Crystal structures of multicopper oxidase CueO G304K mutant: structural basis of the increased laccase activity

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.

Mutations in the coordination spheres of T1 Cu affect Cu2+-activation of the laccase from Thermus thermophilus

“…Escherichia coli that are activated by addition of exogenous copper [21][22][23]. In addition to classical T1 Cu and T2/T3 Cu sites, these enzymes can fix additional labile coppers in the M rich domain allowing the electron transfer from the substrate to the T1 Cu [24][25][26][27][28][29]. We recently performed an in-depth electrochemical study of the intra-and inter-electron transfers using the LAC of Thermus thermophilus HB27 (Tt-LAC) immobilized on different electrodes [23].…”

Effect of Cu(II) and Conserved Copper Binding Sites on Multicopper Oxidase CopA and Characterization of the BioMnOx

“…The modi cation analysis of CopA by mass spectrometry showed that the loss of Cu( ) in the culture condition reduced the CopA modi cation, the position of the modi cation changed, and nally the protein activity decreased. One explanation is that the addition of Cu( ) 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 (Wang et al 2018). These observations well explain the copper dependence of CopA oxidase activity.…”

“…Recent structural and functional studies found the activity of MCO mutant is higher than wild-type MCO. The reason is that dramatic conformational changes in methionine-rich helix and the relative regulatory loop (R-loop) have occurred (Kataoka et al 2007; Wang et al 2018). Amino acid changes affect the redox potential and steric hindrance of copper ion binding conserved sites (Li et al 2007).…”

Development of an efficient biocatalytic system based on bacterial laccase for the oxidation of selected 1,4-dihydropyridines

“…In addition, intracellular conditions may be significantly different from known in vitro optimal conditions for BliLacc and CueO therefore this should be taken into account when designing a whole-cell biocatalyst. Recently, a set of CueO mutants, including G304 K, with increased laccase and the reduced cuprous oxidase activity have been generated [47]. The crystal structure mutated versions of E. coli CueO revealed the importance of methionine-rich helix and the relative regulatory loop and their interaction and movements when the enzyme is Cu-soaked for the access of the substrate, opening the possibility to further design specific substrate-binding mutants [47,48].…”