Cloning, overexpression, and purification of aminoglycoside antibiotic 3-acetyltransferase-IIIb: conformational studies with bound substrates.
Aminoglycoside 3-acetyltransferase-IIIb (AAC3), which acetylates N3 amine of aminoglycoside antibiotics, was cloned from P. Aeruginosa and purified from overexpressing E. coli BL21 (DE3) cells. Bound conformations of kanamycin A and ribostamycin, in the active site of the enzyme that modifies the essential N3B of aminoglycoside antibiotics, were determined by NMR spectroscopy. Experimentally determined interproton distances were used in a simulated annealing protocol to determine enzyme-bound conformations of both antibiotics. Two conformations, consistent with the NOE restraints, were determined for ribostamycin. The only difference between the two conformers was the orientation of the A ring with respect to the rest of the molecule. The average glycosidic dihedral angles were Phi(1A) = -22 degrees +/- 3 and Psi(1A) = -42 degrees +/- 1 (conformer 1) and Phi(1A) = -67 degrees +/- 0.7 and Phi(1A) = -59 degrees +/- 0.8 (conformer 2). Three conformers were determined for the enzyme-bound kanamycin A. Two conformers of kanamycin A were matched well with the two conformers of ribostamycin when the A and the B rings of the antibiotics were superimposed. Conformations of kanamycin A and ribostamycin were compared to those of other aminoglycosides that are bound to different enzymes and RNA. The results lend further support to our earlier hypothesis that the A and B rings of aminoglycosides adopt a conformation that is recognized not only by the aminoglycoside-modifying enzymes but also by RNA (Serpersu, E. H., Cox, J. R., Digiammarino, E. L., Mohler, M. L., Akal, A., Ekman, D. R., and Owston, M. (2000) Cell Biochem. Biophys. 33, 309-321). These results may be useful in designing new antibiotics to combat the antibiotic resistance against infectious diseases.
Owston
Michael A
MA
The Center of Excellence for Structural Biology and the Department of Biochemistry and Cell and Molecular Biology, The University of Tennessee, Walters Life Sciences building, M407 Knoxville, Tennessee 37996-0840, USA.
Serpersu
Engin H
EH
D000900:Anti-Bacterial Agents / Q000096:biosynthesis / Q000737:chemistry*; D001665:Binding Sites; D003001:Cloning, Molecular / Q000379:methods; D007612:Kanamycin / Q000737:chemistry; D019868:Kanamycin Kinase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D011550:Pseudomonas aeruginosa / Q000201:enzymology / Q000235:genetics; D012329:RNA, Bacterial / Q000737:chemistry; D012271:Ribostamycin / Q000737:chemistry; D013379:Substrate Specificity
D000900:Anti-Bacterial Agents; D012329:RNA, Bacterial; D012271:Ribostamycin; D007612:Kanamycin; D019868:Kanamycin Kinase