Recognition Site Generated by Natural Changes in Erm Proteins Leads to Unexpectedly High Susceptibility to Chymotrypsin

Abstract

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.

Plausible Minimal Substrate for Erm Protein

“…The expression vector (pHJJ105) and E. coli strain ( E. coli HJJ105) overexpressing ErmS and those (pHJJ202 and E. coli HJJ202) for ErmE were obtained in previous studies ( 15 , 20 ). For constructing an ErmB expression vector, pVA838 plasmid DNA ( 38 ) was used as a DNA template to obtain the mature protein-coding region.…”

“…Almost 40 different erm methyltransferase genes have been isolated and characterized from diverse sources, ranging from pathogens to antibiotic-producing actinomycetes ( 1 , 2 , 13 , 14 ). Despite the diversity of sources, the significant homology among Erm proteins and their common functional role in methylating the specific adenine residue at position N 6 suggest that they could assume similar structures ( 11 , 15 ) and originate from a common ancestor ( 2 , 16 ). Furthermore, the methylatable adenine (A2058) is located in the peptidyl transferase circle in domain V of 23S rRNA, which has been highly conserved over the course of evolution, presumably because of its functional role as the active site of the ribosome ( 17 , 18 ).…”

Characteristics of the ErmK Protein of Bacillus halodurans C-125

“…Both ErmC′ and ErmK could be overexpressed in sufficient quantity to purify and evaluate its methylation activity in vitro . ErmN was successfully expressed in E. coli BL21(DE3) in a previous study ( 20 ), and it could be expressed with and without IPTG induction in soluble form (data not shown).…”

Potential Target Site for Inhibitors in MLSB Antibiotic Resistance

“…From the Erm family, a total of 44 sequences were aligned, where 41 of them could be retrieved from CARD (comprehensive antibiotic resistance database; [ 22 ]) and ErmI [ 23 , 24 ], Erm50 [ 25 ] and Erm51 [ 26 ] were added to them. To the authors’ knowledge, these are all Erm proteins reported at the time of submission of the paper.…”