrational frequencies of the optimized structures
for MTA in the GS and at the TS. The optimized structure of MTA in the GS was generated
without constraints using water as an implicit solvent (polarizable continuum model) and was
identical for all KIE calculations.
5
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Previous studies on the TS structures of MTAN variants from E. coli, S. pneumonia, and
7
N. meningitides found MTA hydrolysis proceeds through a D *A process wherein C1′–N9
N
N
bond cleavage and C1′–Ow bond formation occur in a stepwise manner (Figure 3). Accordingly,
initial efforts to model the Rv0091 TS structure considered two possible D *A manifolds: (1) a
N
N
‡
N
D *A mechanism in which cleavage of the C1′–N9 ribosidic bond to form an oxocarbenium
N
‡
intermediate is rateꢀlimiting, and (2) a D *AN mechanism in which the oxocarbenium
N
intermediate is in equilibrium with MTA and nucleophilic attack of water is rateꢀlimiting
(Figure 3).
‡
N
To examine the D *A mechanism, TS structures were generated by fixing the C1′–N9
N
bond of MTA at increasing lengths from 1.6 to 3.0 Å in 0.2 Å increments. Predicted KIEs were
calculated and TS structures yielding close matches to the intrinsic values were refined by
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varying the C1′–N9 bond along the reaction coordinate in 0.01 Å steps. The TS structure
providing the closest match to the intrinsic KIEs was identified at a C1′–N9 distance of 2.78 Å,
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although this model matched only the primary 1′ꢀ C KIE within experimental error (Figure S2).
The influence of charge accumulation on the leaving group was investigated by a parallel set of
calculations to those detailed above in which the N7 position of adenine was protonated at the
TS. Protonation at the N7 position activates the adenine for scission of the C1′–N9 bond by
neutralizing negative charge buildꢀup on the leaving group, and is a common TS feature among
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N-ribosyltransferases, including E. coli and N. meningitides MTANs. In this TS structure
search, the best match to intrinsic KIEs was found at a C1′–N9 distance of 2.42 Å (Figure S3).
Relative to the TS structures in which adenine was modeled as an anionic leaving group, the TS
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4
structure including N7 protonation provided a better match to intrinsic KIEs. The primary 1′ꢀ C
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and 9ꢀ N KIEs were well within experimental error of the intrinsic values, and the αꢀsecondary
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′ꢀ H and βꢀsecondary 7ꢀ N were just outside these boundaries (Figure S3).
‡
N
The feasibility of a D *A mechanism was also considered, in which the oxocarbenium
N
intermediate is in equilibrium with MTA prior to the rateꢀlimiting addition of water (Figure 3).
‡
N
For D *A mechanisms, observed KIEs are the product of the equilibrium isotope effect (EIE)
N
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8
for the first step and the KIE for the second step (Figure 3). The EIEs for oxocarbenium
formation were calculated for both neutral (N7 protonated) and anionic (N7 deprotonated) forms
of adenine (Figure S4). Predicted KIEs were then calculated for TS structures involving
nucleophilic attack of water on the oxocarbenium intermediate with C1′–Ow bond distances
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5
from 1.7 to 2.7 Å. Interestingly, the predicted 9ꢀ N EIEs for both neutral and anionic forms of
adenine were within experimental error of the intrinsic KIEs (Figure S4). However, the product
of the EIEs and KIEs (for the nucleophilic attack of water) did not agree with the intrinsic values
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for the 1′ꢀ C or 1′ꢀ H KIEs (Figure S4).
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Figure 3. General mechanisms for the hydrolysis of MTA by Rv0091. The Rv0091 reaction is
represented as two possible mechanisms in which the transformations are considered as their elementary
steps. For MTA hydrolysis, the A step refers to the association of the water nucleophile and the D step
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N
refers to dissociation of the adenine leaving group. If the reaction proceeds through a concerted
bimolecular TS, the mechanism is termed A D . If dissociation of the leaving group precedes association
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N
of the nucleophile and the reaction proceeds through a stepwise process via discrete transition states, the
reaction is termed D *A . For D *A processes, a superscript “‡” is used to denote the rateꢀlimiting step,
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N
N
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‡
e.g., D *A or D *A . MTA, 5′ꢀmethylthioadenosine; MTR, 5′ꢀmethylthioribose.
