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Cloning, overexpression, and purification of aminoglycoside antibiotic 3-acetyltransferase-IIIb: conformational studies with bound substrates.
- Source :
-
Biochemistry [Biochemistry] 2002 Sep 03; Vol. 41 (35), pp. 10764-70. - Publication Year :
- 2002
-
Abstract
- 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.
- Subjects :
- Anti-Bacterial Agents biosynthesis
Binding Sites
Cloning, Molecular methods
Kanamycin chemistry
Kanamycin Kinase biosynthesis
Kanamycin Kinase chemistry
Nuclear Magnetic Resonance, Biomolecular
Protein Conformation
Pseudomonas aeruginosa enzymology
Pseudomonas aeruginosa genetics
RNA, Bacterial chemistry
Ribostamycin chemistry
Substrate Specificity
Anti-Bacterial Agents chemistry
Kanamycin Kinase genetics
Kanamycin Kinase isolation & purification
Subjects
Details
- Language :
- English
- ISSN :
- 0006-2960
- Volume :
- 41
- Issue :
- 35
- Database :
- MEDLINE
- Journal :
- Biochemistry
- Publication Type :
- Academic Journal
- Accession number :
- 12196014
- Full Text :
- https://doi.org/10.1021/bi0261241