enol functionality in contributing to the nucleophilicity of the
double bond of PEP in the enzyme-catalysed reaction.
to those found in PEP were found to be effective inhibitors,
whilst the sulfate-containing SEP was ineffective. Interestingly,
fosmidomycin, bearing both a formylhydroxamate and phospho-
nate group, was also found to inhibit DAH7PS. The inhibitory
potency of compounds against DAH7PS was found to be sensitive
to minor variations in compound structure, with changes such
as stereochemical inversion and alkene isomerisation providing
large differences in binding affinity. The further investigation of
these compounds against DAH7P synthase and KDO8P synthase
continues in our laboratory, in addition these results are guiding
the design of more potent DAH7P synthase inhibitors.
By examining the proposed mechanism of DAH7P synthase,
and focussing on the enzyme’s PEP subsite, this study has
discovered several molecules that inhibit DAH7P synthase.
The most potent of these molecules, vinyl phosphonate 4
(Ki: 4.7 mM) was designed to mimic the geometry and bond order
of the PEP-portion of the proposed oxocarbenium intermediate.
The success of this mechanism-based approach can be verified by
comparison with the isomeric allylic phosphonate 8, a substrate-
based mimic of PEP with a much weaker ability to inhibit DAH7P
synthase (Ki: 270 mM). While this difference in binding affinity
between these two compounds has been ascribed here to the
mimicry of the oxocarbenium ion intermediate 1, the potency of
vinyl phosphonate 4 may also have an entropic contribution. Vinyl
phosphonate 4, by virtue of its (E)-configured alkene, is locked into
a similar conformation to that of PEP in several DAH7P synthase
X-ray crystal structures. Due to this conformational locking, the
binding of vinyl phosphonate 4 to DAH7P synthase perhaps incurs
a smaller entropic disadvantage than the binding of more flexible
phosphonates such as allylic phosphonate 8.41
Notes and references
1 R. Bentley, Crit. Rev. Biochem. Mol. Biol., 1990, 25, 307–384.
2 J. R. Coggins, C. Abell, L. B. Evans, M. Frederickson, D. A. Robinson,
A. W. Roszak and A. P. Lapthorn, Biochem. Soc. Trans., 2003, 31,
548–552.
3 M. D. Toscano, M. Frederickson, D. P. Evans, J. R. Coggins, C. Abell
and C. Gonzalez-Bello, Org. Biomol. Chem., 2003, 1, 2075–2083.
4 E. M. M. Bulloch, M. A. Jones, E. J. Parker, A. P. Osborne, E. Stephens,
G. M. Davies, J. R. Coggins and C. Abell, J. Am. Chem. Soc., 2004,
126, 9912–9913.
The enantiomeric phospholactates (R)-6 and (S)-6 were chosen
as potential mimics of the proposed phosphohemiketal interme-
diate 2. The stereochemistry of the lactates had a strong effect on
inhibition, with the (S)-isomer displaying a greater than 10-fold
larger inhibition constant than its (R)-counterpart. The origin of
this significant difference in affinity of the two stereoisomers for
DAH7P synthase is intriguing, and is expected to be related to
configuration at C2 of the phosphohemiketal intermediate 2. This
effect is under further investigation in our laboratory.
5 I. A. Shumilin, R. H. Kretsinger and R. H. Bauerle, Structure
(London), 1999, 7, 865–875.
6 I. A. Shumilin, R. Bauerle and R. H. Kretsinger, Biochemistry, 2003,
42, 3766–3776.
7 T. Wagner, I. A. Shumilin, R. Bauerle and R. H. Kretsinger, J. Mol.
Biol., 2000, 301, 389–399.
8 I. A. Shumilin, R. Bauerle, J. Wu, R. W. Woodard and R. H. Kretsinger,
J. Mol. Biol., 2004, 341, 455–466.
9 L. R. Schofield, B. F. Anderson, M. L. Patchett, G. E. Norris, G. B.
Jameson and E. J. Parker, Biochemistry, 2005, 44, 11950–11962.
