No abstract

Erm proteins methylate a specific adenine residue (A2058, Escherichia coli coordinates) conferring MLSB (macrolide–lincosamide–streptogramin B) antibiotic resistance on a variety of microorganisms ranging from antibiotic producers to pathogens. To identify the minimal motif required to be recognized and methylated by Erm protein, various RNA substrates from 23S rRNA were constructed, and the substrate activity of these constructs was studied using three Erm proteins: ErmB from Firmicutes and ErmE and ErmS from Actinobacteria. The shortest motif of 15 nt could be recognized and methylated by ErmS, consisting of A2051 to the methylatable adenine (A2058) and its base-pairing counterpart strand presumably assuming a quite similar structure to that in 23S rRNA, an unpaired target adenine immediately followed by an irregular double-stranded RNA region. This observation confirms the ultimate end of each side in stem 73 for methylation, determined by the approaches described above, and could reveal the mechanism behind the binding, recognition, induced fit, methylation, and conformational change for product release in the minimal context of substrate, presumably with the help of structural determination of protein–RNA complex. In the course of determining the minimal portion of substrate from domain V, protein-specific features could be observed among the Erm proteins in terms of the methylation of RNA substrate and cooperativity and/or allostery between the region in stem 73 furthest away from the target adenine and the large portion of domain V above the methylatable adenine.

Erms are proteins that methylate the adenine (A2058) in Escherichia coli 23S rRNA, which results in resistance to macrolide, lincosamide, and streptogramin B antibiotics. In a previous report, ErmN appeared to be more susceptible to contaminating proteases in DNase I. To determine the underlying mechanism, cleavage with chymotrypsin over time was investigated. ErmN possesses unusually high-susceptibility recognition site (F45) as evidenced by a band (band 1) that represented greater than 80% of the total band intensity at 30 s. The exposure rate of the hydrophobic core was more than 67-fold and 104-fold faster in ErmN than those in ErmS and ErmE, respectively. After cleavage at F45, some of the hydrophobic interactions were disrupted. Further digestion of band 1 occurred through the exposed F163 with a half-life of 3.18 min. After 30 min, less than 1% of ErmN remained. On the basis of the structure of ErmC′, the location of F45 was presumed to be in an α helix at the bottom of a cavity. Both substitution of most common amino acids such as isoleucine, valine, or leucine with phenylalanine (ErmH, ErmI, ErmN, and ErmZ out of the 37 known Erms) and the apparent added flexibility, which could result from the additional loop region attached to phenylalanine that is four to nine amino acids longer (ErmI, ErmN, and ErmZ, which form one cluster in the phylogenetic tree), could cause unusually high susceptibility. The unexpectedly high susceptibility among the homologous proteins could indicate that caution should be taken not to misinterpret the observations when conducting any procedure in which protease or protease contamination is involved.

Macrolide–lincosamide–streptogramin B antibiotic resistance occurs through the action of erythromycin ribosome methylation (Erm) family proteins, causing problems due to their prevalence and high minimal inhibitory concentration, and feasibilities have been sought to develop inhibitors. Erms exhibit high conservation next to the N-terminal end region (NTER) as in ErmS, 64SQNF67. Side chains of homologous S, Q and F in ErmC’ are surface-exposed, located closely together and exhibit intrinsic flexibility; these residues form a motif X. In S64 mutations, S64G, S64A and S64C exhibited 71%, 21% and 20% activity compared to the wild-type, respectively, conferring cell resistance. However, mutants harboring larger side chains did not confer resistance and retain the methylation activity in vitro. All mutants of Q65, Q65N, Q65E, Q65R, and Q65H lost their methyl group transferring activity in vivo and in vitro. At position F67, a size reduction of side-chain (F67A) or a positive charge (F67H) greatly reduced the activity to about 4% whereas F67L with a small size reduction caused a moderate loss, more than half of the activity. The increased size by F67Y and F67W reduced the activity by about 75%. In addition to stabilization of the cofactor, these amino acids could interact with substrate RNA near the methylatable adenine presumably to be catalytically well oriented with the SAM (S-adenosyl-L-methionine). These amino acids together with the NTER beside them could serve as unique potential inhibitor development sites. This region constitutes a divergent element due to the NTER which has variable length and distinct amino acids context in each Erm. The NTER or part of it plays critical roles in selective recognition of substrate RNA by Erms and this presumed target site might assume distinct local structure by induced conformational change with binding to substrate RNA and SAM, and contribute to the specific recognition of substrate RNA.