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Journal of Bacteriology, July 2009, p. 4133-4143, Vol. 191, No. 13
0021-9193/09/$08.00+0     doi:10.1128/JB.00149-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

The Crystal Structures of Substrate and Nucleotide Complexes of Enterococcus faecium Aminoglycoside-2''-Phosphotransferase-IIa [APH(2'')-IIa] Provide Insights into Substrate Selectivity in the APH(2'') Subfamily ^,

Paul G. Young,¹ Rupa Walanj,¹ Vendula Lakshmi,¹ Laura J. Byrnes,^2, Peter Metcalf,¹ Edward N. Baker,¹ Sergei B. Vakulenko,³ and Clyde A. Smith^2*

School of Biological Sciences, University of Auckland, Auckland, New Zealand,¹ Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, California,² Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana³

Received 3 February 2009/ Accepted 28 April 2009

Aminoglycoside-2''-phosphotransferase-IIa [APH(2'')-IIa] is one of a number of homologous bacterial enzymes responsible for the deactivation of the aminoglycoside family of antibiotics and is thus a major component in bacterial resistance to these compounds. APH(2'')-IIa produces resistance to several clinically important aminoglycosides (including kanamycin and gentamicin) in both gram-positive and gram-negative bacteria, most notably in Enterococcus species. We have determined the structures of two complexes of APH(2'')-IIa, the binary gentamicin complex and a ternary complex containing adenosine-5'-(β,-methylene)triphosphate (AMPPCP) and streptomycin. This is the first crystal structure of a member of the APH(2'') family of aminoglycoside phosphotransferases. The structure of the gentamicin-APH(2'')-IIa complex was solved by multiwavelength anomalous diffraction methods from a single selenomethionine-substituted crystal and was refined to a crystallographic R factor of 0.210 (R_free, 0.271) at a resolution of 2.5 Å. The structure of the AMPPCP-streptomycin complex was solved by molecular replacement using the gentamicin-APH(2'')-IIa complex as the starting model. The enzyme has a two-domain structure with the substrate binding site located in a cleft in the C-terminal domain. Gentamicin binding is facilitated by a number of conserved acidic residues lining the binding cleft, with the A and B rings of the substrate forming the majority of the interactions. The inhibitor streptomycin, although binding in the same pocket as gentamicin, is orientated such that no potential phosphorylation sites are adjacent to the catalytic aspartate residue. The binding of gentamicin and streptomycin provides structural insights into the substrate selectivity of the APH(2'') subfamily of aminoglycoside phosphotransferases, specifically, the selectivity between the 4,6-disubstituted and the 4,5-disubstituted aminoglycosides.

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* Corresponding author. Mailing address: Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CA. Phone: (650) 926-8544. Fax: (650) 926-3292. E-mail: csmith{at}slac.stanford.edu

Published ahead of print on 1 May 2009.

Supplemental data for this article may be found at http://jb.asm.org/.

Present address: Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY.

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Journal of Bacteriology, July 2009, p. 4133-4143, Vol. 191, No. 13
0021-9193/09/$08.00+0     doi:10.1128/JB.00149-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.