Nathaniel J. Cosper1, David L. Bienvenue2, Jacob E.
Shokes1, Danuta M. Gilner2, Takashi
Tsukamoto3, Robert A. Scott1, and Richard C.
Holz2
1Department of Chemistry, University of Georgia, Athens, Georgia
30602-2556
Bacterial infections, such as tuberculosis, have been identified as a
world-wide problem leading to the deaths of millions of people each year. The
importance of developing new drugs to fight infectious disease caused by these
pathogenic organisms is underscored by the emergence of several bacterial
strains that are resistant to all currently available
antibiotics.1-4 Antibiotics, such as b-lactams, succeed by targeting vital cellular functions
either killing the organism or hindering their multiplication. However, through
evolution, bacteria will most likely develop resistance to these drugs
rendering them useless. Targeting new bacterial-specific pathways is a valid
approach to combating this problem.
The meso-diaminopimelate (mDAP)/lysine biosynthetic pathway offers
several potential anti-bacterial targets that have yet to be explored.5-7 Since both products of this pathway, mDAP and lysine,
are essential components for the synthesis of the peptidoglycan cell wall in
Gram-negative and some Gram-positive bacteria, inhibitors of enzymes within
this pathway may provide a new class of antibiotics.3 The fact that there are no similar pathways in mammals
suggests that inhibitors of enzymes in the mDAP/lysine pathway will provide
selective toxicity against bacteria and have little or no effect on humans.
One of the enzymes in this pathway,8 the
dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE),
catalyzes the hydrolysis of N-succinyl-L,L-diaminopimelate to
L,Ldiaminopimelate and succinate.9 It has been
shown that deletion of the gene encoding DapE is lethal to Helicobacter
pylori and
Mycobacterium smegmatis.10,11 Therefore, DapEs are essential
for cell growth and proliferation making them potential molecular targets for a
new class of antibiotics.
Rational design of inhibitors for DapE relies on an understanding of the active
site structure and mechanism. DapE is known to be a metallohydrolase utilizing
Zn at its active site, but no crystallographic information is available. We
have used Zn K-edge Extended X-ray Absorption Fine Structure (EXAFS) data, of
DapE from Haemophilus influenzae in the presence of one or two
equivalents of Zn(II) (i.e. [Zn_(DapE)] and [ZnZn(DapE)]), to provide
structural information about the active site.12 The Fourier transforms of the Zn
EXAFS are dominated by a peak at ca. 2.0 Å, which can be fit for both
[Zn_(DapE)] and [ZnZn(DapE)] assuming ca. 5 (N,O) scatterers at 1.96 and
1.98 Å, respectively. A secondshell feature at ca. 3.34 Å appears only
in the [ZnZn(DapE)] FT, demonstrating that DapE contains a dinuclear Zn(II)
active site.
In addition, Zn EXAFS data for DapE incubated with two competitive inhibitors,
2- carboxyethylphosphonic acid (CEPA) and 5-mercaptopentanoic acid, establish
the binding modes of phosphonate- and thiolate-containing inhibitors. The
structural data obtained for CEPA bound to [ZnZn(DapE)] also provides an
initial understanding of the transition state for the hydrolysis reaction
catalyzed by DapE. Since most pharmaceuticals target the transition state of
enzymatic reactions, the structural aspects of [ZnZn(DapE)]-CEPA are
particularly important for the rational design of new potent inhibitors of DapE
enzymes.
References:
2Department of Chemistry and Biochemistry, Utah State University,
Logan, Utah 84322-0300
3Guilford Pharmaceuticals Inc., 6611 Tributary Street,
Baltimore, Maryland 21224
