Journal of Biological Chemistry
Volume 274, Issue 43, 22 October 1999, Pages 30697-30706
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ENZYMOLOGY
The COOH Terminus of Aminoglycoside Phosphotransferase (3′)-IIIa Is Critical for Antibiotic Recognition and Resistance*

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The aminoglycoside phosphotransferases (APHs) are widely distributed among pathogenic bacteria and are employed to covalently modify, and thereby detoxify, the clinically relevant aminoglycoside antibiotics. The crystal structure for one of these aminoglycoside kinases, APH(3′)-IIIa, has been determined in complex with ADP and analysis of the electrostatic surface potential indicates that there is a large anionic depression present adjacent to the terminal phosphate group of the nucleotide. This region also includes a conserved COOH-terminal α-helix that contains the COOH-terminal residue Phe264. We report here mutagenesis and computer modeling studies aimed at examining the mode of aminoglycoside binding to APH(3′)-IIIa. Specifically, seven site mutants were studied, five from the COOH-terminal helix (Asp261, Glu262, and Phe264), and two additional residues that line the wall of the anionic depression (Tyr55 and Arg211). Using a molecular modeling approach, six ternary complexes of APH(3′)-IIIa·ATP with the antibiotics, kanamycin, amikacin, butirosin, and ribostamycin were independently constructed and these agree well with the mutagenesis data. The results obtained show that the COOH-terminal carboxylate of Phe264 is critical for proper function of the enzyme. Furthermore, these studies demonstrate that there exists multiple binding modes for the aminoglycosides, which provides a molecular basis for the broad substrate- and regiospecificity observed for this enzyme.

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*

This work was supported in part by Medical Research Council of Canada Grants MT-13536 (to G. D. W.) and MT-13107 (to A. M. B.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recipient of a Natural Sciences and Engineering Research Council of Canada graduate scholarship.

Recipient of a Natural Sciences and Engineering Research Council of Canada graduate scholarship.

Recipient of a Pharmaceutical Association of Canada Health Research Foundation Research Career Award.