Expression, purification and crystallization of adenosine 1408 aminoglycoside-resistance rRNA methyltransferases for structural studies

• Type of Material: Article
• Authors: Natalia Zelinskaya, Emory University; C. Robert Rankin, Emory University; Rachel Macmaster, Emory University; Miloje Savic, Emory University; Graeme L Conn, Emory University
• Language: English
• Date: 2011-01
• Publisher: Elsevier
• Publication Version: Author Accepted Manuscript (After Peer Review)
• Copyright Statement: © 2010 Elsevier Inc. All rights reserved.
• License: Creative Commons Attribution-NonCommerical-NoDerivs 3.0 Unported
• Final Published Version (URL): http://dx.doi.org/10.1016/j.pep.2010.07.005
• Title of Journal or Parent Work: Protein Expression and Purification
• ISSN: 1046-5928
• Volume: 75
• Issue: 1
• Start Page: 89
• End Page: 94
• Grant/Funding Information: This work was supported by the National Institutes of Health [AI088025] and in part by laboratory start-up funds from the Department of Biochemistry, Emory University School of Medicine.; We thank Dr. Ewan W. Blanch for management of the Wellcome Trust grant [079242] providing salary support for RM.
• Research Categories: Chemistry, Biochemistry; Biology, Cell

Abstract

High-level resistance to a broad spectrum of aminoglycoside antibiotics can arise through either N7-methyl guanosine 1405 (m7G1405) 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 E. 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.