Recombinant uracil phosphoribosyltransferase from the thermophile Bacillus caldolyticus: expression, purification, and partial characterization.
The upp gene encoding the major uracil phosphoribosyltransferase (UPRT) of the thermophile Bacillus caldolyticus was cloned by complementation of an Escherichia coli upp mutation. The nucleotide sequence of the cloned DNA revealed an open reading frame of 630 bp encoding a polypeptide of 209 amino acids (M(r) 22,817) with 84% amino acid sequence identity to the deduced upp gene product of Bacillus subtilis. Primer extension analysis indicated that the transcriptional start site of the cloned gene was positioned 37 or 38 bp upstream of the coding region. When over-expressed in E. coli, the recombinant UPRT represented approximately 18% of the soluble cellular proteins. The enzyme was purified to homogeneity by two sequential precipitations with 50 mM Na-phosphate, pH 7.0. Gel filtration chromatography indicated that the native enzyme existed as a dimer at high protein concentrations but that it dissociated to a monomeric form on dilution. In dilute solutions the enzyme is highly unstable but can be stabilized by addition of bovine serum albumin. In concentrated solution (> 5 mg/ml) the enzyme is stable for months at 4 degrees C, even in the absence of bovine serum albumin. By comparing the UPRT activity of crude extracts of B. subtilis and B. caldolyticus it was found that the enzyme from B. caldolyticus was considerably more stable toward thermal inactivation than the homologous enzyme from B. subtilis.
Jensen
H K
HK
Center for Enzyme Research, University of Copenhagen, Denmark.
Mikkelsen
N
N
Neuhard
J
J
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology*; D001483:Base Sequence; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005976:Glutaral / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010430:Pentosyltransferases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D012995:Solubility; D013329:Structure-Activity Relationship; D014158:Transcription, Genetic
D003432:Cross-Linking Reagents; D011994:Recombinant Proteins; D010430:Pentosyltransferases; C023269:uracil phosphoribosyltransferase; D005976:Glutaral