title: 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 (kcat/Km). The kcatwas selectively enhanced up to three orders of magnitude, while the Kmwas 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.
authors.affiliation: Department of Biochemistry, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand; Inter-Department Program of Biomedical Science, Faculty of Graduate School, Chulalongkorn University, Bangkok 10330, Thailand.
authors.forename: Patsarawadee
authors.initials: P
authors.lastname: Paojinda
authors.affiliation: Department of Basic Medical Science, Faculty of Medicine Vajira Hospital, Navamindradhiraj University, Bangkok 10300, Thailand.
authors.forename: Waranya
authors.initials: W
authors.lastname: Imprasittichai
authors.affiliation: Unit of Biochemistry, Department of Medical Science, Faculty of Science, Rangsit University, Patumthani 12000, Thailand.
authors.forename: Sudaratana R
authors.initials: SR
authors.lastname: Krungkrai
authors.affiliation: Department of Molecular Protozoology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Nirianne Marie Q
authors.initials: NMQ
authors.lastname: Palacpac
authors.affiliation: Department of Molecular Protozoology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Toshihiro
authors.initials: T
authors.lastname: Horii
authors.affiliation: Department of Biochemistry, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand. Electronic address: jerapan.k@chula.ac.th.
authors.forename: Jerapan
authors.initials: J
authors.lastname: Krungkrai
mesh_terms: D019677:Artificial Gene Fusion; D004926:Escherichia coli / Q000235:genetics; D050939:Gene Fusion; D000074041:Microorganisms, Genetically-Modified / Q000235:genetics; D009962:Orotate Phosphoribosyltransferase / Q000235:genetics / Q000378:metabolism*; D009964:Orotidine-5'-Phosphate Decarboxylase / Q000235:genetics / Q000378:metabolism*; D010963:Plasmodium falciparum / Q000201:enzymology* / Q000235:genetics*; D015800:Protozoan Proteins / Q000378:metabolism*
chemical_list: D015800:Protozoan Proteins; D009962:Orotate Phosphoribosyltransferase; D009964:Orotidine-5'-Phosphate Decarboxylase
keywords: Bifunctional fused enzyme; Catalytic efficiency; Orotate phosphoribosyltransferase; Orotidine 5′-monophosphate decarboxylase; Plasmodium falciparum