<abstract xmlns="http://www.w3.org/1999/xhtml"><p> 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.</p></abstract>

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.

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

Asian Biomedicine

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License.

Background: Human malaria parasite Plasmodium falciparum operates de novo pyrimidine biosynthetic pathway. The fifth and sixth enzymes of the pathway form a...

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

Publié en ligne: 13 avr. 2018

Pages: 297 - 306

DOI: https://doi.org/10.2478/abm-2010-0037

Mots clés
Co-expression, co-transformation, enzyme complex, orotate phosphoribosyltransferase (OPRT), orotidine 5’-monophosphate decarboxylase (OMPDC), Plasmodium falciparum

© 2018

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License.

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

Publié en ligne: 13 avr. 2018

Pages: 297 - 306

DOI: https://doi.org/10.2478/abm-2010-0037

Mots clésCo-expression, co-transformation, enzyme complex, orotate phosphoribosyltransferase (OPRT), orotidine 5’-monophosphate decarboxylase (OMPDC), Plasmodium falciparum

© 2018

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License.

Mots clés
Co-expression, co-transformation, enzyme complex, orotate phosphoribosyltransferase (OPRT), orotidine 5’-monophosphate decarboxylase (OMPDC), Plasmodium falciparum