Abstract
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.
Highlights
The multicopper oxidase CueO is involved in copper homeostasis and copper (Cu) tolerance in Escherichia coli
Similar to most of the reported crystal structures of CueO, the loop near type 1 Cu centre (T1) Cu atom is disordered in the G304K structure, suggesting that it is flexible (Fig. 1A)
One of the most obvious features is the presence of several new Cu-binding sites only found on the surface of G304K structure after soaked with Cu ions
Summary
The multicopper oxidase CueO is involved in copper homeostasis and copper (Cu) tolerance in Escherichia coli. 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. CueO, an MCOs from Escherichia coli (E. coli), together with CopA, a P-type ATPase, comprises a copper efflux (cue) system that resists copper stress under aerobic conditions[6,7] Expression of both enzymes is stimulated by exogenous Cu ions via the cytosolic metalloregulatory protein CueR8,9. Within domain III, a unique 42-residue methionine-rich region made up mainly of methionine-rich helix (thereafter, MR helix) may hinder the access of bulky organic substrates to the T1 Cu site[10] This region near the T1 Cu position was later found to coordinate a labile fifth Cu atom, a novel feature of CueOs that differs from other MCOs11. CueO can catalyze the oxidation of cuprous ion (Cu(I)) to the less toxic cupric ion (Cu(II)) in vivo[12]
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