Expression and bifunctional characterization of Plasmodium falciparum orotidine 5′-monophosphate decarboxylase/orotate phosphoribosyltransferase fused enzyme

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 some organisms. Here, we produced gene fusions of Plasmodium falciparum OMPDC-OPRT, and expressed as the bifunctional enzyme in Escherichia coli. The enzyme was purified to near homogeneity using affinity and anion-exchange chromatography, exhibited enzymatic activities and functioned as a dimer. The enzymatic 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 turnover number (kcat) was selectively enhanced up to three orders of magnitude, while the Michaelis constant (Km) was not much affected and remained at low µM levels, when compared to the enzymes in the monofunctional and in the complex forms, as published earlier. 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.