Mutational analysis of human DNase I at the DNA binding interface: implications for DNA recognition, catalysis, and metal ion dependence.
Human deoxyribonuclease I (DNase I), an enzyme used to treat cystic fibrosis patients, has been systematically analyzed by site-directed mutagenesis of residues at the DNA binding interface. Crystal structures of bovine DNase I complexed with two different oligonucleotides have implicated the participation of over 20 amino acids in catalysis or DNA recognition. These residues have been classified into four groups based on the characterization of over 80 human DNase I variants. Mutations at any of the four catalytic amino acids His 134, His 252, Glu 78, and Asp 212 drastically reduced the hydrolytic activity of DNase I. Replacing the three putative divalent metal ion-coordinating residues Glu 39, Asp 168, or Asp 251 led to inactive variants. Amino acids Gln 9, Arg 41, Tyr 76, Arg 111, Asn 170, Tyr 175, and Tyr 211 were also critical for activity, presumably because of their close proximity to the active site, while more peripheral DNA interactions stemming from 13 other positions were of minimal significance. The relative importance of these 27 positions is consistent with evolutionary relationships among DNase I across different species, DNase I-like proteins, and bacterial sphingomyelinases, suggesting a fingerprint for a family of DNase I-like proteins. Furthermore, we found no evidence for a second active site that had been previously implicated in Mn2+-dependent DNA degradation. Finally, we correlated our mutational analysis of human DNase I to that of bovine DNase I with respect to their specific activity and dependence on divalent metal ions.
Pan
C Q
CQ
Department of Protein Engineering, Genentech, Inc., South San Francisco, California 94080, USA.
Ulmer
J S
JS
Herzka
A
A
Lazarus
R A
RA
D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D002413:Cations, Divalent; D002417:Cattle; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000737:chemistry / Q000378:metabolism*; D006639:Histidine / Q000737:chemistry; D006801:Humans; D006860:Hydrogen Bonding; D008670:Metals; D008958:Models, Molecular; D010957:Plasmids; D011994:Recombinant Proteins; D013045:Species Specificity; D013329:Structure-Activity Relationship
D002413:Cations, Divalent; D004268:DNA-Binding Proteins; D008670:Metals; D011994:Recombinant Proteins; D006639:Histidine; D003850:Deoxyribonuclease I