Expression in Escherichia coli of a recombinant adenosine kinase from Saccharomyces cerevisiae: purification, kinetics and substrate analyses.
The Saccharomyces cerevisiae ADO1 gene is known to encode a homologue of eukaryotic adenosine kinases. This gene was expressed in Escherichia coli as a recombinant protein fused to a polyhistidine tag by using the rhamnose-inducible bacterial promoter rhaB. The recombinant protein was purified to apparent homogeneity and its ability to phosphorylate different substrates was evaluated. Adenosine (Km 3 microM) is its primary substrate. In addition, it also phosphorylates, albeit less efficiently, 3'-deoxyadenosine (cordycepin; Km 1.84 mM) and 3'-amino-3'-deoxyadenosine (Km 0.26 mM). Other kinetic properties of the recombinant enzyme have also been determined.
Barrado
Patricia
P
Centro de Biología Molecular Severo Ochoa, Departamento de Biología Molecular (CSIC/UAM), Universidad Autónoma Madrid, Cantoblanco 28049 Madrid, Spain.
Rodríguez
María José
MJ
Jiménez
Antonio
A
Fernández Lobato
María
M
D000241:Adenosine / Q000378:metabolism*; D000248:Adenosine Kinase / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D015153:Blotting, Western; D002846:Chromatography, Affinity; D003839:Deoxyadenosines / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics
D003839:Deoxyadenosines; D011994:Recombinant Proteins; C019247:3'-amino-3'-deoxyadenosine; D000248:Adenosine Kinase; C058120:cordycepin; D000241:Adenosine