title: Functional roles in S-adenosyl-L-methionine binding and catalysis for active site residues of the thiostrepton resistance methyltransferase.
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.
authors.affiliation: Department of Chemistry, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada.
authors.forename: Cullen L
authors.initials: CL
authors.lastname: Myers
authors.affiliation: Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322, USA.
authors.forename: Emily G
authors.initials: EG
authors.lastname: Kuiper
authors.affiliation: Department of Chemistry, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada.
authors.forename: Pei C
authors.initials: PC
authors.lastname: Grant
authors.affiliation: Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322, USA.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Hernandez
authors.affiliation: Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322, USA.
authors.forename: Graeme L
authors.initials: GL
authors.lastname: Conn
authors.affiliation: Department of Chemistry, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada. Electronic address: jhonek@uwaterloo.ca.
authors.forename: John F
authors.initials: JF
authors.lastname: Honek
mesh_terms: D000900:Anti-Bacterial Agents; D001120:Arginine / Q000378:metabolism; D001216:Asparagine / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D020134:Catalytic Domain; D002942:Circular Dichroism; D024881:Drug Resistance, Bacterial; D008780:Methyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D012436:S-Adenosylmethionine / Q000378:metabolism*; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics; D013883:Thiostrepton
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D001216:Asparagine; D012436:S-Adenosylmethionine; D001120:Arginine; D008780:Methyltransferases; D013883:Thiostrepton
keywords: Enzyme kinetics; Isothermal titration calorimetry; S-adenosyl-l-methionine; SPOUT methyltransferase; Thiostrepton