Preparative 2'-reduction of ATP catalyzed by ribonucleotide reductase purified by liquid-liquid extraction.
Recombinant Lactobacillus leichmannii ribonucleosidetriphosphate reductase (RTPR, E.C.1.17.4.2) constitutively expressed by E. coli HB101 pSQUIRE has been purified from sonicated cell material in a one-step procedure by PEG 4000 (16% (w/w))/phosphate (7% (w/w)) liquid-liquid extraction. A high yield of 75.1% RTPR in the top phase and a partitioning of 8.5:1 between total RTPR activity in top and bottom phase were obtained in this preparative system. The RTPR-containing top phase was used to reduce ATP in the 2'-position on a gram scale with high final conversion and yield proving the ribonucleotide reductase approach feasible for the preparative synthesis of 2'-deoxyribonucleotides. High concentrations of sodium acetate in the reaction served to substitute for allosteric effectors of RTPR. 1,4-Dithio-DL-threitol was used as an artificial reducing agent for RTPR.
Brunella
A
A
Outcomes International, Basel, Switzerland.
Abrantes
M
M
Ghisalba
O
O
D000255:Adenosine Triphosphate / Q000737:chemistry* / Q000378:metabolism; D000645:Ammonium Sulfate; D004926:Escherichia coli / Q000201:enzymology; D007202:Indicators and Reagents; D007700:Kinetics; D007778:Lactobacillus / Q000201:enzymology; D010084:Oxidation-Reduction; D011092:Polyethylene Glycols; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012264:Ribonucleotide Reductases / Q000302:isolation & purification / Q000378:metabolism*
D007202:Indicators and Reagents; D011994:Recombinant Proteins; D011092:Polyethylene Glycols; D000255:Adenosine Triphosphate; D012264:Ribonucleotide Reductases; D000645:Ammonium Sulfate