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.
Highlights
Microorganisms protect themselves against the action of antibiotics through three major mechanisms [1]: (1) they modify the target site for antibiotics that related antibiotics could not work on [2]; (2) the antibiotic access to the targets is prevented [3]; (3) the chemical modification of antibiotics could be carried out to inactivate them [4]
The recent elucidation of the functions of the unconventional carbon-oxygen (CH . . . O) hydrogen bond between the transferrable methyl group of SAM and oxygen from certain active site residue(s) of the enzyme, which contribute to high-affinity SAM binding, transition state stabilization and limitation of the motion of the SAM methyl group to maintain the alignment of the methyl group for optimal transfer geometry, could provide the two distinct strategies for the specific inhibitor development of MTases: (1) to design the ligands to act as CH . . . O hydrogen bond acceptors instead of SAM or (2) to mimic SAM CH . . . O hydrogen-bond donors using cofactor or transition state analogs [14]
The first amino acid could be represented as G in most erythromycin ribosome methylation (Erm) proteins: 26 including Erm37 and Erm41 of 44 Erms
Summary
Microorganisms protect themselves against the action of antibiotics through three major mechanisms [1]: (1) they modify the target site for antibiotics that related antibiotics could not work on [2]; (2) the antibiotic access to the targets is prevented [3]; (3) the chemical modification of antibiotics could be carried out to inactivate them [4]. Using and modifying KsgA (BsKsgA) from Bacillus subtilis based on structural and phylogenetic analysis, specificity could be switched from that of KsgA, dimethylation at two adjacent adenines A1518 and A1519 in the 3 -end of 16S rRNA, to that of Erm, A2058 of 23S rRNA by either swapping the two loops alone or C-terminal domain together or two loops but truncating C-terminal domain From these observations, the target region of protein-RNA interaction for inhibitor development in Erm proteins could be narrowed down. In that motif, some consensus G(S/T)-Q-N(H)-F(L, Y) in which Q is highly conserved among almost all members of the families of Erm, and another sequence might be altered to amino acid(s) in parenthesis, could be identified with Erm families and some other slight variation in that consensus could be found with KsgA/Dim (see below) These amino acids were observed to be in immediate adjacency to SAM, this region was exhibiting different conformations when ErmC’ bound to different ligands [SAM, S-adenosyl-Lhomocysteine (SAH) and sinefungin), presumably suggesting intrinsic flexibility [13] and probable functional importance. The effects of each mutation were characterized in vitro and in the cell
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