P. J. Tonge, D. S. Tan et al.
MenB, the next downstream enzyme in the menaquinone bio-
synthesis pathway (Scheme 1).[8,71,79] This coupled assay is
based on that described earlier for evaluating the inhibition of
MenB, except that the concentrations of MenE and MenB are
adjusted to ensure that the MenE-catalyzed reaction is rate-
limiting. Assays for saMenE and mtMenE utilized M. tuberculosis
MenB (mtMenB) as the coupling enzyme, while ecMenE was
assayed with E. coli MenB (ecMenB). ecMenE, ecMenB, and
mtMenB were expressed and purified as described previous-
ly,[8,79] while saMenE and mtMenE were cloned and expressed
with N-terminal His6 tags in E. coli (BL21) cells, then purified to
homogeneity using nickel-affinity chromatography (see the
Supporting Information for full details). Reactions were initiat-
ed by adding MenE (final concentration 50–100 nm) to a solu-
tion containing MenB (5–10 mm), ATP (240 mm), CoA (240 mm),
OSB (120–240 mm) and inhibitor (0–200 mm). Formation of
DHNA-CoA was monitored at 392 nm, and IC50 values were de-
termined by fitting the initial velocity data to the standard
dose response equation (Table 1).[71]
3 of the enzyme concentrations used in the assay, thus meet-
ing the experimental criterion for tight-binding inhibitors.[80] To
provide additional information on the mechanism of enzyme
inhibition, Kai pp values were determined using the Morrison
equation[81,82] as a function of substrate concentration to pro-
vide the absolute Ki values for enzyme inhibition. Sulfamate 4
was found to be a competitive inhibitor of mtMenE with re-
spect to ATP (Ki =5.4Æ0.1 nm) and a noncompetitive inhibitor
with respect to OSB (Ki =11.2Æ0.9 nm). These data are consis-
tent with the knowledge that mtMenE follows a Bi Uni Uni Bi
Ping-Pong kinetic mechanism in which the addition of ATP and
OSB is ordered, with ATP binding first (data not shown). A simi-
lar experiment demonstrated that 4 is a competitive inhibitor
of ecMenE with respect to OSB with a Ki value of 128Æ5 nm),
consistent with the knowledge that OSB binds first to ecMenE
(data not shown). Such a mechanism is consistent with studies
on other ligase enzymes.[72,83,84] Although the dependence of
Kai pp on substrate concentration was not determined for the
inhibition of saMenE by 4, fitting the IC50 data to the Morrison
equation gave a value for Kai pp of 22Æ8 nm.
We were gratified to find that the free carboxylate ana-
logues 4–6 proved to be excellent inhibitors of all three en-
Active site recognition of OSB-AMP and MenE in-
hibitors
Table 1. Inhibition of the MenE enzymes from M. tuberculosis, S. aureus, and E. coli.[a]
IC50 [mm] of MenE from
The increased potency of the aromatic carboxylate
analogues 4–6 compared to all previously reported
MenE inhibitors suggests that the OSB carboxylate
functionality may be recognized specifically by one
or more basic side chains in the active site. While
cocrystal structures of MenE with substrates or inhibi-
tors have not yet been reported, a crystal structure
of the unliganded form of saMenE (PDB ID: 3IPL) has
been deposited in the Protein Data Bank by the New
York Structural Genomics Research Center.[87] We
identified the putative active site in saMenE by com-
parison to two other acyl-CoA synthetases that have
been crystallized with their cognate acyl-AMP inter-
mediates bound (Figure 1).[85,86] This binding site is
also conserved across other members of the ANL
family.[30,88–93] Upon examination of residues within
12 ꢁ of the center of this binding pocket, we identi-
fied a basic residue, Arg222, that may interact with
the aromatic carboxylate of OSB (Figure S1A and B in
the Supporting Information). Notably, this residue
Inhibitor
M. tuberculosis
S. aureus
E. coli
1, MeOSB-AMS
2, MeOSB-AMSN
3, MeOSB-AVSN
14.2Æ3.3
23.5Æ1.0
117Æ12
24.6Æ3.5
38.0Æ3.0[b]
>200
45.7Æ2.8
34.1Æ2.8[b]
5.7Æ0.7[b]
4, OSB-AMS
5, OSB-AMSN
6, OSB-AVSN
0.049Æ0.007[c]
0.060Æ0.005[d]
0.24Æ0.01
0.21Æ0.16[e]
0.63Æ0.14
0.57Æ0.06
0.20Æ0.02
0.16Æ0.05
0.33Æ0.05
8, MeOCPB-AMSN
9, MeOCPB-AVSN
>200
>200
>200
>200
>200
>200
11, OCPB-AMSN
12, OCPB-AVSN
101Æ14
106Æ10
85Æ17
n.d.[f]
31.6Æ5.5
54.4Æ2.3
[a] Assays were performed with mtMenE (50 nm), saMenE (100 nm), or ecMenE
(100 nm). [b] Data from ref. [71]. [c] Competitive inhibitor with respect to ATP (Ki =
5.4Æ0.1 nm) and noncompetitive inhibitor with respect to OSB (Ki =11.2Æ0.9 nm).
[d] Kai pp =22Æ8 nm. [e] Competitive inhibitor with respect to OSB (Ki =128Æ5 nm).
[f] n.d.=not determined.
zymes (Table 1), and were more potent than the original
methyl ester analogues 1–3. Notably, the sulfamate 4 (OSB-
AMS) was the most potent inhibitor in the carboxylate series
for all three enzymes, in contrast to the mixed structure-activi-
ty relationship trends observed for the methyl ester series.[71]
Further, while neither of the des-keto methyl ester analogues 8
or 9 exhibited appreciable activity, the corresponding des-keto
carboxylates 11 and 12 were moderate inhibitors. Thus, dele-
tion of the OSB ketone functionality appears to result in de-
creased inhibitory activity by approximately 2–3 orders of mag-
nitude (5 vs. 11, 6 vs. 12).
was not readily identified in sequence alignments guided by
the other acyl-CoA synthetase structures, because it lies one
extra turn toward the C terminus of helix B3/4 compared to
binding pocket residues in the previously determined struc-
tures (data not shown).
To evaluate this hypothesis, AutoDock 3.05[94] was used to
generate an enzyme–ligand complex of OSB-AMP bound to
saMenE. Importantly, the OSB carboxylate is within 5 ꢁ of
Arg222 in this docked structure. Similarly, when OSB alone is
docked into the active site, it places the aromatic carboxylate
within 3 ꢁ of Arg222 (Figure S1C and D). In addition, Ser302 is
within 3 ꢁ of the OSB ketone oxygen in both docked struc-
tures, suggesting a possible hydrogen-bonding interaction.
The IC50 values for the inhibition of M. tuberculosis mtMenE,
saMenE, and ecMenE by 4 (OSB-AMS) are within a factor of 2–
132
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ChemBioChem 2012, 13, 129 – 136