title: Structure-based redesign of the catalytic/metal binding site of Cfr10I restriction endonuclease reveals importance of spatial rather than sequence conservation of active centre residues.
abstract: According to the crystal structure of Cfr10I restriction endonuclease the acidic residues D134, E71 and E204 are clustered together and presumably chelate metal ion(s) at the active site. Indeed, investigation of the DNA cleavage properties of substitutional mutants of Cfr10I D134A, E71Q, E71A and E204Q reveals that D134, E71 and E204 residues are essential for cleavage activity, supporting their active site function. Structural comparison indicates that the D134 residue of Cfr10I spatially overlaps with aspartate residues D91 and D74, from the invariant active site motifs 90PDX19EAK and 73PDX15DIK of EcoRI and EcoRV, respectively. However, structural studies in conjunction with mutational analyses suggest that the sequence motif 133PDX55KX13E corresponds to the active site of Cfr10I, but differs from canonical active site motifs of EcoRI and EcoRV. According to the crystal structure of Cfr10I the serine S188 residue from the 188SVK sequence motif is a spatial equivalent of the acidic residue from the (E/D)XK-part of the active site motif, which is conserved between EcoRI and EcoRV. Site-directed mutagenesis experiments of Cfr10I, however, revealed that the S188 was not so important for catalysis while the E204 residue located 2.8 A away indeed was essential for cleavage, suggesting that the glutamate E204 rather than the S188 residue contributes to the metal binding site in Cfr10I. In addition, model-building studies suggest that mutual interchange of the E204 and S188 residues should lead only to minor positional differences of the carboxylate residues of glutamate side-chains. The double mutant S188E/E204S was therefore prepared by site-directed mutagenesis where the active site motif 133PDX55KX13E of Cfr10I was changed to a canonical motif 133PDX53EVK, which is similar to that of EcoRI and EcoRV. Interestingly, the double mutant S188E/E204S of Cfr10I with redesigned active site structure, exhibited 10% of Wt cleavage activity in a gamma DNA cleavage assay. Thus, structure guided redesign of the catalytic/metal binding site of Cfr10I, provides novel experimental evidence to suggest that spatial rather than sequence conservation plays the dominant role in the formation of restriction enzyme active sites.
authors.affiliation: Institute of Biotechnology, Vilnius, Lithuania.
authors.forename: R
authors.initials: R
authors.lastname: Skirgaila
authors.forename: S
authors.initials: S
authors.lastname: Grazulis
authors.forename: D
authors.initials: D
authors.lastname: Bozic
authors.forename: R
authors.initials: R
authors.lastname: Huber
authors.forename: V
authors.initials: V
authors.lastname: Siksnys
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D017124:Conserved Sequence; D017931:DNA Primers / Q000235:genetics; D015246:Deoxyribonuclease EcoRI / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D008670:Metals / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation
chemical_list: D017931:DNA Primers; D008670:Metals; D015246:Deoxyribonuclease EcoRI; C066244:endodeoxyribonuclease Cfr10I; D015252:Deoxyribonucleases, Type II Site-Specific; C027440:GATATC-specific type II deoxyribonucleases