title: Cloning, overexpression, and purification of aminoglycoside antibiotic nucleotidyltransferase (2'')-Ia: conformational studies with bound substrates.
abstract: Aminoglycoside nucleotidyltransferase (2'')-Ia [ANT (2'')-Ia] was cloned from Pseudomonas aeruginosa and purified from overexpressing Escherichia coli BL21(DE3) cells. The first enzyme-bound conformation of an aminoglycoside antibiotic in the active site of an aminoglycoside nucleotidyltransferase was determined using the purified aminoglycoside nucleotidyltransferase (2' ')-Ia. The conformation of the aminoglycoside antibiotic isepamicin, a psuedo-trisaccharide, bound to aminoglycoside nucleotidyltransferase (2' ')-Ia has been determined using NMR spectroscopy. Molecular modeling, employing experimentally determined interproton distances, resulted in two different enzyme-bound conformations (conformer 1 and conformer 2) of isepamicin. Conformer 1 was by far the major conformer defined by the following average glycosidic dihedral angles: PhiBC = -65.26 +/- 1.63 degrees and PsiBC = -54.76 +/- 4.64 degrees. Conformer 1 was further subdivided into one major (conformer 1a) and two minor components (conformers 1b and 1c) based on the comparison of glycosidic dihedral angles PhiAB and PsiAB. The arrangement of substrates in the enzyme.metal-ATP.isepamicin complex was determined on the basis of the measured effect of the paramagnetic substrate analogue Cr(H2O)4ATP on the relaxation rates of substrate protons which were used to determine relative distances of isepamicin protons to the Cr3+. Both conformers of isepamicin yielded arrangements that satisfied the NOE restraints and the observed paramagnetic effects of Cr(H2O)4ATP. It has been suggested that aminoglycosides use both electrostatic interactions and hydrogen bonds in binding to RNA and that the contacts made by the A and B rings to RNA are the most important for binding [Fourmy, D., Recht, M. I., Blanchard, S. C., and Puglisi, J. D. (1996) Science 274, 1367-1371]. Comparisons based on the determined conformations of enzyme-bound aminoglycoside antibiotics also suggested that interactions of rings A and B with enzymes may be the major determinant in aminoglycoside binding to enzymes [Serpersu, E. H., Cox, J. R., DiGiammarino, E. L., Mohler, M. L., Ekman, D. R., Akal-Strader, A., and Owston, M. (2000) Cell Biochem. Biophys. (in press)]. The conformation of isepamicin bound to the aminoglycoside nucleotidyltransferase (2' ')-Ia, determined in this work, lent further support to this theory. Furthermore, comparison of enzyme-bound conformations of isepamicin to the RNA-bound conformation of gentamycin C1a also showed remarkable similarities between the enzyme-bound and RNA-bound aminoglycoside antibiotic conformations. These studies should aid in the design of effective inhibitors possessing a broad range of aminoglycoside-modifying enzymes as targets.
authors.affiliation: Department of Biochemistry and Cellular and Molecular Biology, The University of Tennessee, Walters Life Sciences Building, M407, Knoxville, Tennessee 37996-0840, USA.
authors.forename: D R
authors.initials: DR
authors.lastname: Ekman
authors.forename: E L
authors.initials: EL
authors.lastname: DiGiammarino
authors.forename: E
authors.initials: E
authors.lastname: Wright
authors.forename: E D
authors.initials: ED
authors.lastname: Witter
authors.forename: E H
authors.initials: EH
authors.lastname: Serpersu
mesh_terms: D001426:Bacterial Proteins; D001665:Binding Sites; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005839:Gentamicins / Q000737:chemistry / Q000378:metabolism; D007612:Kanamycin / Q000737:chemistry* / Q000378:metabolism; D007613:Kanamycin Resistance; D019906:Nuclear Magnetic Resonance, Biomolecular; D009713:Nucleotidyltransferases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D011485:Protein Binding; D011487:Protein Conformation; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics; D012329:RNA, Bacterial / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D005839:Gentamicins; D012329:RNA, Bacterial; D007612:Kanamycin; C428104:Ant(4')-Ia protein, Staphylococcus aureus; D009713:Nucleotidyltransferases; C016927:isepamicin