10 C. J. Webby, H. M. Baker, J. S. Lott, E. N. Baker and E. J. Parker,
J. Mol. Biol., 2005, 353, 927–939.
Based on the superficial similarities between DXR substrate
DXP and the DAH7P synthase substrate E4P, we trialled fos-
midomycin as an inhibitor of DAH7P synthase. Fosmidomycin
was successful as an inhibitor of DAH7P synthase; however,
surprisingly fosmidomycin was a competitive inhibitor of DAH7P
synthase with respect to PEP, with a relatively potent binding
constant of 35 mM. Given the approximately 1000-fold poorer
inhibition of DAH7P synthase by fosmidomycin compared with
DXR, it seems likely that the inhibition of DAH7P synthase
by fosmidomycin is of little consequence to the anti-microbial
effects of fosmidomycin in vivo. The competitive inhibition of
DAH7P synthase by fosmidomycin with relation to PEP suggests
fosmidomycin occupies the PEP binding region of the active site,
rather than the E4P binding region as initially hypothesised. The
observed metal dependence of this inhibition may suggest that
fosmidomycin is also capable of interacting with the metal cofactor
of the DAH7P synthase active site.
The failure of sulfoenolpyruvate 7 to act as either a substrate
or inhibitor of DAH7P synthase is a surprising finding. One
major difference between PEP and SEP is the charge state of
each compound; whereas PEP can be expected to be in both di-
or tri-anionic forms at pH 6.8, sulfoenolpyruvate can only be a
dianion. The failure of SEP to inhibit or be processed by DAH7P
synthase may suggest that PEP and analogues bind to DAH7P
synthase exclusively as trianionic species.
11 V. Konig, A. Pfeil, G. H. Braus and T. R. Schneider, J. Mol. Biol., 2004,
337, 675–690.
12 G. Y. Sheflyan, D. L. Howe, T. L. Wilson and R. W. Woodard, J. Am.
Chem. Soc., 1998, 120, 11027–11032.
13 R. M. Williamson, A. L. Pietersma, G. B. Jameson and E. J. Parker,
Bioorg. Med. Chem. Lett., 2005, 15, 2339–2342.
14 M. Ahn, A. L. Pietersma, L. R. Schofield and E. J. Parker, Org. Biomol.
Chem., 2005, 3, 4046–4049.
15 D. L. Howe, A. K. Sundaram, J. Wu, D. L. Gatti and R. W. Woodard,
Biochemistry, 2003, 42, 4843–4854.
16 C. Furdui, L. Zhou, R. W. Woodard and K. S. Anderson, J. Biol. Chem.,
2004, 279, 45618–45625.
17 A. B. DeLeo and D. B. Sprinson, Biochem. Biophys. Res. Commun.,
1968, 32, 873–877.
18 D. K. Onderka and H. G. Floss, Biochem. Biophys. Res. Commun.,
1969, 35, 801–804.
19 D. K. Onderka and H. G. Floss, J. Am. Chem. Soc., 1969, 91, 5894–
5896.
20 H. G. Floss, D. K. Onderka and M. Carroll, J. Biol. Chem., 1972, 247,
736–744.
21 S. R. Walker and E. J. Parker, Bioorg. Med. Chem. Lett., 2006, 16,
2951–2954.
22 L. Kaustov, S. Kababya, V. Belakhov, T. Baasov, Y. Shoham and A.
Schmidt, J. Am. Chem. Soc., 2003, 125, 4662–4669.
23 V. Belakhov, E. Dovgolevsky, E. Rabkin, S. Shulami, Y. Shoham and
T. Baasov, Carbohydr. Res., 2004, 339, 385–392.
24 X. Xu, J. Wang, C. Grison, S. Petek, P. Coutrot, M. R. Birck, R. W.
Woodard and D. L. Gatti, Drug Design and Discovery, 2003, 18, 91–99.
25 J. Wang, H. S. Duewel, R. W. Woodard and D. L. Gatti, Biochemistry,
2001, 40, 15676–15683.
26 V. D. Romanenko and V. P. Kukhar, Chem. Rev. (Washington, DC,
US), 2006, 106, 3868–3935.
27 T. Kuzuyama, T. Shimizu, S. Takahashi and H. Seto, Tetrahedron Lett.,
1998, 39, 7913–7916.
In conclusion, the ability of the PEP site of DAH7P synthase
to bind a series of inhibitors of varying structure has been
investigated. Inhibitors bearing both a carboxylate group and
a phosphonate or phosphate separated by a similar distance
28 U. Wong and R. J. Cox, Angew. Chem., Int. Ed., 2007, 46, 4926–
4929.
3034 | Org. Biomol. Chem., 2009, 7, 3031–3035
This journal is
The Royal Society of Chemistry 2009
©