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N
The preliminary D *A model for MTA hydrolysis by Rv0091 (Figure S3) was refined
N
through addition of low bond order to the nucleophilic water. The TS structure providing the best
match to intrinsic values (Table 2) had a C1′–N9 distance of 2.45 Å and a C1′–Ow distance of
2.70 Å (Figure 4). Interestingly, the C1′–Ow distance of 2.70 Å in this TS model is similar to
the distance between the 1′ position of the aminoribitol ring and the bound active site water
(2.75 Å) in the binary complex of 5′ꢀmethylthioꢀDADMeꢀimmucillinA (MTꢀDADMeꢀImmA)
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and E. coli MTAN (PDB: 1Y6Q; Figure S5). The slight discrepancy between the predicted
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ꢀ N KIE and corresponding intrinsic value (Table 2) likely results from hydrogen bonding
interactions with Asp220, which is predicted by homology modeling to interact with the N7
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position of adenine , but was not included in this TS model. This final model is most consistent
with a dissociative A D TS displaying significant loss of C1′–N9 bond order, low bond order to
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N
the incoming nucleophile, and substantial oxocarbenium character of the ribosyl ring. The
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C1′–N9 distance of 2.45 Å corresponds to a 0.74 loss of bond order at the TS, and the
nucleophilic water shares a bond order of 0.074 with C1′ (Table S1). Oxocarbenium character of
the ribosyl ring at the TS is supported by a short O4′–C1′ bond (1.42 Å in MTA versus 1.27 Å at
the TS), which corresponds to an increase of 0.468 in bond order relative to the GS (Table S1).
In addition, natural bond orbital (NBO) analysis indicates the relative positive charge increases
by 0.182 for C1′ and 0.178 for O4′ at the TS (Table S2).
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Figure 4. Calculated model for the Rv0091 TS structure. TS structure model of MTA hydrolysis
catalyzed by Rv0091 was generated by matching KIEs predicted for calculated transition states
(
RB3LYP/6ꢀ31g(d) theory) to intrinsic KIEs. Features of the Rv0091 TS structure include a C1′–N9 bond
distance of 2.45 Å, a C1′–Ow bond distance of 2.70 Å, protonation at the N7 position of adenine, and
significant oxocarbenium character. TS, transition state; MTA, 5′ꢀmethylthioadenosine; KIEs, kinetic
isotope effects; Ow, water.
Previous reports indicate the MTAN reaction for other species proceeds via highly
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dissociative (E. coli and S. pneumonia ) and early (N. meningitides ) D *A transition states
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(Figure 3). As such, inclusion of the nucleophilic water in the Rv0091 TS model (Figure 4)
distinguishes it from that of other MTAN variants, though dissociative A D mechanisms are
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common among Nꢀribosyltransferases.
A feature of note in the Rv0091 TS model is the
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Ow–C1´–N9 angle is 150.4º rather than the 180º (Figure 4), which would be predicted for
optimal orbital overlap in a pure S 2 reaction. Accordingly, the partial bond orders present in
N
this model dictate a nonꢀlinear TS for the most favorable orbital overlap. The term “nucleophileꢀ
assisted S 1” has been invoked to describe S 1 mechanisms in which low bond order must be
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included to the nucleophile in order to match predicted KIEs with experimental values. As the
C1′–Ow bond order is low (0.074; Table S1) in the Rv0091 TS model, it is plausible the active
site water could be preassociated in the TS structure via stabilizing interactions with the
oxocarbenium species, but not directly participating in a concerted displacement reaction.
Preassociation of a nucleophilic water within electronꢀreorganization distance of the C1′ reactive
center is consistent with mass spectrometry experiments carried out with MTꢀDADMeꢀImmA
and E. coli MTAN, where the enzyme, inhibitor, and nucleophilic water remain associated in the
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gas phase.
TS Analogues are Picomolar Inhibitors of Rv0091
The firstꢀ and secondꢀgeneration immucillins were developed as TS analogue inhibitors for
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Nꢀribosyltransferase reactions.
