Structure and Function of the Engineered Multicopper Oxidase CueO from Escherichia coli—Deletion of the Methionine-Rich Helical Region Covering the Substrate-Binding Site

Abstract

CueO is a multicopper oxidase involved in the homeostasis of Cu in Escherichia coli, and functions as the sole cupric oxidase ever found. Differing from other multicopper oxidases, the substratebinding site of CueO is deeply buried under a methionine-rich helical region including α-helices 5, 6, and 7 that interfere the access of organic substrates. We deleted this region, Pro357-His406 and replaced it with a Gly-Gly linker. Crystal structures of the truncated mutant in the presence and absence of excess Cu(II) indicated that the scaffold of the CueO molecule and the metal binding sites were reserved in comparison with those of CueO. In addition, the high thermostability of the protein molecule and spectroscopic and magnetic properties due to the four Cu centers were also conserved after the truncation. As for functions, the cuprous oxidase activity of the mutant was reduced to ca.10% of that of recombinant CueO owning 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 the changes in accessibilities of these organic substrates towards the type I Cu site. The present engineering of CueO indicates that the methionine-rich α-helices function as a barrier to interfere with the access of bulky organic substrates to provide CueO with the specificity as cuprous oxidase.Keywords: CueO; multicopper oxidase; homeostasis; truncated mutant; X-ray crystal structure A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT3 IntroductionMulticopper oxidases (MCOs) are enzymes containing a multiple copper center to catalyze the oxidation of a variety of substrates such as polyphenols, aromatic polyamines, L-ascorbate, and metal ions concomitantly with the four-electron reduction of dioxygen to water. [1][2][3][4][5] Laccase, the largest subfamily of MCOs, shows multiple functions including lignin degradation, pigmentation, and pathogenesis in fungi as well as lignin biosynthesis and wound healing in plants. [6][7][8][9][10] Therefore, structures and functions of new MCOs such as Escherichia coli CueO (formerly called YacK) [11][12][13] andBacillus subtilis CotA, 14,15 have been discussed in comparison with those of laccase.CueO is a 53.4-kDa periplasmic protein involved in the Cu efflux system, together with CopA, the P-type ATPase. 16 CueO is responsible for the oxidation of cuprous ion to less toxic cupric ion and the oxidation of enterobactin to prevent the copper-catalyzed Fenton reaction so as not to sequester iron from the environment. 17,18 CueO also catalyzes the oxidation of organic compounds including 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), p-phenylenediamine (p-PD), and 2,6-dimethoxyphenol (2,6-DMP). Oxidase activities of CueO toward these substrates are considerably low, but are fairly enhanced in the presence of an excess Cu(II) ions. 11,12 That is, the enzymatic activity of CueO is regulated by Cu ions in ...

Crystal Structures of Multicopper Oxidase CueO Bound to Copper(I) and Silver(I)

“…1). The methionine-rich insert provides for substrate discrimination because it buries the T1 site; its deletion results in a protein with decreased Cu(I) oxidase activity and an increased ability to oxidize organic substrates in the absence of added copper, presumably through increased access to the T1 site (44).…”

Heterologous laccase production and its role in industrial applications

“…Heterologous expression of laccases in bacteria, yeasts, filamentous fungi and plants has been reported, along with examples of homologous expression. [25][26][27][28][29][30] Laccase heterologous expression in Escherichia coli has been often used as a strategy to get around the problem of obtaining laccases not easily producible in natural hosts. The recombinant expression of Bacillus subtilis CotA in E. coli has allowed its deep characterization, structure solving, and functional evolution.…”

Study on dioxygen reduction by mutational modifications of the hydrogen bond network leading from bulk water to the trinuclear copper center in bilirubin oxidase

“…and of substrates at 440 nm ( = 68.5 mM -1 cm -1 ) and 440 nm ( = 49.5 mM -1 cm -1 ), respectively [27].…”