The multicopper oxidase CueO is involved in copper homeostasis and copper (Cu) tolerance in Escherichia coli. The laccase activity of CueO G304K mutant is higher than wild-type CueO. To explain this increase in activity, we solved the crystal structure of G304K mutant at 1.49 Å. Compared with wild-type CueO, the G304K mutant showed dramatic conformational changes in methionine-rich helix and the relative regulatory loop (R-loop). We further solved the structure of Cu-soaked enzyme, and found that the addition of Cu ions induced further conformational changes in the R-loop and methionine-rich helix as a result of the new Cu-binding sites on the enzyme’s surface. We propose a mechanism for the enhanced laccase activity of the G304K mutant, where movements of the R-loop combined with the changes of the methionine-rich region uncover the T1 Cu site allowing greater access of the substrate. Two of the G304K double mutants showed the enhanced or decreased laccase activity, providing further evidence for the interaction between the R-loop and the methionine-rich region. The cuprous oxidase activity of these mutants was about 20% that of wild-type CueO. These structural features of the G304K mutant provide clues for designing specific substrate-binding mutants in the biotechnological applications.
T:G base pair arising from spontaneous deamination of 5mC or polymerase errors is a great challenge for DNA repair of hyperthermophilic archaea, especially Crenarchaea. Most strains in this phylum lack the protein homologues responsible for the recognition of the mismatch in the DNA repair pathways. To investigate whether Cren7, a highly conserved chromatin protein in Crenarchaea, serves a role in the repair of T:G mispairs, the crystal structures of Cren7-GTAATTGC and Cren7-GTGATCGC complexes were solved at 2.0 Å and 2.1 Å. In our structures, binding of Cren7 to the AT-rich DNA duplex (GTAATTGC) induces opening of T2:G15 but not T10:G7 base pair. By contrast, both T:G mispairs in the GC-rich DNA duplex (GTGATCGC) retain the classic wobble type. Structural analysis also showed DNA helical changes of GTAATTGC, especially in the steps around the open T:G base pair, as compared to GTGATCGC or the matched DNAs. Surface plasmon resonance assays revealed a 4-fold lower binding affinity of Cren7 for GTAATTGC than that for GTGATCGC, which was dominantly contributed by the decrease of association rate. These results suggested that binding of Cren7 to DNA leads to T:G mispair opening in a sequence dependent manner, and therefore propose the potential roles of Cren7 in DNA repair.
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