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.

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dc.contributor.author Young, Paul en
dc.contributor.author Walanj, Rupa en
dc.contributor.author Lakshmi, V en
dc.contributor.author Byrnes, LJ en
dc.contributor.author Metcalf, Peter en
dc.contributor.author Baker, Edward en
dc.contributor.author Vakulenko, SB en
dc.contributor.author Smith, CA en
dc.date.accessioned 2012-03-21T02:56:35Z en
dc.date.issued 2009 en
dc.identifier.citation JOURNAL OF BACTERIOLOGY 191(13):4133-4143 01 Jul 2009 en
dc.identifier.issn 0021-9193 en
dc.identifier.uri http://hdl.handle.net/2292/14899 en
dc.description.abstract 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'-(beta,gamma-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 A. 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. en
dc.description.uri http://jb.asm.org/cgi/content/full/191/13/4133?view=long&pmid=19429619 en
dc.publisher American Society for Microbiology en
dc.relation.ispartofseries Journal of Bacteriology en
dc.rights Items in ResearchSpace are protected by copyright, with all rights reserved, unless otherwise indicated. Previously published items are made available in accordance with the copyright policy of the publisher. Details obtained from: http://www.sherpa.ac.uk/romeo/issn/0021-9193/ en
dc.rights.uri https://researchspace.auckland.ac.nz/docs/uoa-docs/rights.htm en
dc.title 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. en
dc.type Journal Article en
dc.identifier.doi 10.1128/​JB.00149-09 en
pubs.issue 13 en
pubs.begin-page 4133 en
pubs.volume 191 en
dc.rights.holder Copyright: American Society for Microbiology en
dc.identifier.pmid 19429619 en
pubs.end-page 4143 en
dc.rights.accessrights http://purl.org/eprint/accessRights/RestrictedAccess en
pubs.subtype Article en
pubs.elements-id 86558 en
pubs.org-id Academic Services en
pubs.org-id Examinations en
pubs.org-id Science en
pubs.org-id Biological Sciences en
pubs.org-id Science Research en
pubs.org-id Maurice Wilkins Centre (2010-2014) en
pubs.record-created-at-source-date 2010-09-01 en
pubs.dimensions-id 19429619 en


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