title: Fine tuning of the catalytic activity of colicin E7 nuclease domain by systematic N-terminal mutations.
abstract: The nuclease domain of colicin E7 (NColE7) promotes the nonspecific cleavage of nucleic acids at its C-terminal HNH motif. Interestingly, the deletion of four N-terminal residues (446-449 NColE7 = KRNK) resulted in complete loss of the enzyme activity. R447A mutation was reported to decrease the nuclease activity, but a detailed analysis of the role of the highly positive and flexible N-terminus is still missing. Here, we present the study of four mutants, with a decreased activity in the following order: NColE7  >> KGNK > KGNG ∼ GGNK > GGNG. At the same time, the folding, the metal-ion, and the DNA-binding affinity were unaffected by the mutations as revealed by linear and circular dichroism spectroscopy, isothermal calorimetric titrations, and gel mobility shift experiments. Semiempirical quantum chemical calculations and molecular dynamics simulations revealed that K446, K449, and/or the N-terminal amino group are able to approach the active centre in the absence of the other positively charged residues. The results suggested a complex role of the N-terminus in the catalytic process that could be exploited in the design of a controlled nuclease.
authors.affiliation: Department of Inorganic and Analytical Chemistry, University of Szeged, 6720, Szeged, Hungary; Department of Physical Chemistry and Material Sciences, University of Szeged, 6720, Szeged, Hungary.
authors.forename: Eszter
authors.initials: E
authors.lastname: Németh
authors.forename: Tamás
authors.initials: T
authors.lastname: Körtvélyesi
authors.forename: Peter W
authors.initials: PW
authors.lastname: Thulstrup
authors.forename: Hans E M
authors.initials: HE
authors.lastname: Christensen
authors.forename: Milan
authors.initials: M
authors.lastname: Kožíšek
authors.forename: Kyosuke
authors.initials: K
authors.lastname: Nagata
authors.forename: Anikó
authors.initials: A
authors.lastname: Czene
authors.forename: Béla
authors.initials: B
authors.lastname: Gyurcsik
mesh_terms: D055162:Biocatalysis; D002151:Calorimetry; D002942:Circular Dichroism; D003087:Colicins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D018360:Crystallography, X-Ray; D004789:Enzyme Activation; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D009154:Mutation / Q000235:genetics*; D009696:Nucleic Acids / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000235:genetics; D011789:Quantum Theory
chemical_list: C508089:ColE7 protein, E coli; D003087:Colicins; D009696:Nucleic Acids
keywords: DNA cleavage; Zn2+; binding; flow linear dichroism; isothermal calorimetry; positively charged N-terminal residues