Research Group Heussler (Malaria I) - Bernhard-Nocht-Institut für ...

Project Description and Results
AdoMetDCs are highly regulated at the transcriptional,
at the translational as well as at the posttranslational
level. The catalytic activity of mammalian AdoMetDC,
e.g., is stimulated by putrescine which is thought to be
a regulatory mechanism to relate putrescine abundance
with the synthesis of higher polyamines in order
to avoid unbalanced consumption of S-adenosylmethionine.
AdoMetDCs from the nematodes O. volvulus and
C. elegans resemble to a large extent the biochemical
and biophysical properties of their mammalian counterparts.
However, while the human AdoMetDC exhibits a
high basic activity even in the absence of the activator
putrescine, both nematode enzymes are nearly inactive
under these conditions (Figure 1). We conclude that
they highly depend on an activator to achieve significant
catalytic activity. Accordingly, activation of the C.
elegans and O. volvulus AdoMetDC by putrescine is
much more pronounced (maximal 350- and 63-fold, respectively)
than of the human enzyme (maximal 3-fold).
Moreover, the human AdoMetDC responses exclusively
to putrescine. In contrast to that, the enzymatic activity
of the nematode AdoMetDC increases also in the presence
of a number of other polyamines including the
naturally occuring spermidine and spermine (Figure 1).
The recently resolved crystal structure of the human
AdoMetDC revealed the topology of the putrescinebinding
site responsible for the putrescine stimulation.
Interestingly, the amino acid residues that were found
to be involved in putrescine binding are well conserved
in the nematode enzymes despite their lower specificity
for stimulator molecules. Mutagenic studies indicate that
at least three of these residues most likely also contribute
to activator binding of C. elegans AdoMetDC. Furthermore,
the C. elegans AdoMetDC mutant Glu 194Gln
has a 100-fold enhanced basic activity in the absence
of any stimulator, suggesting that this mutant protein
mimics the conformational change naturally induced by
activator molecules.
Figure 2: Synthetic polyamine analogue BW-1 evaluated for
inhibitory activity against O. volvulus, C.elegans and human
AdoMetDC in vitro.
Usually, AdoMetDCs are translated as pro-proteins, that
are subsequently cleaved by an autocatalytic process
resulting in a heteroterameric enzyme complex. Both
recombinantly expressed nematode AdoMetDC are
correctly posttranslationally processed in Escherichia
coli resulting in the formation of a small β- and a large
α-subunit. Mutations of amino acid residues that are
part of the putative activator binding site affect this
cleavage step, indicative for a putrescine responsiveness
of this process like it has been reported also for
mammalian AdoMetDC.
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Parasitology Section
As shown above, at least two features distinguish nematode
AdoMetDCs from their mammalian counterparts.
First, the unstimulated enzyme exhibits a very low specific
activity. Second, its polyamine binding pocket has
a lower specificity for activator binding. To examine if
these differences might be exploitable to specifically inhibit
nematode AdoMetDC activity, we looked for polyamine
analogues that bind to the pocket but do not stimulate
the enzyme. The effects of several new polyamine
analogues were tested on the O. volvulus, C. elegans
and human AdoMetDC activity. Tetramines with a 3-7-3
backbone and terminal bulky ring systems like BW-1
(Figure 2) do not stimulate nematode AdoMetDC. On the
contrary, they reduce the basic activity of the unstimulated
AdoMetDCs and, in addition, have an inhibitory effect
on the maximal stimulated enzymes (Table 1). At
this, both nematode enzymes are generally more sensitive
than the human AdoMetDC. Future studies will elucidate
whether the found unspecificity of nematode
AdoMetDCs towards stimulator molecules may be exploitable
for the development of novel chemotherapeutic
agents (polyamine analogues) against nematodes.
O. volvulus C. elegans H. sapiens
Addition Specific activity
2 mM [nmol min-1 mg-1]
Putrescine 79 ± 3 240 ± 24 410 ± 34
Putrescine
+ BW-1
0.3 ± 0.1 0.5 ± 0.4 63 ± 6
Table 1: Inhibition of putrescine-stimulated O. volvulus, C. elegans
and human AdoMetDC by the polyamine analogue BW-1.
Specific activities were determined under standard assay conditions
in the presence of 2 mM putrescine with or without the
addition of 2 mM of the polyamine analogue. Results are given
as the mean (± SD) of at least three independent duplicate determinations.
Selected Publications
• Ndjonka D et al. (2003) Biol Chem 384: 1195-201
• Ndjonka D et al. (2003) Biol Chem 384: 83-91
Funding
• Deutsche Forschungsgemeinschaft
• Deutscher Akademischer Austauschdienst
Cooperating Partners
• P. Woster, Wayne State University, Detroit, USA
• A. Da’dara, Harvard School of Public Health, Boston,
USA
Investigators
• Kai Lüersen
• Dieudonné Ndjonka
• Rolf D. Walter