title: Structural insights into DNA sequence recognition by Type ISP restriction-modification enzymes.
abstract: Engineering restriction enzymes with new sequence specificity has been an unaccomplished challenge, presumably because of the complexity of target recognition. Here we report detailed analyses of target recognition by Type ISP restriction-modification enzymes. We determined the structure of the Type ISP enzyme LlaGI bound to its target and compared it with the previously reported structure of a close homologue that binds to a distinct target, LlaBIII. The comparison revealed that, although the two enzymes use almost a similar set of structural elements for target recognition, the residues that read the bases vary. Change in specificity resulted not only from appropriate substitution of amino acids that contacted the bases but also from new contacts made by positionally distinct residues directly or through a water bridge. Sequence analyses of 552 Type ISP enzymes showed that the structural elements involved in target recognition of LlaGI and LlaBIII were structurally well-conserved but sequentially less-conserved. In addition, the residue positions within these structural elements were under strong evolutionary constraint, highlighting the functional importance of these regions. The comparative study helped decipher a partial consensus code for target recognition by Type ISP enzymes.
authors.affiliation: Division of Biology, Indian Institute of Science Education and Research, Pune 411008, India.
authors.forename: Manasi
authors.initials: M
authors.lastname: Kulkarni
authors.affiliation: Division of Biology, Indian Institute of Science Education and Research, Pune 411008, India.
authors.forename: Neha
authors.initials: N
authors.lastname: Nirwan
authors.affiliation: DNA-Protein Interactions Unit, School of Biochemistry, Medical Sciences Building, University of Bristol, Bristol BS8 1TD, UK.
authors.forename: Kara
authors.initials: K
authors.lastname: van Aelst
authors.affiliation: DNA-Protein Interactions Unit, School of Biochemistry, Medical Sciences Building, University of Bristol, Bristol BS8 1TD, UK.
authors.forename: Mark D
authors.initials: MD
authors.lastname: Szczelkun
authors.affiliation: Division of Biology, Indian Institute of Science Education and Research, Pune 411008, India saikrishnan@iiserpune.ac.in.
authors.forename: Kayarat
authors.initials: K
authors.lastname: Saikrishnan
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry*; D001483:Base Sequence; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry; D004262:DNA Restriction Enzymes / Q000737:chemistry*; D006860:Hydrogen Bonding; D013294:Lactococcus lactis / Q000201:enzymology; D008958:Models, Molecular; D011485:Protein Binding; D000072756:Protein Conformation, alpha-Helical; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D004247:DNA; D004262:DNA Restriction Enzymes