Amino acid residues in the GIY-YIG endonuclease II of phage T4 affecting sequence recognition and binding as well as catalysis
2008 (English)In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 190, no 16, 5533-5544 p.Article in journal (Refereed) Published
Phage T4 endonuclease II (EndoII), a GIY-YIG endonuclease lackinga carboxy-terminal DNA-binding domain, was subjected to site-directedmutagenesis to investigate roles of individual amino acids insubstrate recognition, binding, and catalysis. The structureof EndoII was modeled on that of UvrC. We found catalytic rolesfor residues in the putative catalytic surface (G49, R57, E118,and N130) similar to those described for I-TevI and UvrC; inaddition, these residues were found to be important for substraterecognition and binding. The conserved glycine (G49) and arginine(R57) were essential for normal sequence recognition. Our resultsare in agreement with a role for these residues in forming theDNA-binding surface and exposing the substrate scissile bondat the active site. The conserved asparagine (N130) and an adjacentproline (P127) likely contribute to positioning the catalyticdomain correctly. Enzymes in the EndoII subfamily of GIY-YIGendonucleases share a strongly conserved middle region (MR,residues 72 to 93, likely helical and possibly substitutingfor heterologous helices in I-TevI and UvrC) and a less stronglyconserved N-terminal region (residues 12 to 24). Most of theconserved residues in these two regions appeared to contributeto binding strength without affecting the mode of substratebinding at the catalytic surface. EndoII K76, part of a conservedNUMOD3 DNA-binding motif of homing endonucleases found to overlapthe MR, affected both sequence recognition and catalysis, suggestinga more direct involvement in positioning the substrate. Ourdata thus suggest roles for the MR and residues conserved inGIY-YIG enzymes in recognizing and binding the substrate.
Place, publisher, year, edition, pages
2008. Vol. 190, no 16, 5533-5544 p.
phage T4, GIY-YIG endonuclease, nicking, structure modeling, sequence recognition, information content
Biochemistry and Molecular Biology
IdentifiersURN: urn:nbn:se:uu:diva-97870DOI: 10.1128/JB.00094-08ISI: 000258274400001OAI: oai:DiVA.org:uu-97870DiVA: diva2:172963