Linked Life Data

8720144

Source:http://linkedlifedata.com/resource/pubmed/id/8720144

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
6
pubmed:dateCreated
1996-10-10
pubmed:abstractText
Orotate phosphoribosyltransferase (OPRTase, EC2.4.2.10) plays a role in de novo synthesis of pyrimidine nucleotide and transfers orotate to 5-phosphoribosyl-1-pyrophosphate (PRPP) to form orotidine-5'-monophosphate (OMP). To obtain heat-stable OPRTase and to elucidate the mechanism of heat stability, this enzyme from Thermus thermophilus was expressed in Escherichia coli and purified. The pyrE gene of T. thermophilus which encodes OPRTase, contains an open reading frame of 549 base pairs with 69% G+C content. Since this gene expressed itself inefficiently in E. coli, the 5' and 3' ends of the coding regions were replaced with synonymous codons which contain more A+T and corresponds to major codons for E. coli. Introduction of the modified gene fragments into a plasmid having a tac promoter resulted in production of a polypeptide of molecular weight (M(r)) 20,000 in the presence of isopropyl-beta-D-thiogalactopyranoside (IPTG) in E. coli. This protein represented as much as 16% of the bacterial total protein and showed the OPRTase activity. Three purification steps, consisting of heat treatment at 65 degrees C, 40% ammonium sulfate fractionation, and KCl gradient elution from DEAE-Sephadex A-50, resulted in highly purified single polypeptide. The optimum activity of the purified OPRTase was observed at 150 mM KCl, pH 9.0, 75-80 degrees C, and in the presence of 100 microM PRPP. The activation energy of this enzyme reaction was 20.3 kJ/mol. The Km of this enzyme for orotate as a substrate was 75 microM and the maximum specific activity was 300 units/mg protein under the optimum conditions. The purified OPRTase was stable for 20 min at 85 degrees C.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Dec
pubmed:issn
0021-924X
pubmed:author
pubmed:issnType
Print
pubmed:volume
118
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
1261-7
pubmed:dateRevised
2008-11-21
pubmed:meshHeading
pubmed:year
1995
pubmed:articleTitle
Orotate phosphoribosyltransferase from Thermus thermophilus: overexpression in Escherichia coli, purification and characterization.
pubmed:affiliation
Department of Biological and Chemical Engineering, Gunma University.
pubmed:publicationType
Journal Article, Comparative Study, Research Support, Non-U.S. Gov't