Substrate requirements for ErmC' methyltransferase activity.
ErmC' is a methyltransferase that confers resistance to the macrolide-lincosamide-streptogramin B group of antibiotics by catalyzing the methylation of 23S rRNA at a specific adenine residue (A-2085 in Bacillus subtilis; A-2058 in Escherichia coli). The gene for ErmC' was cloned and expressed to a high level in E. coli, and the protein was purified to virtual homogeneity. Studies of substrate requirements of ErmC' have shown that a 262-nucleotide RNA fragment within domain V of B. subtilis 23S rRNA can be utilized efficiently as a substrate for methylation at A-2085. Kinetic studies of the monomethylation reaction showed that the apparent Km of this 262-nucleotide RNA oligonucleotide was 26-fold greater than the value determined for full-size and domain V 23S rRNA. In addition, the Vmax for this fragment also rose sevenfold. A model of RNA-ErmC' interaction involving multiple binding sites is proposed from the kinetic data presented.
Zhong
P
P
Pharmaceutical Products Division, Abbott Laboratories, Abbott Park, Illinois 60064, USA.
Pratt
S D
SD
Edalji
R P
RP
Walter
K A
KA
Holzman
T F
TF
Shivakumar
A G
AG
Katz
L
L
D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D015964:Gene Expression Regulation, Bacterial; D007700:Kinetics; D008745:Methylation; D008780:Methyltransferases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D010458:Peptidyl Transferases / Q000235:genetics / Q000378:metabolism; D012329:RNA, Bacterial / Q000378:metabolism; D012338:RNA, Ribosomal, 23S / Q000378:metabolism; D013379:Substrate Specificity
D009838:Oligodeoxyribonucleotides; D012329:RNA, Bacterial; D012338:RNA, Ribosomal, 23S; D008780:Methyltransferases; C035181:rRNA (adenosine-O-2'-)methyltransferase; D010458:Peptidyl Transferases