The Plasmodium falciparum Bifunctional Ornithine Decarboxylase, S-Adenosyl-l-methionine Decarboxylase, Enables a Well Balanced Polyamine Synthesis without Domain-Domain Interaction

Abstract

In the human malaria parasite Plasmodium falciparum (Pf), polyamines are synthesized by a bifunctional enzyme that possesses both ornithine decarboxylase (ODC) and S-adenosyl-L-methionine decarboxylase (AdoMetDC) activities. The mature enzyme consists of the heterotetrameric N-terminal AdoMetDC and the Cterminal dimeric ODC, which results in the formation of a heterotetrameric complex. For the native bifunctional protein a half-life longer than 2 h was determined, which is in contrast to the extreme short half-life of its mammalian monofunctional counterparts. The biological advantage of the plasmodial bifunctional ODC/ AdoMetDC might be that the control of polyamine synthesis is achieved by only having to regulate the abundance and activity of one protein. An interesting feature in the regulation of the bifunctional protein is that putrescine inhibits PfODC activity ϳ10-fold more efficiently than the mammalian ODC activity, and in contrast to the mammalian AdoMetDC the activity of the PfAdoMetDC domain is not stimulated by the diamine. To analyze post-translational processing, polymerization, and domain-domain interactions, several mutant proteins were generated that have single mutations in either the PfODC or PfAdoMetDC domains. The exchange of amino acids essential for the activity of one domain had no effect on the enzyme activity of the other domain. Even prevention of the post-translational cleavage of the AdoMetDC domain or ODC dimerization and thus the interference with the folding of the protein hardly affected the activity of the partner domain. In addition, inhibition of the activity of the PfODC domain had no effect on the activity of the PfAdoMetDC domain and vice versa. These results demonstrate that no domain-domain interactions occur between the two enzymes of the bifunctional PfODC/AdoMetDC and that both enzymatic activities are operating as independent catalytic sites that do not affect each other.The polyamines putrescine, spermidine, and spermine are ubiquitous and essential cellular components involved in various metabolic processes including the proliferation and differentiation of bacteria, plants, and animals (1, 2). The two rate-limiting enzymes of polyamine synthesis are ornithine decarboxylase (ODC, 1 EC 4.1.1.17) and S-adenosyl-L-methionine decarboxylase (AdoMetDC, EC 4.1.1.50), which provide putrescine and decarboxylated S-adenosyl-L-methionine (AdoMet) for the synthesis of spermidine. Interference with polyamine biosynthesis is considered as antitumor and antiparasitic strategy, although the success of polyamine-related cancer therapeutic approaches has been modest (3-5). However, 2-difluoromethylornithine (DFMO), an inhibitor of ODC originally designed as an anticancer agent, has proved to be clinically successful in the treatment of African sleeping sickness caused by the protozoan Trypanosoma gambiense (6). The selective toxicity of DFMO on T. gambiense is complex and discussed to depend on various metabolic differences between parasite and mammalian host including the presence ...

Divergent polyamine metabolism in the Apicomplexa

“…In this study, we demonstrate that T. gondii lacks ODC and ADC activity, and therefore is auxotrophic for polyamines. These results agree with the observed growth requirement of T. gondii for host polyamines reported by others (Moraes et al, 2004;Seabra et al, 2004), but distinguish T. gondii from other members of the apicomplexa that have the ability to synthesize polyamines (Keithly et al, 1997;Wrenger et al, 2001). T. gondii has previously been shown to have a highaffinity putrescine transporter, demonstrating the ability to scavenge host-derived putrescine (Seabra et al, 2004).…”

“…The various members of the apicomplexa have different methods for polyamine biosynthesis: E. tenella and P. falciparum have an active ODC (Keithly et al, 1997;Wrenger et al, 2001), whereas C. parvum has an ADC (Keithly et al, 1997); these differences may be the result of different intracellular localizations of these parasites. Most information is available for the malaria parasite P. falciparum, which has been shown to possess a bifunctional ODC-adenosylmethionine decarboxylase that is responsible for polyamine biosynthesis (Wrenger et al, 2001). The activity of the P. falciparum enzyme has been shown to be significantly blocked by putrescine analogues that exhibited 1300-fold higher activity toward the parasite compared with DFMO (Das Gupta et al, 2005).…”

“…By data-mining various apicomplexan genome sequences, we failed to detect any ODC and ADC homologues encoded by the T. gondii genome (ToxoDB, release 3.0, with 106 coverage), but we identified ODC genes from P. falciparum which has a well characterized bifunctional ODC/Sadenosyl-L-methionine decarboxylase (Wrenger et al, 2001) and E. tenella which has ODC activity (Keithly et al, 1997; The Sanger Institute, with >86 coverage). No ADC homologues were detected from the T. gondii, P. falciparum and E. tenella genomes, except for the lysine decarboxylase (LDC) homologues that typically display significant sequence similarity with ADC and ODC.…”

Biochemical characterisation and novel classification of monofunctional S-adenosylmethionine decarboxylase of Plasmodium falciparum

“…Synonymous codons were chosen to ensure that the positional codon frequency of low/intermediate and high usage codons are similar to the frequency used by that of the E. coli host [17]. Codon-harmonised nucleotides 1-1461 (GeneArt, Regensburg, Germany) encoding the core of PfAdoMetDC (487 residues) [11] [9]), which encodes half of the hinge region (wtPfAdoMetDC-hinge, 660 residues) with an N-terminal Strep-tag. In addition, expression of the full-length PfAdoMetDC/ODC proteins from the pASK-IBA3 constructs containing harmA/wtO and wtA/wtO [8] were also compared.…”

“…Both of these decarboxylase activities function independently of each other within the bifunctional complex [9], although it was shown that inter-and intradomain interactions modulate both activities [10]. In contrast to their single-enzyme orthologues in other organisms, the PfODC activity in the bifunctional enzyme is strongly feedback-inhibited by putrescine, whereas PfAdoMetDC activity is not stimulated by putrescine [9,11]. In the absence of known regulatory mechanisms that control polyamine levels in other organisms, the presence of a single PfAdoMetDC/ODC polypeptide has been suggested to allow regulation of polyamine pools in plasmodia [9].…”