Characterization of Mycobacterium tuberculosis mycothiol S-conjugate amidase
Characterization of Mycobacterium tuberculosis mycothiol S-conjugate amidase
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DOI：
10.1021/bi030080u
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发表时间：
2003-10-21
期刊：
影响因子：
2.9
通讯作者：
Fahey, RC
中科院分区：
文献类型：
--
作者：
Steffek, M;Newton, GL;Fahey, RC
真菌硫醇由N-乙酰半胱氨酸（AcCys）酰胺与1D-肌肌醇2-氨基-2-脱氧-α-D-吡喃葡萄糖苷（GlcN-Ins）连接组成，是大多数放线菌中发现的主要硫醇。真菌硫醇S-缀合物酰胺酶（Mca）切割各种烷化剂和外源性物质的真菌硫醇S-缀合物的酰胺键，产生GlcN-Ins和可从细胞分泌的巯基尿酸。结核分枝杆菌（Mycobacterium tuberculosis，Rv 1082）Mca基因在大肠杆菌中得到了表达。纯化后蛋白质含有1.4 +/-0.1当量的锌，表明Mca是以锌作为天然金属的金属蛋白。与14个底物的Mca活性的动力学研究表明，Mca是高度特异性的真菌硫醇S-共轭物的真菌硫醇部分和相对非特异性的硫连接的共轭物的结构。Mca与GlcNAc-Ins的脱乙酰酶活性小但显著，并且在高达2 mM GlcNAc-Ins时未能饱和，表明当GlcNAc-Ins水平高时，Mca可能适度地有助于GlcN-Ins的产生。Mca的多功能性可以在其与广泛的真菌硫醇S-缀合物反应的能力中看出，包括两种不同类别的抗生素。利福霉素S的真菌硫醇S-缀合物在生理相关条件下产生，并且被证明是氧化和还原形式的Mca的底物。显着的活性也被视为与抗生素浅蓝菌素的真菌硫醇S-共轭物作为Mca的底物。
Mycothiol is comprised of N-acetylcysteine (AcCys) amide linked to 1D-myo-inosityl 2-amino-2-deoxy-alpha-D-glucopyranoside (GlcN-Ins) and is the predominant thiol found in most actinomycetes. Mycothiol S-conjugate amidase (Mca) cleaves the amide bond of mycothiol S-conjugates of a variety of alkylating agents and xenobiotics, producing GlcN-Ins and a mercapturic acid that can be excreted from the cell. Mca of Mycobacterium tuberculosis (Rv1082) was cloned and expressed as a soluble protein in Escherichia coli. The protein contained 1.4 +/- 0.1 equiv of zinc after purification, indicating that Mca is a metalloprotein with zinc as the native metal. Kinetic studies of Mca activity with 14 substrates demonstrated that Mca is highly specific for the mycothiol moiety of mycothiol S-conjugates and relatively nonspecific for the structure of the sulfur-linked conjugate. The deacetylase activity of Mca with GlcNAc-Ins is small but significant and failed to saturate at up to 2 mM GlcNAc-Ins, indicating that Mca may contribute modestly to the production of GlcN-Ins when GlcNAc-Ins levels are high. The versatility of Mca can be seen in its ability to react with a broad range of mycothiol S-conjugates, including two different classes of antibiotics. The mycothiol S-conjugate of rifamycin S was produced under physiologically relevant conditions and was shown to be a substrate for Mca in both oxidized and reduced forms. Significant activity was also seen with the mycothiol S-conjugate of the antibiotic cerulenin as a substrate for Mca.