Purification and properties of gentamicin nucleotidyltransferase from Escherichia coli: nucleotide specificity, pH optimum, and the separation of two electrophoretic variants.
Gentamicin nucleotidyltransferase, AAD 2", catalyzes the transfer of a nucleotide to many aminoglycoside antibiotics, which are the drugs of choice in the treatment of gram-negative bacterial infections. The transfer is accompanied by the production of pyrophosphate, which is coupled to three other enzymes so that an increase in absorbance at 340 nm of NADPH can be monitored continuously as a quantitative assay of activity. A purification method was developed for this enzyme using all common principles of protein purification. These include selection of a desirable source of enzyme (choice of plasmid pMY 10), maximizing cellular yield of enzyme (controlled and monitored growth of Escherichia coli pMY 10/W677), selective extraction of protein (modified osmotic shock), removal of nucleic acids (precipitation with streptomycin sulfate), concentration of protein (precipitation with ammonium sulfate), removal of low-molecular-weight impurities (chromatography on Bio-Gel P-2), separation of proteins on the basis of charge (ion-exchange chromatography on DEAE-Bio-Gel A), separation of proteins according to a biospecific property (affinity chromatography on gentamicin-Affi-Gel), and separation of proteins according to size (gel filtration on Ultrogel AcA 54). Purification to near-homogeneity revealed the presence of two related forms of enzyme. The first had a specific activity of 0.134 units/mg, bound rapidly and tightly to gentamicin-Affi-Gel, eluted as a function of ionic strength from Ultrogel, and migrated faster during electrophoresis in both the presence and absence of sodium dodecyl sulfate. It has an isoelectric point of 5.7 +/- 0.2 and consists of a single polypeptide of 32,500 Da. Kinetic characterization showed a pH optimum of 9.5 and Michaelis constants of 2.76 +/- 0.35 microM for tobramycin, 404 +/- 28 microM for Mg-ATP, 2008 +/- 260 microM for Mg-CTP, 30 +/- 3 microM for Mg-dATP and Mg-dGTP, and 90 +/- 7 microM for Mg-dCTP and Mg-dTTP. The second form had a specific activity of 0.274 unit/mg. It also bound tightly to gentamicin-Affi-gel but the onset of binding was time dependent. This form migrated slower during polyacrylamide gel electrophoresis in both the presence and absence of sodium dodecyl sulfate. It has an isoelectric point of 6.0 +/- 0.2 and consists of a single polypeptide of 31,500 Da. The exact relationship between the two forms has not been elucidated. It is probable that they have a recent common ancestor or are the same polypeptide because the amino acid compositions and polypeptide chain lengths are essentially identical.(ABSTRACT TRUNCATED AT 400 WORDS)
D055598:Chemical Phenomena; D002621:Chemistry; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007700:Kinetics; D009713:Nucleotidyltransferases / Q000302:isolation & purification* / Q000378:metabolism; D011815:R Factors; D013379:Substrate Specificity
D009713:Nucleotidyltransferases; C020565:gentamicin 2''-nucleotidyltransferase