BIOLOGICALLY IMPORTANT CONFORMATIONS OF AMINOGLYCOSIDE ANTIBIOTICS BOUND TO AN AMINOGLYCOSIDE 3'-PHOSPHOTRANSFERASE AS DETERMINED BY TRANSFERRED NUCLEAR OVERHAUSER EFFECT SPECTROSCOPY
Jr. Cox et Eh. Serpersu, BIOLOGICALLY IMPORTANT CONFORMATIONS OF AMINOGLYCOSIDE ANTIBIOTICS BOUND TO AN AMINOGLYCOSIDE 3'-PHOSPHOTRANSFERASE AS DETERMINED BY TRANSFERRED NUCLEAR OVERHAUSER EFFECT SPECTROSCOPY, Biochemistry, 36(9), 1997, pp. 2353-2359
NMR spectroscopy has been used to study the interaction of aminoglycos
ide antibiotics with an aminoglycoside antibiotic 3'-phosphotransferas
e [APH(3')-IIIa]. APH(3')IIIa is an enterococcal enzyme that is respon
sible for the ATP-dependent O-phosphorylation of a broad range of amin
oglycoside antibiotics. The NMR method of transferred nuclear Overhaus
er effect spectroscopy (TRNOESY) was used to detect intra- and inter-r
ing NOEs for butirosin A and amikacin in their respective ternary comp
lexes with APH(3')-IIIa and ATP. NOE-derived distance constraints were
used in energy minimization and dynamics routines to yield enzyme-bou
nd structures for butirosin A. These structures suggest that the 2,6-d
iamino-2,6-dideoxy-D-glucose and D-xylose rings have restricted motion
s and are in a stacking arrangement. The TRNOE spectra for amikacin su
ggest that the 6-amino-6-deoxy-D-glucose ring is flexible when the ant
ibiotic is bound to APH(3')-IIIa. The N-15 resonances of butirosin A w
ere assigned and the pK(a) values of the amino groups of butirosin A a
nd amikacin were determined by N-15 NMR spectroscopy. The N3 amino gro
ups of butirosin A and amikacin have lowered pK(a) values, which is at
tributed to the (S)-4-amino-2-hydroxybutyryl (AHB) group of the antibi
otics. This work provides an insight into the geometrical and electros
tatic nature of aminoglycoside antibiotics bound to a modifying enzyme
and will provide a basis for the design of inhibitors of APH(3')-IIIa
.