The gene for orotate phosphoribosyltransferase from Saccharomyces cerevisiae has been subcloned into an Escherichia coli overexpression vector and the enzyme has been produced in large quantities, thus simplifying the purification to one step. We were able to repeat the published (J. Victor, L. B. Greenberg, and D. L. Sloan J. Biol. Chem. 254, 2647-2655, 1979). 32PPi/5-phosphorylribose 1-alpha-diphosphate exchange experiments and could demonstrate the exchange by magnetization inversion transfer NMR experiments as well. However, when contaminating orotidine 5'-monophosphate (OMP) was eliminated with OMP decarboxylase, any evidence of magnetization transfer vanished. Consequently, it is concluded that a ping pong mechanism is not operable and that a previously proposed oxocarbocation intermediate along the pathway to OMP does not persist long enough in the catalytic cycle of this enzyme to be recognized by NMR exchange experiments.
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http://dx.doi.org/10.1006/abbi.1998.0971 | DOI Listing |
Structure
December 2024
Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, 2200 Copenhagen, Denmark. Electronic address:
Gene variants resulting in insertions or deletions of amino acid residues (indels) have important consequences for evolution and are often linked to disease, yet, compared to missense variants, the effects of indels are poorly understood and predicted. We developed a sensitive protein folding sensor based on the complementation of uracil auxotrophy in yeast by circular permutated orotate phosphoribosyltransferase (CPOP). The sensor reports on the folding of disease-linked missense variants and de-novo-designed proteins.
View Article and Find Full Text PDFInt J Biol Macromol
February 2024
Departamento de Bioquímica, Facultad de Química, Universidad Nacional Autónoma de México, Ciudad de México 04510, Mexico. Electronic address:
In higher eukaryotes and plants, the last two sequential steps in the de novo biosynthesis of uridine 5'-monophosphate (UMP) are catalyzed by a bifunctional natural chimeric protein called UMP synthase (UMPS). In higher plants, UMPS consists of two naturally fused enzymes: orotate phosphoribosyltransferase (OPRTase) at N-terminal and orotidine-5'-monophosphate decarboxylase (ODCase) at C-terminal. In this work, we obtained the full functional recombinant protein UMPS from Coffea arabica (CaUMPS) and studied its structure-function relationships.
View Article and Find Full Text PDFSensors (Basel)
February 2023
Graduate School of Pharmaceutical Sciences, Nagasaki International University, 2825-7 Huis Ten Bosch, Sasebo 859-3298, Japan.
Orotate phosphoribosyltransferase (OPRT) exists as a bifunctional enzyme, uridine 5'-monophosphate synthase, in mammalian cells and plays an important role in pyrimidine biosynthesis. Measuring OPRT activity has been considered important for understanding biological events and development of molecular-targeting drugs. In this study, we demonstrate a novel fluorescence method for measuring OPRT activity in living cells.
View Article and Find Full Text PDFJ Biol Chem
March 2023
The Hormel Institute, University of Minnesota, Austin, Minnesota, USA. Electronic address:
Human uridine 5'-monophosphate synthase (HsUMPS) is a bifunctional enzyme that catalyzes the final two steps in de novo pyrimidine biosynthesis. The individual orotate phosphoribosyl transferase and orotidine monophosphate domains have been well characterized, but little is known about the overall structure of the protein and how the organization of domains impacts function. Using a combination of chromatography, electron microscopy, and complementary biophysical methods, we report herein that HsUMPS can be observed in two structurally distinct states, an enzymatically active dimeric form and a nonactive multimeric form.
View Article and Find Full Text PDFPhys Chem Chem Phys
January 2023
Quantum and Molecular Engineering Laboratory, Department of Chemical Engineering, Indian Institute of Technology Kharagpur, Kharagpur 721302, India.
Orotate phosphoribosyltransferase (OPRT) catalyses the reversible phosphoribosyl transfer from α-D-5-phosphoribosyl-1-pyrophosphate (PRPP) to orotic acid (OA) to yield orotidine 5'-monophosphate (OMP) during the synthesis of nucleotides. Numerous studies have reported the inhibition of this reaction as a strategy to check diseases like tuberculosis, malaria and cancer. Insight into the inhibition of this reaction is, therefore, of urgent interest.
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