Rational engineering of sequence specificity in R.MwoI restriction endonuclease

R.MwoI is a Type II restriction endonucle-ases enzyme (REase), which specifically recognizes a palindromic interrupted DNA sequence 50-GCNNNNNNNGC-30 (where N indicates any nu-cleotide), and hydrolyzes the phosphodiester bond in the DNA between the 7th and 8th base in both strands. R.MwoI exhibits remote sequence similarity to R.BglI, a REase with known structure, which recognizes an interrupted palindromic target 50-GCCNNNNNGGC-30. A homology model of R.MwoI in complex with DNA was constructed and used to predict functionally important amino acid residues that were subsequently targeted by mutagenesis. The model, together with the supporting experimen-tal data, revealed regions important for recognition of the common bases in DNA sequences recognized by R.BglI and R.MwoI. Based on the bioinfor-matics analysis, we designed substitutions of the S310 residue in R.MwoI to arginine or glutamic acid, which led to enzyme variants with altered sequence selectivity compared with the wild-type enzyme. The S310R variant of R.MwoI preferred the 50-GCCNNNNNGGC-30 sequence as a target, similarly to R.BglI, whereas the S310E variant pref-erentially cleaved a subset of the MwoI sites, depending on the identity of the 3rd and 9th nucleo-tide residues. Our results represent a case study of a REase sequence specificity alteration by a single amino acid substitution, based on a theoretical model in the absence of a crystal structure