Parasite-specific inserts in the bifunctional S-adenosylmethionine decarboxylase/ornithine decarboxylase of Plasmodium falciparum modulate catalytic activities and domain interactions

Abstract

Polyamine biosynthesis of the malaria parasite, Plasmodium falciparum, is regulated by a single, hinge-linked bifunctional PfAdoMetDC/ODC [ P. falciparum AdoMetDC (S-adenosylmethionine decarboxylase)/ODC (ornithine decarboxylase)] with a molecular mass of 330 kDa. The bifunctional nature of AdoMetDC/ODC is unique to Plasmodia and is shared by at least three species. The PfAdoMetDC/ODC contains four parasite-specific regions ranging in size from 39 to 274 residues. The significance of the parasite-specific inserts for activity and protein-protein interactions of the bifunctional protein was investigated by a single- and multiple-deletion strategy. Deletion of these inserts in the bifunctional protein diminished the corresponding enzyme activity and in some instances also decreased the activity of the neighbouring, non-mutated domain. Intermolecular interactions between AdoMetDC and ODC appear to be vital for optimal ODC activity. Similar results have been reported for the bifunctional P. falciparum dihydrofolate reductase-thymidylate synthase [Yuvaniyama, Chitnumsub, Kamchonwongpaisan, Vanichtanankul, Sirawaraporn, Taylor, Walkinshaw and Yuthavong (2003) Nat. Struct. Biol. 10, 357-365]. Co-incubation of the monofunctional, heterotetrameric approximately 150 kDa AdoMetDC domain with the monofunctional, homodimeric ODC domain (approximately 180 kDa) produced an active hybrid complex of 330 kDa. The hinge region is required for bifunctional complex formation and only indirectly for enzyme activities. Deletion of the smallest, most structured and conserved insert in the ODC domain had the biggest impact on the activities of both decarboxylases, homodimeric ODC arrangement and hybrid complex formation. The remaining large inserts are predicted to be non-globular regions located on the surface of these proteins. The large insert in AdoMetDC in contrast is not implicated in hybrid complex formation even though distinct interactions between this insert and the two domains are inferred from the effect of its removal on both catalytic activities. Interference with essential protein-protein interactions mediated by parasite-specific regions therefore appears to be a viable strategy to aid the design of selective inhibitors of polyamine metabolism of P. falciparum.

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

“…7) [11,39]. It has furthermore been shown that interdomain interactions occur between PfAdoMetDC and PfODC in the bifunctional complex and are mediated by parasite-specific inserts [10]. Inserts are speciesspecific, non-homologous regions that diverge rapidly and may contain low-complexity segments with prevalence for positively-charged residues [40].…”

“…Inserts are speciesspecific, non-homologous regions that diverge rapidly and may contain low-complexity segments with prevalence for positively-charged residues [40]. Furthermore, these inserts may form nonglobular, flexible loops that protrude on the surface of the protein [10]. Since the relative positions of these parasite-specific inserts in the quaternary structure of PfAdoMetDC are unknown [11], their potential role in dimerisation (or oligomerisation) cannot be excluded (Fig.…”

“…Protein purification using Strep-Tactin affinity matrix (IBA) was performed as described previously [10]. Protein bands separated by SDS-PAGE and visualised with Colloidal…”

“…The monofunctional proteins A c c e p t e d M a n u s c r i p t separated with SDS-PAGE were also identified with Western immunodetection using monoclonal Strep-tag II mouse antiserum conjugated to horseradish peroxidase (Acris antibodies) [10].…”

“…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].…”