Bifunctional activity of fused Plasmodium falciparum orotate phosphoribosyltransferase and orotidine 5'-monophosphate decarboxylase.Parasitol Int. 2018 Feb; 67(1):79-84.PI
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 (kcat/Km). The kcat was selectively enhanced up to three orders of magnitude, while the Km 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.