Potential Target Site for Inhibitors in MLSB Antibiotic Resistance

Abstract

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.

Inhibition of Erythromycin and Erythromycin-Induced Resistance among Staphylococcus aureus Clinical Isolates

“…Molecular modeling studies showed that quinine, ketoprofen, and fosfomycin formed H -bond and hydrophobic interactions with key amino acids at the SAM-binding site of the ErmC’ protein ( Table S7 ). This binding inhibits N -6 methylation of A2058 at the macrolides-binding site of 23S rRNA in 50S ribosomal subunit, thus allowing clindamycin to bind to its binding site on the ribosome and decreasing inducible clindamycin resistance [ 90 ]. In brief, quinine achieved the highest binding affinity, indicating that quinine had the highest inhibitory activity on inducible clindamycin resistance.…”

Characteristics of the ErmK Protein of Bacillus halodurans C-125

“…However, the NdeI site could be found in ermK structural gene and site directed mutagenesis of the ermK gene without any amino acid change ( G C A TATG TG , which code 158AYV160 but as could be seen in underlined letters, it contains NdeI recognition site, so it changed to C T TATG as denoted in italicized letter.) was carried out using a sequential PCR method, known as the “splicing by overlap extension” ( 31 , 32 ). In brief, to obtain the DNA fragment encompassing the 5′ end region, including the mutagenizing site, PCR was carried out using Bacillus halodurans C-125 chromosomal DNA as a template and the oligonucleotides oligo-1 and oligo-2 ( Table 2 ) as the forward and reverse primers.…”

“…The activity of each Erm protein expressed in E. coli was evaluated as described in the previous report with slight modifications ( 32 ). Briefly, each E. coli culture ( E. coli HJJ402, E. coli HJJ502, and E. coli HJJ602) was spread on quartered LB agar plate.…”

Nitrogen-Containing Flavonoids─Preparation and Biological Activity

“…The protein MLS_B (also known as ERM_B) was predicted using the artificial intelligence program AlphaFold. Following the studies by Lee et al, 44 the N-terminal portion (NTERM) was identified as the region primary interest for the activities of proteins encoded by ERMs. The following approach was used to construct the spatial grid box the region of interest was identified between amino acids 8TQNF11, delimited within the region defined as the X-motif.…”