Biochemical and structural characterization of Salmonella typhimurium glyoxalase II:: New insights into metal ion selectivity
Biochemical and structural characterization of Salmonella typhimurium glyoxalase II:: New insights into metal ion selectivity
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DOI：
10.1021/bi7007245
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发表时间：
2007-10-02
期刊：
影响因子：
2.9
通讯作者：
Vila, Alejandro J.
中科院分区：
文献类型：
--
作者：
Campos-Bermudez, Valeria A.;Leite, Ney Ribeiro;Vila, Alejandro J.
乙二醛酶II是乙二醛酶系统的水解酶部分，负责利用谷胱甘肽解毒几种细胞毒性化合物。乙二醛酶II属于金属- β -内酰胺酶超家族，具有保守的基元，能够在活性位点结合两种金属离子，通常是锌。相反，几种真核乙二醛酶已被表征为不同比例的铁，锌和锰离子。我们从鼠伤寒沙门氏菌中表达了该酶超家族的一个假定成员的基因编码，根据其活性，我们证明了它是一种乙二醛酶，命名为GloB。在大肠杆菌中表达的重组GloB用不同量的铁、锌和锰纯化。所有形式都表现出类似的活性，这可以从补充了特定金属离子的最小培养基中的蛋白质表达中看出。在1.4埃处解析的GloB晶体结构显示出与真核同源物相似的蛋白质折叠和活性位点。核磁共振和EPR实验也揭示了金属位点的保守电子结构。因此，GloB能够容纳这些不同的金属离子，并在所有情况下以相似的效率进行水解反应。该酶的金属混杂性（与同一超家族的其他成员相比）可以通过作为第二壳配体的保守Asp残基的存在来解释，该残基预计会增加金属结合位点的硬度，因此有利于乙二醛酶II中的铁摄取。
Glyoxalase II is a hydrolytic enzyme part of the glyoxalase system, responsible for detoxifying several cytotoxic compounds employing glutathione. Glyoxalase II belongs to the superfamily of metallo-beta-lactamases, with a conserved motif able to bind up to two metal ions in their active sites, generally zinc. Instead, several eukaryotic glyoxalases II have been characterized with different ratios of iron, zinc, and manganese ions. We have expressed a gene coding for a putative member of this enzyme superfamily from Salmonella typhimurium, that we demonstrate, on the basis of its activity, to be a glyoxalase II, named GloB. Recombinant GloB expressed in Escherichia coli was purified with variable amounts of iron, zinc, and manganese. All forms display similar activities, as can be shown from protein expression in minimal medium supplemented with specific metal ions. The crystal structure of GloB solved at 1.4 angstrom shows a protein fold and active site similar to those of its eukaryotic homologues. NMR and EPR experiments also reveal a conserved electronic structure at the metal site. GloB is therefore able to accommodate these different metal ions and to carry out the hydrolytic reaction with similar efficiencies in all cases. The metal promiscuity of this enzyme (in contrast to other members of the same superfamily) can be accounted for by the presence of a conserved Asp residue acting as a second-shell ligand that is expected to increase the hardness of the metal binding site, therefore favoring iron uptake in glyoxalases II.