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KMID : 0364820120480020079
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Korean Journal of Microbiology
2012 Volume.48 No. 2 p.79 ~ p.86
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Site-directed Mutagenesis Analysis Elucidates the Role of 223/227 Arginine in 23S rRNA Methylation,Which Is in ‘Target Adenine Binding Loop' Region of ErmSF
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Jin Hyung-Jong
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Abstract
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ErmSF is one of the Erm family proteins which catalyze S-adenosyl-L-methionine dependent modification of a
specific adenine residue (A2058, E. coli numbering) in bacterial 23S rRNA, thereby conferring resistance to
clinically important macrolide, lincosamide and streptogramin B (MLSB) antibiotics. 222FXPXPXVXS230 (ErmSF
numbering) sequence appears to be a consensus sequence among the Erm family. This sequence was supposed to be
involved in direct interaction with the target adenine from the structural studies of Erm protein ErmC'. But in DNA
methyltarnsferase M. Taq I, this interaction have been identified biochemically and from the complex structure with
substrate. Arginine 223 and 227 in this sequence are not conserved among Erm proteins, but because of the basic
nature of residues, it was expected to interact with RNA substrates. Two amino acid residues were replaced with Ala
by site-directed mutagenesis. Two mutant proteins still maintained its activity in vivo and resistant to the antibiotic
erythromycin. Compared to the wild-type ErmSF, R223A and R227A proteins retained about 50% and 88% of
activity in vitro, respectively. Even though those arginine residues are not essential in the catalytic step, with their
positive charge they may play an important role for RNA binding.
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KEYWORD
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in vivo, in vitro activity test, MLSB (macrolide-lincosamide-streptogramin B) antibiotic resistance factorprotein, overexpression, site-directed mutagenesis
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