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

Journal of Molecular Biology
Remigijus SkirgailaVirginijus Siksnys

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 ...Continue Reading

References

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Citations

Sep 15, 2001·Nucleic Acids Research·A Pingoud, A Jeltsch
Oct 14, 2004·Nucleic Acids Research·Claire L MiddletonCharles S Bond
May 31, 2008·Nucleic Acids Research·Eva SisákováMark D Szczelkun
Aug 15, 2008·Nucleic Acids Research·Pete W DuntenNancy C Horton
Oct 2, 2009·Nucleic Acids Research·Rachel M SmithMark D Szczelkun
Sep 24, 2010·Nucleic Acids Research·Malgorzata FirczukMatthias Bochtler
Jul 19, 2005·Biochimica Et Biophysica Acta·Edita KriukieneArvydas Lubys
May 9, 2002·FEBS Letters·Gintautas TamulaitisVirginijus Siksnys
Feb 6, 2002·The Journal of Biological Chemistry·Vera PingoudAlfred Pingoud
Feb 13, 2020·Angewandte Chemie·Cangsong Liao, Florian P Seebeck
Oct 26, 2018·Nucleic Acids Research·Thomas A McMurroughDavid R Edgell
Nov 18, 2018·Nucleic Acids Research·Giedre TamulaitieneVirginijus Siksnys
Jan 2, 2001·The Journal of Biological Chemistry·R Rimseliene, A Janulaitis
Jan 1, 2017·Nucleic Acids Research·Giedre TamulaitieneVirginijus Siksnys
Jan 23, 2002·Biochemistry·Lori H Conlan, Cynthia M Dupureur
Oct 16, 1999·Journal of Molecular Biology·V SiksnysR Huber
Oct 6, 1999·Current Opinion in Chemical Biology·R A Kovall, B W Matthews
Aug 2, 2002·Trends in Biochemical Sciences·Annabel E ToddJanet M Thornton
Nov 17, 2001·Chemical Reviews·T M Penning, J M Jez
Nov 3, 2004·Biochemistry·Brett ChevalierBarry L Stoddard
Aug 18, 2009·Journal of Molecular Biology·Vera PingoudAlfred Pingoud

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