Abstract
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 alpha-helix that contains the COOH-terminal residue Phe(264). 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 (Asp(261), Glu(262), and Phe(264)), and two additional residues that line the wall of the anionic depression (Tyr(55) and Arg(211)). 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 Phe(264) 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.
Highlights
The aminoglycoside antibiotics constitute an important class of anti-infective agents which find use in the treatment of bacterial and some protozoal infections [1]
The C-terminal helix of aminoglycoside phosphotransferases (APHs)(3Ј)-IIIa contains the amino acid sequence, Leu260-Asp-Glu-Leu-Phe-COOH, a sequence which is highly conserved among the 3Ј-aminoglycoside kinases [6]
The studies presented here demonstrate the importance of COOH-terminal residues in aminoglycoside recognition in the aminoglycoside antibiotic resistance enzyme APH(3Ј)-IIIa
Summary
The aminoglycoside antibiotics constitute an important class of anti-infective agents which find use in the treatment of bacterial and some protozoal infections [1]. We have studied the aminoglycoside kinase, APH1(3Ј)-IIIa, which is harbored by Gram-positive cocci such as the enterococci and staphylococci, both highly relevant human pathogens, especially in clinical institutions. This enzyme has a broad aminoglycoside substrate specificity with the demonstrated capacity to phosphorylate aminoglycosides at position 3Ј, and for aminoglycosides with a pentose linked to position 6 of the 2-deoxyaminocyclitol ring, position 5Љ (see Fig. 1 for representative structures and ring numbering) [3, 4]. The x-ray structure of the APH(3Ј)-IIIa1⁄7ADP binary complex has been determined to 2.2 Å and demonstrates striking similarity with the Ser/Thr/Tyr protein kinase family [7] This observation is paralleled by work which indicates that APH(3Ј)-IIIa has a similar chemical mechanism as protein kinases [8]. Our results demonstrate that the COOH-terminal residue of the enzyme is critical for efficient substrate binding and indicate that the bulk of the aminoglycoside specificity lies in the COOH-terminal domain, but that there are many binding modes for aminoglycoside substrates
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