A unique insertion of low complexity amino acid sequence underlies protein-protein interaction in human malaria parasite orotate phosphoribosyltransferase and orotidine 5'-monophosphate decarboxylase

OBJECTIVE@#To investigate the multienzyme complex formation of human malaria parasite Plasmodium falciparum (P. falciparum) orotate phosphoribosyltransferase (OPRT) and orotidine 5'-monophosphate decarboxylase (OMPDC), the fifth and sixth enzyme of the de novo pyrimidine biosynthetic pathway. Previously, we have clearly established that the two enzymes in the malaria parasite exist physically as a heterotetrameric (OPRT)2(OMPDC)2 complex containing two subunits each of OPRT and OMPDC, and that the complex have catalytic kinetic advantages over the monofunctional enzyme.@*METHODS@#Both enzymes were cloned and expressed as recombinant proteins. The protein-protein interaction in the enzyme complex was identified using bifunctional chemical cross-linker, liquid chromatography-mass spectrometric analysis and homology modeling.@*RESULTS@#The unique insertions of low complexity region at the α 2 and α 5 helices of the parasite OMPDC, characterized by single amino acid repeat sequence which was not found in homologous proteins from other organisms, was located on the OPRT-OMPDC interface. The structural models for the protein-protein interaction of the heterotetrameric (OPRT)2(OMPDC)2 multienzyme complex were proposed.@*CONCLUSIONS@#Based on the proteomic data and structural modeling, it is surmised that the human malaria parasite low complexity region is responsible for the OPRT-OMPDC interaction. The structural complex of the parasite enzymes, thus, represents an efficient functional kinetic advantage, which in line with co-localization principles of evolutional origin, and allosteric control in protein-protein-interactions.

Co-expression of human malaria parasite Plasmodium falciparum orotate phosphoribosyltransferase and orotidine 5?-monophosphate decarboxylase as enzyme complex in Escherichia coli: a novel strategy for drug development

Background: Human malaria parasite Plasmodium falciparum operates de novo pyrimidine biosynthetic pathway. The fifth and sixth enzymes of the pathway form a heterotetrameric complex, containing two molecules each of orotate phosphoribosyltransferase (OPRT) and orotidine 5’-monophosphate decarboxylase (OMPDC). Objective: Define the function of OPRT-OMPDC enzyme complex of P. falciparum by co-expressing the enzymes in Escherichia coli. Methods: The constructed plasmids containing either P. falciparum OPRT or OMPDC were cloned in E. coli by co-transformation. Both genes were co-expressed as OPRT-OMPDC enzyme complex and the complex was purified by chromatographic techniques, including N2+-NTA affinity, Hi Trap Q HP anion-exchange, uridine 5’- monophosphate affinity, and Superose 12 gel-filtration columns. Physical and kinetic properties of the enzyme complex were analyzed for its molecular mass. Results: Co-transformation of PfOPRT and PfOMPDC plasmids in E. coli were achieved with a clone containing DNA ratio of 1:2, respectively. Both plasmids remained stable and were functionally expressed in the E. coli cell for at least 20 weeks. The P. falciparum OPRT-OMPDC enzyme complex were co-expressed and the complex was co-eluted in all chromatographic columns during purification and physical analysis. The molecular mass of the complex was 130 kDa, whereas the PfOPRT and PfOMPDC component were 35.6 and 41.5 kDa, respectively. The enzymatic activities of the complex were competitively inhibited by their products of each enzyme component. Conclusion: P. falciparum OPRT and OMPDC in E. coli as an enzyme complex were co-transformed and functionally co-expressed. These have similar properties to the native enzyme purified directly from P. falciparum, and this character is different from that of the human host organism. The enzyme complex would be suitable as new target to research selective inhibitors as suitable drugs to better control this disease.