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Aminoglycoside O-Nucleotidyltransferases

There are five members of the Aminoglycoside O-Nucleotidyltransferases (ANT) family of enzymes, ANT(2 ), ANT(4 ),
ANT(3 ), ANT(6 ) and ANT(9).

ANT(2 )-Ia is a highly prevalent cause of gentamicin resistance in North America and is one of the most clinically significant members of this family of enzymes [1], and it is widespread among Gram-negatives. The ant(2 )-Ia gene has been found on small nonconjugative plasmids, conjugative plasmids, transposons and integrons. The enzyme follows a kinetic mechanism in which the Mg–ATP complex first binds to ANT(2 )-Ia followed by the binding of the aminoglycoside, leading to the transfer of the adenyl moiety to the aminoglycoside. The rate-limiting step of this reaction is the release of the adenylylated aminoglycoside [2-4].

The NMR structure of the isepamicin bound to the enzyme together with a Cr–ATP complex has been reported [5]. The staphylococcal
enzyme ANT(4 )-Ia has been crystallized, and the structure has been solved in the presence of substrates [6]. The X-ray structure of this enzyme revealed it to be an obligate dimer, and each subunit interacts with the substrates in an asymmetric fashion.

One subunit contributes the residues required for binding ATP and Mg2+, and the other subunit provides Lys149 required for transfer the adenyl moiety to the target aminoglycoside. Both subunits are involved in aminoglycoside binding, and the intersection of the two subunits
generates a negatively charged pattern on the surface of ANT(4 )-Ia that is involved in recruiting the positively charged aminoglycoside to the active-site binding pocket of the enzyme. The structure of this enzyme also has significant structural homology to NMP-transferases, specifically rat DNA-polymerase b [7], another nucleotidylyltransferase.

 


1. Miller, G. H.; Sabatelli, F. J.; Naples, L.; Hare, R. S.; Shaw, K. J.; J. Chemother. 1995, 7 Suppl. 2, 17.

2. Gates, C. A.; Northrop, D. B. Biochemistry 1988, 27, 3820.

3. Gates, C. A.; Northrop, D. B. Biochemistry 1988, 27, 3826.

4. Gates, C. A.; Northrop, D. B. Biochemistry 1988, 27, 3834.

5. Ekman, D. R.; DiGiammarino, E. L.; Wright, E.; Witter, E. D.; Serpersu, E. H. Biochemistry 2001, 40, 7017.

6. Pedersen, L. C.; Benning, M. M.; Holden, H. M. Biochemsitry 1995, 34, 13305.

7. Holm, L.; Sander, C. Trends Biol. Chem. 1995, 20, 345.

 

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