Abstract
Erythromycin-resistance methyltransferases are SAM dependent Rossmann fold methyltransferases that convert A2058 of 23S rRNA to m62A2058. This modification sterically blocks binding of several classes of antibiotics to 23S rRNA, resulting in a multidrug-resistant phenotype in bacteria expressing the enzyme. ErmC is an erythromycin resistance methyltransferase found in many Gram-positive pathogens, whereas ErmE is found in the soil bacterium that biosynthesizes erythromycin. Whether ErmC and ErmE, which possess only 24% sequence identity, use similar structural elements for rRNA substrate recognition and positioning is not known. To investigate this question, we used structural data from related proteins to guide site-saturation mutagenesis of key residues and characterized selected variants by antibiotic susceptibility testing, single turnover kinetics, and RNA affinity-binding assays. We demonstrate that residues in α4, α5, and the α5-α6 linker are essential for methyltransferase function, including an aromatic residue on α4 that likely forms stacking interactions with the substrate adenosine and basic residues in α5 and the α5-α6 linker that likely mediate conformational rearrangements in the protein and cognate rRNA upon interaction. The functional studies led us to a new structural model for the ErmC or ErmE-rRNA complex.
Highlights
Posttranscriptional modification of RNA is ubiquitous throughout the three domains of life
Formation of m62A2058 on 23S rRNA by erythromycin-resistance methyltransferases (Erm) confers the MLSbK multidrug phenotype named for the macrolide, lincosamide, streptogramin B, and ketolide antibiotics (Fig. 1A)
Extensive mutagenesis of ErmC identified only three residues essential for erythromycin resistance (Asn-101, Tyr-104, and Arg-134), two of which are conserved in ErmE (Fig. 1B and Fig. S1) [17, 18]
Summary
Posttranscriptional modification of RNA is ubiquitous throughout the three domains of life. We have used site-directed mutagenesis to construct two of the three corresponding mutants in ErmE (Fig. S1), and in the case of Y134A have performed detailed in vitro studies to identify the mechanism of the erythromycin-sensitive mutant. Both Escherichia coli RsmA (KsgA) and human TFB1M, members of the same protein family as Erm, do, and their structures bound to substrate suggest the basic residue on a5 forms a key interaction with RNA (Fig. 1, C and E).
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