The ornithine decarboxylase domain of the bifunctional ornithine decarboxylase/S-adenosylmethionine decarboxylase of Plasmodium falciparum: recombinant expression and catalytic properties of two different constructs.
The polyamines putrescine, spermidine and spermine play an essential role in cell differentiation and proliferation. Inhibition of the rate-limiting enzymes of polyamine biosynthesis, ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC), has been proposed as a therapeutic strategy against cancer and parasitic infections. In the case of Plasmodium falciparum, the causative agent of malaria tropica, this approach is especially interesting, because here both key enzymes, ODC and AdoMetDC, are combined in a bifunctional protein, ODC/AdoMetDC. This arrangement has not been found in any other organism investigated so far. We report the cloning and recombinant expression of the ODC domain of P. falciparum in Escherichia coli. First, we expressed the mere recombinant ODC domain (rPfODC). Secondly, we expressed the recombinant ODC domain in conjunction with the preceding part of the hinge region of the bifunctional ODC/AdoMetDC (rPfHinge-ODC). K(m) values for L-ornithine were 47.3 microM for the rPfHinge-ODC and 161. 5 microM for the rPfODC. Both recombinant enzymes were inhibited by putrescine, but the K(i) value for the rPfHinge-ODC was 50.4 microM (IC(50)=157 microM), whereas the IC(50) for the rPfODC was 500 microM. Spermidine was a weak inhibitor in both cases. alpha-Difluoromethylornithine inhibited the rPfHinge-ODC with a K(i) value of 87.6 microM. For two novel ODC inhibitors, CGP52622A and CGP54619A, the K(i) values of the rPfHinge-ODC were in the nanomolar range.
Krause
T
T
Bernhard Nocht Institute for Tropical Medicine, Biochemical Parasitology, Bernhard-Nocht-Str. 74, D-20359 Hamburg, Germany.
Lüersen
K
K
Wrenger
C
C
Gilberger
T W
TW
Müller
S
S
Walter
R D
RD
D012437:Adenosylmethionine Decarboxylase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D009955:Ornithine Decarboxylase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D065108:Ornithine Decarboxylase Inhibitors; D010963:Plasmodium falciparum / Q000201:enzymology*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D017931:DNA Primers; D065108:Ornithine Decarboxylase Inhibitors; D011994:Recombinant Proteins; D009955:Ornithine Decarboxylase; D012437:Adenosylmethionine Decarboxylase