Identification of amino acids important for target recognition by the DNA:m5C methyltransferase M.NgoPII by alanine-scanning mutagenesis of residues at the protein-DNA interface.
DNA:m(5)C MTases comprise a catalytic domain with conserved residues of the active site and a strongly diverged TRD with variable residues involved in DNA recognition and binding. To date, crystal structures of 2 DNA:m(5)C MTases complexed with the substrate DNA have been obtained; however, for none of these enzymes has the importance of the whole set of DNA-binding residues been comprehensively studied. We built a comparative model of M.NgoPII, a close homologue and isomethylomer of M.HaeIII, and systematically analyzed the effect of alanine substitutions for the complete set of amino acid residues from its TRD predicted to be important for DNA binding and target recognition. Our data demonstrate that only 1 Arg residue is indispensable for the MTase activity in vivo and in vitro, and that mutations of only a few other residues cause significant reduction of the activity in vitro, with little effect on the activity in vivo. The identification of dispensable protein-DNA contacts in the wild-type MTase will serve as a platform for exhaustive combinatorial mutagenesis aimed at the design of new contacts, and thus construction of enzyme variants that retain the activity but exhibit potentially new substrate preferences.
Radlinska
Monika
M
Institute of Microbiology, Warsaw University, Warsaw, Poland.
Kondrzycka-Dada
Aneta
A
Piekarowicz
Andrzej
A
Bujnicki
Janusz M
JM
D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D001120:Arginine / Q000737:chemistry; D020134:Catalytic Domain; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry*; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000378:metabolism; D005822:Genetic Vectors; D008780:Methyltransferases / Q000737:chemistry*; D008958:Models, Molecular; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D009344:Neisseria gonorrhoeae / Q000201:enzymology; D009841:Oligonucleotides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry*; D040901:Proteomics / Q000379:methods*; D017386:Sequence Homology, Amino Acid
D000596:Amino Acids; D009841:Oligonucleotides; D011506:Proteins; D004247:DNA; D001120:Arginine; D008780:Methyltransferases; C072271:m(5)C rRNA methyltransferase; D000409:Alanine