Biochemical and Computational Analysis of the Substrate Specificities of Cfr and RlmN Methyltransferases

Cfr and RlmN methyltransferases both modify adenine 2503 in 23S rRNA (Escherichia colinumbering). RlmN methylates position C2 of adenine while Cfr methylates position C8, andto a lesser extent C2, conferring antibiotic resistance to peptidyl transferase inhibitors. Cfrand RlmN show high sequence homology and may be evolutionarily linked to a commonancestor. To explore their individual specificity and similarity we performed two sets ofexperiments. We created a homology model of Cfr and explored the C2/C8 specificity usingdocking and binding energy calculations on the Cfr homology model and an X-ray structureof RlmN. We used a trinucleotide as target sequence and assessed its positioning at theactive site for methylation. The calculations are in accordance with different poses of the trinucleotidein the two enzymes indicating major evolutionary changes to shift the C2/C8specificities. To explore interchangeability between Cfr and RlmN we constructed variouscombinations of their genes. The function of the mixed genes was investigated by RNAprimer extension analysis to reveal methylation at 23S rRNA position A2503 and by MICanalysis to reveal antibiotic resistance. The catalytic site is expected to be responsible forthe C2/C8 specificity and most of the combinations involve interchanging segments at thissite. Almost all replacements showed no function in the primer extension assay, apart froma few that had a weak effect. Thus Cfr and RlmN appear to be much less similar thanexpected from their sequence similarity and common target.