Structural and Biochemical Characterization of a Copper-Binding Mutant of the Organomercurial Lyase MerB: Insight into the Key Role of the Active Site Aspartic Acid in Hg–Carbon Bond Cleavage and Metal Binding Specificity,Biochemistry

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Structural and Biochemical Characterization of a Copper-Binding Mutant of the Organomercurial Lyase MerB: Insight into the Key Role of the Active Site Aspartic Acid in Hg–Carbon Bond Cleavage and Metal Binding Specificity
Biochemistry ( IF 2.9 ) Pub Date : 2016-02-11 00:00:00 , DOI: 10.1021/acs.biochem.5b01298
Haytham M. Wahba ₁ , Lauriane Lecoq , Michael Stevenson ₂ , Ahmed Mansour , Laurent Cappadocia , Julien Lafrance-Vanasse , Kevin J. Wilkinson , Jurgen Sygusch , Dean E. Wilcox ₂ , James G. Omichinski

Affiliation

In bacterial resistance to mercury, the organomercurial lyase (MerB) plays a key role in the detoxification pathway through its ability to cleave Hg–carbon bonds. Two cysteines (C96 and C159; Escherichia coli MerB numbering) and an aspartic acid (D99) have been identified as the key catalytic residues, and these three residues are conserved in all but four known MerB variants, where the aspartic acid is replaced with a serine. To understand the role of the active site serine, we characterized the structure and metal binding properties of an E. coli MerB mutant with a serine substituted for D99 (MerB D99S) as well as one of the native MerB variants containing a serine residue in the active site (Bacillus megaterium MerB2). Surprisingly, the MerB D99S protein copurified with a bound metal that was determined to be Cu(II) from UV–vis absorption, inductively coupled plasma mass spectrometry, nuclear magnetic resonance, and electron paramagnetic resonance studies. X-ray structural studies revealed that the Cu(II) is bound to the active site cysteine residues of MerB D99S, but that it is displaced following the addition of either an organomercurial substrate or an ionic mercury product. In contrast, the B. megaterium MerB2 protein does not copurify with copper, but the structure of the B. megaterium MerB2–Hg complex is highly similar to the structure of the MerB D99S–Hg complexes. These results demonstrate that the active site aspartic acid is crucial for both the enzymatic activity and metal binding specificity of MerB proteins and suggest a possible functional relationship between MerB and its only known structural homologue, the copper-binding protein NosL.

中文翻译：

结构和生化特征的有机结合裂解酶MerB的铜结合突变体：深入了解活性位点天冬氨酸在汞-碳键裂解和金属结合特异性中的关键作用

在对汞的细菌抗性中，有机汞裂解酶（MerB）通过其裂解Hg-碳键的能力在排毒途径中起着关键作用。已鉴定出两个半胱氨酸（C96和C159；大肠杆菌MerB编号）和天冬氨酸（D99）是关键的催化残基，除四个已知的MerB变体（其中天冬氨酸被a取代）外，这三个残基在所有其他变体中均保守。丝氨酸。为了了解活性位点丝氨酸的作用，我们表征了用丝氨酸替代D99（MerB D99S）以及在丝氨酸残基中含有丝氨酸残基的天然MerB变体之一的大肠杆菌MerB突变体的结构和金属结合特性。活性位点（巨大芽孢杆菌MerB2）。出人意料的是，MerB D99S蛋白与结合的金属共纯化，该结合的金属经UV-vis吸收，电感耦合等离子体质谱，核磁共振和电子顺磁共振研究确定为Cu（II）。X射线结构研究表明，Cu（II）与MerB D99S的活性位点半胱氨酸残基结合，但是在添加有机汞底物或离子汞产物后，Cu（II）被置换了。相反，巨大芽孢杆菌MerB2蛋白不与铜共纯化，而是巨大芽孢杆菌的结构MerB2-Hg复合物与MerB D99S-Hg复合物的结构高度相似。这些结果表明，活性位点天冬氨酸对于MerB蛋白的酶促活性和金属结合特异性都至关重要，并暗示了MerB及其唯一已知的结构同源物铜结合蛋白NosL之间的可能功能关系。

更新日期：2016-02-11

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