Rational engineering of sequence specificity in R.MwoI restriction endonuclease.
R.MwoI is a Type II restriction endonucleases enzyme (REase), which specifically recognizes a palindromic interrupted DNA sequence 5'-GCNNNNNNNGC-3' (where N indicates any nucleotide), 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 5'-GCCNNNNNGGC-3'. 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 experimental data, revealed regions important for recognition of the common bases in DNA sequences recognized by R.BglI and R.MwoI. Based on the bioinformatics 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 5'-GCCNNNNNGGC-3' sequence as a target, similarly to R.BglI, whereas the S310E variant preferentially cleaved a subset of the MwoI sites, depending on the identity of the 3rd and 9th nucleotide 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.
Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology, Trojdena 4, 02-109 Warsaw, Poland. krzysiek@genesilico.pl
Krzysztof
K
Skowronek
Michal J
MJ
Boniecki
Boguslaw
B
Kluge
Janusz M
JM
Bujnicki
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015202:Protein Engineering; D016415:Sequence Alignment; D013379:Substrate Specificity
D004247:DNA; C060413:endodeoxyribonuclease MwoI; C041749:endodeoxyribonuclease SfiI; D015252:Deoxyribonucleases, Type II Site-Specific; C018492:GCCNNNNNGGC-specific type II deoxyribonucleases