Expression,purification and crystallization of adenosine 1408 aminoglycoside-resistance rRNA methyltransferases for structural studies |
| |
| Authors: | Natalia Zelinskaya C Robert Rankin Rachel Macmaster Miloje Savic Graeme L Conn |
| |
| Institution: | 1. Department of Biochemistry, Emory University School of Medicine, 1510 Clifton Road NE, Atlanta, GA 30322, USA;2. Biochemistry, Cell and Developmental Biology Program, Emory University School of Medicine, 1510 Clifton Road NE, Atlanta, GA 30322, USA;3. Visiting Researcher from Manchester Interdisciplinary Biocentre, Faculty of Life Sciences, University of Manchester, 131 Princess Street, Manchester M1 7DN, UK;1. Departments of Surgery, Texas Tech University Health Sciences Center, Lubbock, TX, United States;2. Immunology and Molecular Microbiology, Texas Tech University Health Sciences Center, Lubbock, TX, United States;3. TTUHSC Surgery Burn Center of Research Excellence, Texas Tech University Health Sciences Center, Lubbock, TX, United States;1. Nano-Bio Interfacial Research Laboratory (NBIRL), Department of Biotechnology, Siddaganga Institute of Technology, BH Road, Tumkur, 572103, Karnataka, India;2. Department of Molecular Biology and Biotechnology, Rajasthan College of Agriculture, Udaipur, 313001, Rajasthan, India;3. Department of Applied Physiology, Faculty of Medicine, University of Miyazaki, 8891692, Miyazaki, Japan;4. Amity Institute of Biotechnology, Amity University Rajasthan, Kant Kalwar, NH-11C, Jaipur Delhi Highway, Jaipur, 303002, Rajasthan, India;1. Synthesis and Solid State Pharmaceutical Centre (SSPC), School of Chemical and Bioprocess Engineering, University College Dublin, Dublin 4, Ireland;2. APC Ltd, Dublin 4, Ireland |
| |
| 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 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. |
| |
| Keywords: | |
| 本文献已被 ScienceDirect 等数据库收录！ |
|
