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Orotate Phosphoribosyltransferase from Corynebacterium ammoniagenes Lacking a Conserved Lysine▿ †
- Publication Year :
- 2007
- Publisher :
- American Society for Microbiology (ASM), 2007.
-
Abstract
- The pyrE gene, encoding orotate phosphoribosyltransferase (OPRTase), was cloned by nested PCR and colony blotting from Corynebacterium ammoniagenes ATCC 6872, which is widely used in nucleotide production. Sequence analysis shows that there is a lack of an important conserved lysine (Lys 73 in Salmonella enterica serovar Typhimurium OPRTase) in the C. ammoniagenes OPRTase. This lysine has been considered to contribute to the initiation of catalysis. The enzyme was overexpressed and purified from a recombinant Escherichia coli strain. The molecular mass of the purified OPRTase was determined to be 45.4 ± 1.5 kDa by gel filtration. Since the molecular mass for the subunit of the enzyme was 21.3 ± 0.6 kDa, the native enzyme exists as a dimer. Divalent magnesium was necessary for the activity of the enzyme and can be substituted for by Mn 2+ and Co 2+ . The optimal pH for the forward (phosphoribosyl transfer) reaction is 10.5 to 11.5, which is higher than that of other reported OPRTases, and the optimal pH for the reverse (pyrophosphorolysis) reaction is 5.5 to 6.5. The K m values for the four substrates were determined to be 33 μM for orotate, 64 μM for 5-phosphoribosyl-1-pyrophosphate (PRPP), 45 μM for orotidine-5-phosphate (OMP), and 36 μM for pyrophosphate. The K m value for OMP is much larger than those of other organisms. These differences may be due to the absence of Lys 73, which is present in the active sites of other OPRTases and is known to interact with OMP and PRPP.
- Subjects :
- Cations, Divalent
Orotate Phosphoribosyltransferase
Lysine
Molecular Sequence Data
Coenzymes
Gene Expression
Phosphoribosyl Pyrophosphate
Corynebacterium
medicine.disease_cause
Microbiology
Cofactor
chemistry.chemical_compound
Bacterial Proteins
Enzyme Stability
medicine
Escherichia coli
Nucleotide
Cloning, Molecular
Molecular Biology
Conserved Sequence
chemistry.chemical_classification
Orotic Acid
biology
Molecular mass
Phosphoribosyl pyrophosphate
Sequence Analysis, DNA
Hydrogen-Ion Concentration
Molecular biology
Enzymes and Proteins
Recombinant Proteins
Diphosphates
Molecular Weight
Kinetics
Enzyme
chemistry
Biochemistry
biology.protein
Chromatography, Gel
Orotate phosphoribosyltransferase
Uridine Monophosphate
Dimerization
Subjects
Details
- Language :
- English
- Database :
- OpenAIRE
- Accession number :
- edsair.doi.dedup.....20d9dcfe4ecef0c1c7f53478e159bf4a