Modifications of laccase activities of copper efflux oxidase, CueO by synergistic mutations in the first and second coordination spheres of the type I copper center.

The redox potential of type I copper in the Escherichia coli multicopper oxidase CueO was shifted in the positive or negative direction as a result of the single, double, and triple mutations in the first and second coordination spheres: the formation of the NH···S(-)(Cys500 ligand) hydrogen bond, the breakdown of the NH(His443 ligand)···O(-)(Asp439) hydrogen bond, and the substitution of the Met510 ligand for the non-coordinating Leu or coordinating Gln. Laccase activities of CueO were maximally enhanced 140-fold by virtue of the synergistic effect of mild mutations at and at around the ligand groups to type I copper.

Hypochnicium sensu lato (Polyporales, Basidiomycota) from Japan, with descriptions of a new genus and three new species.

Species of Hypochnicium (Polyporales, Basidiomycota) collected from Japan were studied on their taxonomy by morphological and phylogenetic approaches. Phylogenetic analyses based on a nrDNA LSU and ITS dataset including the Japanese specimens and other publicly available ones show that Hypochnicium is polyphyletic. Since the clade containing the type species H. bombycinum was well-supported, we defined this clade as Hypochnicium s. str., and emended Hypochnicium to include restricted taxa with only smooth basidiospores. The new genus Neohypochnicium is proposed to accommodate the remaining taxa excluded from the genus Hypochnicium s. str., which includes both species with smooth basidiospores and ornamented ones. Three new species, Gyrophanopsis japonica, N. asiaticum and N. perlongicystidiosum are described and illustrated based on morphological and phylogenetic analyses using an ITS region dataset. In addition, the following 15 new combinations are proposed: N. albostramineum, N. aotearoae, N. capitulateum, N. cremicolor, N. cystidiatum, N. geogenium, N. guineense, N. huinayense, N. michelii, N. microsporum, N. patagonicum, N. pini, N. punctulatum, N. subrigescens and N. wakefieldiae. An identification key to Japanese species of Bulbillomyces, Gyrophanopsis, Hypochnicium and Neohypochnicium is provided.

Enhancement of laccase activity through the construction and breakdown of a hydrogen bond at the type I copper center in Escherichia coli CueO and the deletion mutant Δα5-7 CueO.

CueO is a multicopper oxidase involved in a copper efflux system of Escherichia coli and has high cuprous oxidase activity but little or no oxidizing activity toward various organic substances. However, its activity toward oxidization of organic substrates was found to be considerably increased by the removal of the methionine-rich helical segment that covers the substrate-binding site (Δα5-7 CueO) [Kataoka, K., et al. (2007) J. Mol. Biol. 373, 141]. In the study presented here, mutations at Pro444 to construct a second NH-S hydrogen bond between the backbone amide and coordinating Cys500 thiolate of the type I copper are shown to result in positive shifts in the redox potential of this copper center and enhanced oxidase activity in CueO. Analogous enhancement of the activity of Δα5-7 CueO has been identified only in the Pro444Gly mutant because Pro444 mutants limit the incorporation of copper ions into the trinuclear copper center. The activities of both CueO and Δα5-7 CueO were also enhanced by mutations to break down the hydrogen bond between the imidazole group of His443 that is coordinated to the type I copper and the ß-carboxy group of Asp439 that is located in the outer sphere of the type I copper center. A synergetic effect of the positive shift in the redox potential of the type I copper center and the increase in enzyme activity has been achieved by the double mutation of Pro444 and Asp439 of CueO. Absorption, circular dichroism, and resonance Raman spectra indicate that the characteristics of the Cu(II)-S(Cys) bond were only minimally perturbed by mutations involving formation or disruption of a hydrogen bond from the coordinating groups to the type I copper. This study provides widely applicable strategies for tuning the activities of multicopper oxidases.