Cloning, overexpression, and purification of aminoglycoside antibiotic nucleotidyltransferase (2 '')-Ia: Conformational studies with bound substrates

Aminoglycoside nucleotidyltransferase (2")-Ia [AN'T (2")-Ia] was cloned fro m Pseudomonas aeruginosa and purified from overexpressing Escherichia coli BL21(DE3) cells. The first enzyme-bound conformation of an aminoglycoside a ntibiotic in the active site of an aminoglycoside nucleotidyltransferase wa s 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 b een determined using NMR spectroscopy. Molecular modeling, employing experi mentally 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 glycosid ic dihedral angles: Phi (BC) = -65.26 +/- 1.63 degrees and psi (BC) = -54.7 6 +/- 4.64 degrees. Conformer 1 was further subdivided into one major (conf ormer 1a) and two minor components (conformers 1b and 1c) based on the comp arison of glycosidic dihedral angles Phi (AB) and psi (AB). 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)(4)ATP on the relaxation rates of substrate protons which were used to determine relative distances of isepamicin protons to the Cr3+. Both confo rmers of isepamicin yielded arrangements that satisfied the NOE restraints and the observed paramagnetic effects of Cr(H2O)(4)ATP. It has been suggest ed that aminoglycosides use both electrostatic interactions and hydrogen bo nds in binding to RNA and that the contacts made by the A and B rings to RN A 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., Co x, J.R., DiGiammarino, E. L., Mohler, M. L., Ekman, D. R., Akal-Strader, A. , and Owston, M. (2000) Cell Biochem. Biophys. (in press)]. The conformatio n 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 co nformation of gentamycin C-1a also showed remarkable similarities between t he enzyme-bound and RNA-bound aminoglycoside antibiotic conformations. Thes e studies should aid in the design of effective inhibitors possessing a bro ad range of aminoglycoside-modifying enzymes as targets.