Bifunctional activity of fused Plasmodium falciparum orotate phosphoribosyltransferase and orotidine 5'-monophosphate decarboxylase

Abstract

Fusion of the last two enzymes in the pyrimidine biosynthetic pathway in the inversed order by having a COOH-terminal orotate phosphoribosyltransferase (OPRT) and an NH₂-terminal orotidine 5'-monophosphate decarboxylase (OMPDC), as OMPDC-OPRT, are described in many organisms. Here, we produced gene fusions of Plasmodium falciparum OMPDC-OPRT and expressed the bifunctional protein in Escherichia coli. The enzyme was purified to homogeneity using affinity and anion-exchange chromatography, exhibited enzymatic activities and functioned as a dimer. The activities, although unstable, were stabilized by its substrate and product during purification and long-term storage. Furthermore, the enzyme expressed a perfect catalytic efficiency (k_cat/K_m). The k_cat was selectively enhanced up to three orders of magnitude, while the K_m was not much affected and remained at low μM levels when compared to the monofunctional enzymes. The fusion of the two enzymes, creating a "super-enzyme" with perfect catalytic power and more flexibility, reflects cryptic relationship of enzymatic reactivities and metabolic functions on molecular evolution.

Keywords: Bifunctional fused enzyme; Catalytic efficiency; Orotate phosphoribosyltransferase; Orotidine 5′-monophosphate decarboxylase; Plasmodium falciparum.