We sought to compare the electrostatics of the Rv0091 TS
structure with those of 5′ꢀmethylthioꢀimmucillinA (MTꢀImmA; a first generation analogue) and
MTꢀDADMeꢀImmA (a second generation analogue; Figure 5). EPS maps extrapolated from
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singleꢀpoint energy calculations (RB3LYP/6ꢀ31g(d) theory) are presented in Figure 5 for the
Rv0091 TS model, and optimized structures of MTꢀImmA and MTꢀDADMeꢀImmA.
Comparison of the EPS maps reveals the geometry and charge localization observed in the
Rv0091 TS (Figure 5a) is more similar to that of MTꢀDADMeꢀImmA (Figure 5b) than to MTꢀ
ImmA (Figure 5c). Consistent with these models, the K for MTꢀDADMeꢀImmA with Rv0091
i
(1.5 nM) was 56ꢀfold more potent than that of MTꢀImmA (K = 85 nM).
i
DADMeꢀImmA TS analogues inhibit MTAN activity in the pM – fM range, and display
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increased binding affinity when functionalized with hydrophobic groups at the C5′ position.
Crystallographic studies of DADMeꢀImmA TS analogues with MTANs from Salmonella
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enterica and H. pylori revealed an elongated hydrophobic binding pocket proximal to the C5′
substituent of the bound immucillin scaffolds. Consistent with these findings, the most potent
inhibition of Rv0091 was observed with DADMeꢀImmA inhibitors bearing long alkyl groups at
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the C5′ position (Figure 6). For example, hexyl thioether 3 inhibited Rv0091 activity with a Ki
of 87 pM, whereas methyl thioether 1 and butyl thioether 2 both displayed K values in the low
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Figure 5. EPS maps for Rv0091 TS structure and immucillin TS analogues. EPS maps (red = partial
negative; blue = partial positive) were extrapolated from singleꢀpoint energy calculations (RB3LYP/6ꢀ
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31g(d) theory) and visualized in GaussView 5.0 (isovalue = 0.04). (a) Energyꢀminimized structure and
EPS map for the TS structure of MTA hydrolysis by Rv0091. (b) Energyꢀminimized structure and EPS
map for MTꢀDADMeꢀImmA. (c) Energyꢀminimized structure and EPS map for MTꢀImmA. EPS,
electrostatic potential surface; TS, transition state; MTA, 5′ꢀmethylthioadenosine; MTꢀDADMeꢀImmA, 5′ꢀ
methylthioꢀDADMeꢀimmucillinꢀA; MTꢀImmA, 5′ꢀmethylthioꢀimmucillinꢀA.
Overall, DADMeꢀImmA TS analogues inhibit Rv0091 less potently than they do other
MTANs. For example, the K values for MTꢀDADMeꢀImmA with E. coli MTAN and Rv0091
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are 2 pM and 1.5 nM (Figure 6), respectively. This difference in binding affinity for
MTꢀDADMeꢀImmA is anticipated from the lower catalytic activity of Rv0091 relative to E. coli
MTAN. Catalytic rate enhancement has been correlated with the affinity of TS analogues for
4,41,42
their enzyme targets by applying the Wolfenden approximation.
As such, the attenuated
binding of DADMeꢀImmA inhibitors with Rv0091 is consistent with the slow turnover of
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Rv0091 indicated by k . In addition, the DADMeꢀImmA scaffold was designed to mimic
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Nꢀribosyltransferase reactions with highly dissociative TS structures.
