Purification and characterization of a hygromycin B phosphotransferase from Streptomyces hygroscopicus.
A hygromycin B phosphotransferase activity from Streptomyces hygroscopicus has been highly purified by ammonium sulphate fractionation followed by affinity column chromatography through Sepharose-6B-hygromycin-B. The combined active fractions showed a single protein band (41 kDa) when subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. When gel electrophoresis was performed under non-denaturing conditions, the single protein band promoted in situ phosphorylation of hygromycin B, indicating that this protein corresponded to the purified hygromycin B phosphotransferase. The enzyme has been purified 236-fold and approximate Km values of 0.56 microM for hygromycin B and ATP, respectively, were deduced.
D019868:Kanamycin Kinase; D007700:Kinetics; D008970:Molecular Weight; D010766:Phosphorylation; D010770:Phosphotransferases / Q000302:isolation & purification* / Q000378:metabolism; D013302:Streptomyces / Q000201:enzymology*
D010770:Phosphotransferases; D019868:Kanamycin Kinase