ARRANGEMENT OF SUBSTRATES AT THE ACTIVE-SITE OF AN AMINOGLYCOSIDE ANTIBIOTIC 3'-PHOSPHOTRANSFERASE AS DETERMINED BY NMR

The arrangements of the antibiotics amikacin and butirosin A at the ac tive site of an aminoglycoside antibiotic 3'-phosphotransferase (APH(3 ')-IIIa), which mediates resistance to a broad spectrum of aminoglycos ide antibiotics, were determined. APH(3')-IIIa phosphorylates a wide r ange of aminoglycoside antibiotics in an ATP-dependent manner. beta,ga mma-Bidentate CrATP, a stable exchange-inert metal-nucleotide analog, was used as a paramagnetic probe to determine the arrangement of amika cin and butirosin A in the respective enzyme CrATP antibiotic complexe s. The paramagnetic effects of Cr3+ on the longitudinal relaxation rat es (1/T-1p) of the H-1 nuclei of amikacin and butirosin A were examine d to determine the distances between enzyme-bound CrATP and various pr otons of these aminoglycoside antibiotics in the ternary APH(3')-IIIa CrATP antibiotic complexes. From these distances, models were construc ted that represent possible enzyme-bound arrangements and conformation s for these aminoglycosides. These models show that amikacin and butir osin A adopt different arrangements at the active site of APH(3')-IIIa . 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 which is c onsistent with its solution structure. This is the first paper to desc ribe the arrangement and conformation of aminoglycoside antibiotics bo und to a modifying enzyme and is an important step in the design of no vel antibiotics and/or enzyme inhibitors.