Linked Life Data

20667473

Source:http://linkedlifedata.com/resource/pubmed/id/20667473

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
1
pubmed:dateCreated
2010-10-29
pubmed:abstractText
High-level resistance to a broad spectrum of aminoglycoside antibiotics can arise through either N7-methyl guanosine 1405 (m?G1405) or N1-methyl adenosine 1408 (m¹A1408) modifications at the drug binding site in the bacterial 30S ribosomal subunit decoding center. Two distinct families of 16S ribosomal RNA (rRNA) methyltransferases that incorporate these modifications were first identified in aminoglycoside-producing bacteria but were more recently identified in both human and animal pathogens. These resistance determinants thus pose a new threat to the usefulness of aminoglycosides as antibiotics, demanding urgent characterization of their structures and activities. Here, we describe approaches to cloning, heterologous expression in Escherichia coli, and purification of two A1408 rRNA methyltransferases: KamB from the aminoglycoside-producer Streptoalloteichus tenebrarius and NpmA identified in a clinical isolate of pathogenic E. coli ARS3. Antibiotic minimum inhibitory concentration (MIC) assays and in vitro analysis of KamB and NpmA using circular dichroism (CD) spectroscopy, S-adenosyl-l-methionine (SAM) binding by isothermal titration calorimetry and 30S subunit methylation assays showed both enzymes were soluble, folded and active. Finally, crystals of each enzyme complexed with SAM were obtained, including selenomethionine-derived KamB, that will facilitate high-resolution X-ray crystallographic analyses of these important bacterial antibiotic-resistance determinants.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jan
pubmed:issn
1096-0279
pubmed:author
pubmed:copyrightInfo
Copyright © 2010 Elsevier Inc. All rights reserved.
pubmed:issnType
Electronic
pubmed:volume
75
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
89-94
pubmed:meshHeading
pubmed:year
2011
pubmed:articleTitle
Expression, purification and crystallization of adenosine 1408 aminoglycoside-resistance rRNA methyltransferases for structural studies.
pubmed:affiliation
Department of Biochemistry, Emory University School of Medicine, 1510 Clifton Road NE, Atlanta, GA 30322, USA.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't, Research Support, N.I.H., Extramural