NMR studies of aminoglycoside antibiotics bound to an aminoglycoside antibiotic 3'-phosphotransferase

Author

James Richard Cox

Date of Award

8-1997

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biochemistry and Cellular and Molecular Biology

Major Professor

Engin H. Serpersu

Committee Members

Elizabeth Howell, Barry Bruce, David Baker

Abstract

Bacterial resistance to antibiotics is currently at a crisis level and has been declared a public health emergency. The once seemingly formidable array of effective antibiotics is declining, thus making previously treatable infections life- threatening. One of the most alarming examples of antibiotic resistance is that in Gram-positive cocci, which are responsible for a large fraction of hospital-acquired infections. Enterococcal and staphylococcal infections are typically treated through the administration of aminoglycoside antibiotics, which are a class of aminocyclitol antibiotics that bind to the bacterial 30S ribosomal subunit. They interfere with normal protein biosynthesis which ultimately leads to cell death. Bacteria have responded to the evolutionary pressure brought about by the use of these drugs by expressing an array of enzymes which covalently modify aminoglycosides, thereby rendering them inoffensive. These modifying enzymes can be classified as O- phosphotransferases (APH), O-nucleotidyltransferases, and N-acetyltransferases. The 3'-phosphotransferases are widely distributed in pathogenic bacteria, and at least seven different isozymes have been identified.

One particular enterococcal 3'-phosphotransferase, APH(3')-Illa, has the broadest substrate specificity of all the isozymes. Recently, this enzyme has been overexpressed and characterized as an ATP-dependent aminoglycoside kinase. A variety of NMR experiments were performed to determine the arrangement and conformation of APH(3')-Illa-bound aminoglycosides.

The complete one-dimensional ¹H assignments of various aminoglycosides were made through the use of various homo- and heteronuclear two-dimensional NMR methods. The complete ¹H NMR assignments were necessary for studies to determine the bound conformations of aminoglycosides.

The ¹⁵N resonances of butirosin A and ribostamycin were assigned, and the pK_a values of the amino groups of amikacin, butirosin A, and ribostamycin were determined by ¹⁵N NMR spectroscopy. The N-3 amino group of butirosin A and amikacin have lowered pK_a values, which are attributed to the (S)-4-amino-2- hydroxybutyryl group (AHB) group of the antibiotics, while the N-1 amino group of ribostamycin has a perturbed pK_a (much lower pK_a) which is attributed to charge repulsion with another amino group at N-3.

β, γ-Bidentate CrATP, a stable exchange-inert metal-nucleotide analog, was used as a paramagnetic probe to determine the arrangement of amikacin and butirosin A in their respective Enzyme•CrATP•Antibiotic complexes. The paramagnetic effects of Cr³⁺ on the longitudinal relaxation rates (1/T_1p) of the ¹H nuclei of amikacin and butirosin A were examined to determine the distances between enzyme-bound CrATP and various protons of these aminoglycoside antibiotics in the ternary APH(3')-Illa•CrATP•Antibiotic complexes. From these distances, models were constructed that represent possible enzyme-bound arrangements and conformations for these aminoglycosides. These models show that amikacin and butirosin A adopt different arrangements at the active site of APH(3')-Illa. The results for butirosin A suggest that the 2,6-diamino-2,6-dideoxy-D-glucose and D-xylose rings are in a stacking arrangement.

The NMR method of transferred nuclear Overhauser effect spectroscopy (TRNOESY) was used to detect intra- and inter-ring TRNOEs for amikacin, butirosin A and ribostamycin in their respective ternary complexes with APH(3')-Illa and ATP. NOE-derived distance restraints were used in energy minimization and dynamics routines to yield enzyme-bound structures for butirosin A and ribostamycin. The results for butirosin A suggest that the 2,6-diamino-2,6-dideoxy- D-glucose and D-xylose rings have restricted motions and are in a stacking arrangement. Two major conformers of enzyme-bound ribostamycin resulted from the energy minimization/molecular dynamics calculations. One of the conformers is similar to the structure of APH(3')-Illa-bound butirosin A with the primed ring (ring A) and the double-primed ring (ring C) in a stacking arrangement. The other conformer is similar to the structure of paromomycin bound to ribosomal RNA (rRNA), that represents the A-site of the small ribosomal subunit, where rings A and C are in a bisecting arrangement. The TRNOESY data for amikacin suggest that the 6-amino-6-deoxy-D-glucose ring is flexible when the antibiotic is bound to APH(3')- Illa.

This work is significant in that it demonstrates that aminoglycosides can use a similar conformation to bind both protein- and RNA-based targets. Additionally, this work provides insight into the geometrical and electrostatic nature of aminoglycoside antibiotics bound to a modifying enzyme and will provide a basis for the design of inhibitors of APH(3')-IIIa.

Recommended Citation

Cox, James Richard, "NMR studies of aminoglycoside antibiotics bound to an aminoglycoside antibiotic 3'-phosphotransferase. " PhD diss., University of Tennessee, 1997.
https://trace.tennessee.edu/utk_graddiss/9462