title: Heterologous Escherichia coli expression, purification and characterization of the GrmA aminoglycoside-resistance methyltransferase.
abstract: The mechanism of resistance to aminoglycosides based on methylation of their target, 16S rRNA, was until recently described only in antibiotic producing microorganisms. However, equivalent methyltransferases have now also been identified among numerous clinical Gram-negative pathogenic isolates. We have cloned, expressed, and purified GrmA, the aminoglycoside-resistance methyltransferase from Micromonospora purpurea, producer of gentamicin complex. Two vectors were created that express protein with an N-terminal 6x histidine tag with and without an enterokinase recognition producing proteins His(6)-EK-GrmA and His(6)-GrmA, respectively. The activity of both recombinant proteins was demonstrated in vivo. After optimized expression and native purification both protein variants proved to be active in in vitro methylation assays. This work lays a foundation for future detailed biochemical, structural and pharmacological studies with this member of an important group of aminoglycoside-resistance enzymes.
authors.affiliation: Institute of Molecular Genetics and Genetic Engineering, Laboratory for Molecular Genetics of Actinomycetes, University of Belgrade, Vojvode Stepe 444a, P.O. Box 23, 11010, Beograd, Serbia.
authors.forename: Ivana
authors.initials: I
authors.lastname: Moric
authors.forename: Sanja
authors.initials: S
authors.lastname: Bajkic
authors.forename: Miloje
authors.initials: M
authors.lastname: Savic
authors.forename: Tatjana Ilic
authors.initials: TI
authors.lastname: Tomic
authors.forename: Graeme L
authors.initials: GL
authors.lastname: Conn
authors.forename: Branka
authors.initials: B
authors.lastname: Vasiljevic
mesh_terms: D000617:Aminoglycosides / Q000494:pharmacology*; D001426:Bacterial Proteins; D003001:Cloning, Molecular / Q000379:methods*; D004351:Drug Resistance; D004926:Escherichia coli / Q000235:genetics*; D006639:Histidine; D008780:Methyltransferases / Q000096:biosynthesis* / Q000302:isolation & purification; D008847:Micromonospora / Q000201:enzymology
chemical_list: D000617:Aminoglycosides; D001426:Bacterial Proteins; D006639:Histidine; D008780:Methyltransferases