Functional roles in S-adenosyl-L-methionine binding and catalysis for active site residues of the thiostrepton resistance methyltransferase

• Type of Material: Article
• Authors: Cullen L. Myers, University of Waterloo; Emily G. Kuiper, Emory University; Pei C. Grant, University of Waterloo; Jennifer Hernandez, Emory University; Graeme Conn, Emory University; John F. Honek, University of Waterloo
• Language: English
• Date: 2015-10-24
• Publisher: Elsevier
• Publication Version: Author Accepted Manuscript (After Peer Review)
• Copyright Statement: © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
• Final Published Version (URL): http://dx.doi.org/10.1016/j.febslet.2015.09.028
• Title of Journal or Parent Work: FEBS Letters
• ISSN: 0014-5793
• Volume: 589
• Issue: 21
• Start Page: 3263
• End Page: 3270
• Grant/Funding Information: Financial support was provided by the Natural Sciences and Engineering Research Council of Canada (JFH), the Ontario Graduate Scholarship program (CLM), the USDA National Institute of Food and Agriculture (Agriculture and Food Research Initiative Competitive Grant No. 2013-67011-21133) (EGK), the National Institutes of Health (R01-AI088025) (GLC), the Howard Hughes Medical Institute Science Education Program (award #52006923) and the National Science Foundation (MRI program grant 104177).
• Keywords: Thiostrepton; Biochemistry & Molecular Biology; ELONGATION-FACTOR-G; Science & Technology; Isothermal titration calorimetry; PROTEIN; SPOUT methyltransferase; Cell Biology; S-adenosyl-L-methionine; Biophysics; METHYLATION; Enzyme kinetics; Life Sciences & Biomedicine; CRYSTAL-STRUCTURE
• Research Categories: Biophysics, Medical; Chemistry, Biochemistry

Abstract

Resistance to the antibiotic thiostrepton, in producing Streptomycetes, is conferred by the S-adenosyl-l-methionine (SAM)-dependent SPOUT methyltransferase Tsr. For this and related enzymes, the roles of active site amino acids have been inadequately described. Herein, we have probed SAM interactions in the Tsr active site by investigating the catalytic activity and the thermodynamics of SAM binding by site-directed Tsr mutants. Two arginine residues were demonstrated to be critical for binding, one of which appears to participate in the catalytic reaction. Additionally, evidence consistent with the involvement of an asparagine in the structural organization of the SAM binding site is presented.