The crystal structure of
MTꢀDADMeꢀImmA in binary complex with E. coli MTAN reveals a distance of 2.8 Å between
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the N1′ position of the 3′ꢀhydroxypyrrolidine ring and C9 of the 9ꢀdeazaadenine moiety. This
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inhibitor geometry is a good match to the TS structure of E. coli MTAN wherein adenine is
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.0 Å from C1′. By comparison, the Rv0091 TS structure is somewhat earlier with a C1′–N9
distance of 2.45 Å (Figure 4). Moreover, the protonated aminoribitol scaffold of the DADMeꢀ
ImmA analogues (Figure 6) is designed to mimic the oxocarbenium species resulting from near
full dissociation of adenine. The earlier TS of Rv0091 (relative to E. coli MTAN) would result in
less electropositive charge at the C1′ position, which may not be as accurately mimicked by the
DADMeꢀImmA scaffold. The EPS maps for the Rv0091 TS structure (Figure 5a) and MTꢀ
DADMeꢀImmA (Figure 5b) reveal greater positive charge is localized at the 1′ position of the
inhibitor. Accordingly, differences in TS geometry and electrostatics between E. coli MTAN and
Rv0091 may also contribute to the lower potency observed for DADMeꢀImmA inhibitors with
the Rv0091 protein.
Figure 6. Inhibition of Rv0091 with DADMe-ImmA TS analogues. Inhibition of Rv0091 activity was
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monitored at 305 nm via a coupled assay with xanthine oxidase in which the adenine product was
converted to 2,8ꢀdihydroxyadenine. The K values were calculated using the Morrison equation for tightꢀ
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binding inhibitors.
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The TDR M. tuberculosis gene function library classifies Rv0091 as a nonꢀessential gene.
This is consistent with the assignment of Rv0091 as a putative MTAN with roles in quorum
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sensing and SAM recycling. With the function of Rv0091 now defined as a 5′ꢀdAdo hydrolase,
the in vivo activity of Rv0091 is more likely coupled to radical SAM reactions that act to
generate radical reaction centers, with 5′ꢀdAdo and methionine as products. Radical SAM
47
enzymes are recognized by the CX CX C motif, and 21 of these proteins have been annotated
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in the M. tuberculosis genome. Preliminary growth experiments with M. tuberculosis indicate
MTꢀDADMeꢀimmucillinA (3) and butylthioꢀDADMeꢀimmucillinA (4) have no effect on growth
of cultured cells.
CONCLUSION
The Rv0091 protein from M. tuberculosis, previously annotated as a putative MTAN, exists
as a dimer in solution and displays substrate specificity for 5′ꢀdAdo relative to MTA and SAH.
These findings suggests the protein is not crucial in quorum sensing pathways, but more likely
plays a role in reactions involving SAM radical enzymes. Intrinsic KIE measurements and
quantum chemical calculations were used to establish a model for the TS structure of Rv0091.
The TS for Rv0091 differs from the transition states of previously reported MTANs. The
Rv0091 TS is characterized by significant decrease in C1′–N9 bond order, substantial
oxocarbenium character of the ribosyl ring, and weak participation of the water nucleophile.
DADMeꢀImmA TS analogues resemble the Rv0091 TS structure and inhibit Rv0091 activity
–
9
–11
with K values in the range of 10 to 10 M. The most potent inhibitor of Rv0091 activity
i
identified in this study was hexylꢀthioꢀDADMeꢀimmucillinA, which exhibited a K of 87 pM.
i
Studies to investigate the in vivo role of Rv0091 in mycobacteria, as well as the potential for
DADMeꢀImmA TS analogues to function as modulators of virulence in M. tuberculosis, are
ongoing.
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METHODS
Full details for all experimental methods are provided in the Supporting Information.
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ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at DOI:
XX.XXXX/acschembio.XXXXX.
Supporting Information Figure S1–S5 and Supporting Information Tables S1–S2.
Complete experimental procedures for expression and purification of Rv0091; kinetic
assays and oligomeric state determination of Rv0091; synthesis of isotopically labeled
MTA substrates; measurement of KIEs and forward commitment; computational
analyses; and determination of inhibition constants.
AUTHOR INFOMRATION
Corresponding Author
*Phone: 718ꢀ430ꢀ2813. Eꢀmail: vern.schramm@einstein.yu.edu.
Author Contributions
†
These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGEMENTS
We thank M. Poulin (Einstein), Z. Wang (Einstein), and A. Gizzi (Einstein) for helpful
discussions. The inhibitor molecules were a generous gift of P. C. Tyler and G. B. Evans from
the Ferrier Research Institute, Victoria University of Wellington, New Zealand. This work was
supported by research grant GM041916 and training grant T32AI070117 from the National
Institutes of Health.
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(
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