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首页> 外文期刊>Biochemistry >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 MutantΔα5-7 CueO
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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 MutantΔα5-7 CueO

机译：通过在大肠杆菌CueO和缺失突变体Δα5-7CueO的I型铜中心的氢键的构建和破坏来增强漆酶活性。

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摘要

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 1 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 Δα 5-7 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 beta-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.
机译：CueO是一种参与大肠杆菌铜外排系统的多铜氧化酶，具有较高的亚铜氧化酶活性，但对多种有机物质的氧化活性很小或没有。然而，发现通过除去覆盖底物结合位点（Δα5-7CueO）的富含蛋氨酸的螺旋链段，其对有机底物氧化的活性大大提高了[Kataoka，K。等人。 （2007）J.Mol。生物学373，141]。在此处提出的研究中，Pro444突变在骨架酰胺和1型铜的配位Cys500硫醇盐之间构建第二个NH-S氢键，表明该铜中心的氧化还原电势发生正移并增强了氧化酶活性在CueO中。仅在Pro444Gly突变体中发现了Δα5-7CueO活性的类似增强，因为Pro444突变体限制了铜离子向三核铜中心的结合。通过突变破坏与I型铜配位的His443的咪唑基团与位于外部的Asp439的β-羧基之间的氢键，突变也增强了CueO和Δα5-7 CueO的活性。 I型铜中心的球体。通过CueO的Pro444和Asp439的双重突变，实现了I型铜中心氧化还原电势正移和酶活性增加的协同效应。吸收，圆二色性和共振拉曼光谱表明，Cu（II）-S（Cys）键的特征仅受到涉及从配位基团到I型铜的氢键形成或破坏的突变的最小扰动。这项研究为调节多铜氧化酶的活性提供了广泛适用的策略。

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