Rapid and Oriented Immobilization of Laccases on Electrodes via a Methionine-Rich Peptide

Abstract

Different from other laccases, copper efflux oxidase (CueO) from Escherichia coli possesses an additional methioninerich segment (MetRich), which is generally considered to be detrimental to its oxidase activity. Herein, we reveal that MetRich plays an important role in rapid immobilization of CueO on electrodes by studying the adsorption and bioelectrocatalysis behaviors of CueO, a truncated CueO (ΔMetRich CueO), and a serine-rich substituted CueO (SerRich CueO). Atomic molecular dynamics (MD) simulations demonstrate that the synergistic effect of π−π stacking and hydrophobic interactions contribute to the high affinity of MetRich to carbon nanotubes. Considering that the location of the electron acceptor (i.e., T1 Cu active site) in the family of laccases is close to the C-terminus, MetRich fused to another bacterial laccase, spore coat protein A (CotA) from Bacillus licheniformics, is found to endow CotA with the properties of rapid and oriented adsorption at the electrode surface. The finding and validation make MetRich a valuable binding motif for the rapid immobilization and high performance of laccases and perhaps other oxidoreductases in bioelectrocatalytic applications.

Electrochemistry of copper efflux oxidase-like multicopper oxidases involved in copper homeostasis

“…In agreement, Climent et al explained the DET process they observed with E. coli CueO on bare gold by the formation of sulfur-gold bonds that place CuT1 at a short distance of the electrode [43]. The role of the Met-rich domain in E. coli CueO was recently rationalized by considering the adsorption and electrocatalytic properties of WT CueO and two mutants, one lacking the Met-rich domain and the other one in which Met in the helix was mutated to Ser residues [29]. Only WT CueO was electroactive towards O2 reduction on gold electrode, although both WT and mutants were proved by SPR measurements to be adsorbed.…”

“…). The additional domain comprises Met and Asp ligands for a 5th copper atom (Cu5), a helical region with 6 Met that covers the CuT1 site and Cu5, and a loosely constrained loop lying across the Met-rich helix and carrying 5 Met and 5 His residues [25][26][27][28][29]. [30].…”

“…All CueOs present an additional domain covering the CuT1 not found in other MCOs. This domain is rich in Met residues, but depending on the organism, it presents a variety of structural organization and a large range of number of Met residues engaged (Figure 2) [15,21,25,[29][30][31][32][33][34].…”

“…Such results were explained based on an adsorption taking place through the Metrich helix and controlled by hydrophobic interactions. [29].…”

Sortase-mediated immobilization of Candida antarctica lipase B (CalB) on graphene oxide; comparison with chemical approach

“…Therefore, the random immobilization often reduces activity and efficiency of the immobilized enzyme. In oriented immobilization, the enzyme is attached to the surface through a specific site already embedded on the enzyme surface by genetic engineering, providing identical orientation of the protein molecules to the environment [16] . In this way, the target area is selected in a manner to have the maximum distance from the active site.…”