Substrate requirements for ErmC' methyltransferase activity

Abstract

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 K m 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 V max for this fragment also rose sevenfold. A model of RNA-ErmC interaction involving multiple binding sites is proposed from the kinetic data presented.Resistance to the macrolide-lincosamide-streptogramin B group of antibiotics in bacteria is mediated through the methylation of 23S rRNA by a series of methyltransferases (methylases) designated Erm (20). In general, Erm methylases confer resistance through the mono-or dimethylation of the N-6 of a specific adenine residue in 23S rRNA (A-2085 in Bacillus subtilis; A-2058 in Escherichia coli), which results in decreased affinity of methylated ribosomes for macrolide-lincosamidestreptogramin B antibiotics (9)(10)(11)20). Meier et al. (12) have shown that clarithromycin (an erythromycin analog)-resistant strains have undergone mutation at the A-2058-equivalent site to each of the other three nucleotides. Though the basis for macrolide-lincosamide-streptogramin B resistance has been known for more than 20 years, neither the mechanism of the methylation reaction nor the structure of the RNA segment that participates in the reaction is well understood. Kovalic et al. (8) and Vester and Douthwaite (22) have shown independently that the domain V (DV) segment of 23S rRNA, corresponding to the 23S rRNA sequences containing A-2085 from B. subtilis or A-2058 from E. coli, could serve as a substrate for in vitro methylation by ErmSF (from Streptomyces fradiae) and ErmE (from Saccharopolyspora erythraea), respectively.One of the better studied Erm methylases is ErmC, a 29,000-Da polypeptide encoded by plasmid pE194, originally found in Staphylococcus aureus (6) but subsequently transferred to B. subtilis (19). A variant, ErmCЈ, encoded by the B. subtilis plasmid pIM13 (13) differs in 5 of the 224 total amino acids and has been shown to be more stable than ErmC (21). In this paper we have cloned and expressed ermCЈ to a high level in E. coli and have purified the protein to virtual homogeneity. Studies on substrate requirements of ErmCЈ have shown that a 262-nucleotide (nt) RNA fragment within DV of B. subtilis 23S rRNA can be utilized efficiently as a substrate for methylation at A-2085. We also compared the kinetics of the monomethylation reaction using various RNAs as the substrates. MATERIALS A...

Plausible Minimal Substrate for Erm Protein

“…1a). In contrast to various domain V constructs used in previous studies (6)(7)(8)19), which contained extra nucleotides before the 5= end of helix 73 and/or after its 3= end, domain V harboring only the exact helix 73 was used in this study, from which gradual truncation was performed to define the end nucleotide or base pair for accepting the methyl group from S-adenosyl-L-methionine (SAM) with the action of Erm proteins (Fig. 1a).…”

Molecular dynamics simulations suggest why the A2058G mutation in 23S RNA results in bacterial resistance against clindamycin

“…Due to methylation, the minimal inhibitory concentrations required to inhibit bacterial growth increase from 2 to 512 μ g/ml for clindamycin and from 16 to 8000 μ g/ml for lincomycin. Bacteria resistant to lincosamides also exhibit resistance to macrolides [ 11 – 13 ] with a similar mechanism, i.e., due to modifications of A2058. In addition to bacterial resistance, lincosamides also have side effects such as diarrhea, nausea, rash, hypersensitivity, and jaundice [ 14 , 15 ], which further limit their efficiency in therapy.…”

Impact of Ribosomal Modification on the Binding of the Antibiotic Telithromycin Using a Combined Grand Canonical Monte Carlo/Molecular Dynamics Simulation Approach

“…Ketolides have largely addressed resistance due to drug efflux and metabolism yet still remain susceptible to ribosomal modification [13], [14], the most clinically relevant of which is modification of A2058 in 23S rRNA ( E. coli numbering throughout) that confers cross-resistance to antibiotics in the macrolide, lincosamide and streptogramin B classes (MLS) [15]–[18]. A2058-based resistance includes mutation of A2058 to G as well as methylation of the exocyclic N6 of A2058 via the expression of erm genes encoding methyltransferases (Erm methyltransferases) that add one or two methyl groups and represent the most effective mechanism of macrolide resistance [19]–[21]. Telithromycin and other ketolides have been found to mitigate macrolide resistance due to the A2058G mutation ( Table S1 in Text S1 ) [22]–[24], though ketolides that are widely effective against Erm methylation-based modifications are not known.…”