Electrochemical characterization of a unique, "neutral" laccase from Flammulina velutipes.

The flac1 gene consisted of 1488 bases encodes a novel laccase (Flac1) from Flammulina velutipes. The deduced amino acid sequence of Flac1 with 496 amino acids shows 58-64% homologies with other fungal laccases. The recombinant Flac1 (rFlac1) was heterologously expressed in Pichia pastoris, with sugars of approximately 4 kDa attached on the protein molecule, which has the calculated molecular mass of 53,532 Da. rFlac1 was shown to be a multi-copper oxidase from spectroscopies. The optimum pHs of rFlac1 for oxidations of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), p-phenylenediamine, and o-aminophenol, were 5.0, 5.0, and 6.0-6.5, respectively, showing higher pH values than those from many other fungal laccases. The slightly acidic or neutral optimum pH that is not strongly dependent on substrates is a unique property of rFlac1. Effective O(2) reduction was realized by the direct electron transfer of rFlac1 at a highly oriented pyrolytic graphite electrode modified with fine carbon particles (Ketjen Black) in O(2)-saturated solution. The pHs showing the maximum ΔE°' [=E°'(enzyme) - E°'(substrate)] coincided well with the optimum pHs shown by rFlac1 under steady-state conditions. The present electrochemical results of rFlac1 indicate that ΔE°' is one of the primary factors to determine the activity of multi-copper oxidases.

Structure and function of the engineered multicopper oxidase CueO from Escherichia coli--deletion of the methionine-rich helical region covering the substrate-binding site.

CueO is a multicopper oxidase (MCO) that is involved in the homeostasis of Cu in Escherichia coli and is the sole cuprous oxidase to have ever been found. Differing from other MCOs, the substrate-binding site of CueO is deeply buried under a methionine-rich helical region including alpha-helices 5, 6, and 7 that interfere with the access of organic substrates. We deleted the region Pro357-His406 and replaced it with a Gly-Gly linker. The crystal structures of a truncated mutant in the presence and in the absence of excess Cu(II) indicated that the scaffold of the CueO molecule and metal-binding sites were reserved in comparison with those of CueO. In addition, the high thermostability of the protein molecule and its spectroscopic and magnetic properties due to four Cu centers were also conserved after truncation. As for functions, the cuprous oxidase activity of the mutant was reduced to ca 10% that of recombinant CueO owing to the decrease in the affinity of the labile Cu site for Cu(I) ions, although activities for laccase substrates such as 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), p-phenylenediamine, and 2,6-dimethoxyphenol increased due to changes in the access of these organic substrates to the type I Cu site. The present engineering of CueO indicates that the methionine-rich alpha-helices function as a barrier to the access of bulky organic substrates, which provides CueO with specificity as a cuprous oxidase.

Mutations at Asp112 adjacent to the trinuclear Cu center in CueO as the proton donor in the four-electron reduction of dioxygen.

Asp112 adjacent to the trinuclear Cu center of a multicopper oxidase, CueO was mutated for Glu, Ala and Asn. Mutations on Asp112 affected not only spectroscopic and magnetic properties derived from the trinuclear Cu center but also enzyme activities. The uncoordinated Asp112 was found to play multiple roles to promote the binding of dioxygen at the trinuclear Cu center and to accelerate the conversion of dioxygen to water molecules by facilitating the supply of H+ to the reaction intermediates.