Catalysis and Inhibition of Mycobacterium tuberculosis Methionine Aminopeptidase
Catalysis and Inhibition of Mycobacterium tuberculosis Methionine Aminopeptidase
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DOI：
10.1021/jm901624n
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发表时间：
2010-02-11
影响因子：
7.3
通讯作者：
Ye, Qi-Zhuang
中科院分区：
文献类型：
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作者：
Lu, Jing-Ping;Chai, Sergio C.;Ye, Qi-Zhuang
甲硫氨肽酶（MetAP）是一种重要的共翻译酶，能切除新生蛋白质的N端甲硫氨酸，是开发抗菌和抗结核药物的潜在靶点。我们克隆了结核分枝杆菌中的两个MetAP之一（来自mapB基因的MtMetAP 1c），并将其纯化为同质的脱辅基酶。其活性需要二价金属离子，Co（II）、Ni（II）、Mn（II）和Fe（II）是该酶的活化剂。Co（II）和Fe（II）具有最紧密的结合，而Ni（II）是催化作用最有效的辅因子。MtMetAP 1c在E.大肠杆菌中表达的MtMetAP 1c补充了大肠杆菌中MetAP的基本功能。大肠杆菌中表达，并支持细胞生长。一组有效的MtMetAP 1c抑制剂被确定，它们分别对Fe（II）-形式，Mn（II）-形式或Co（II）和Ni（II）形式的酶表现出高选择性。这些金属形态选择性抑制剂用于分配细胞MtMetAP 1c的金属形态。只有Fe（II）形式的选择性抑制剂抑制细胞MtMetAP 1c活性并抑制MtMetAP 1c补充的细胞生长的事实表明Fe（II）是MtMetAP 1c在E. coli细胞环境。最后，对MtMetAP 1c与三种金属形态选择性抑制剂复合物的X射线结构进行了分析，结果表明MtMetAP 1c与金属离子和活性位点残基的结合方式不同，相互作用也不同.
Methionine aminopeptidase (MetAP) carries out an important cotranslational N-terminal methionine excision of nascent proteins and represents a potential target to develop antibacterial and antitubercular drugs. We cloned one of the two MetAPs in Mycobacterium tuberculosis (MtMetAP1c from the mapB gene) and purified it to homogeneity as an apoenzyme. Its activity required a divalent metal ion, and Co(II), Ni(II), Mn(II), and Fe(II) were among activators of the enzyme. Co(II) and Fe(II) had the tightest binding, while Ni(II) was the most efficient cofactor for the catalysis. MtMetAP1c was also functional in E. coli cells because a plasmid-expressed MtMetAP1c complemented the essential function of MetAP in E. coli and supported the cell growth. A set of potent MtMetAP1c inhibitors were identified, and they showed high selectivity toward the Fe(II)-form, the Mn(II)-form, or the Co(II) and Ni(II) forms of the enzyme, respectively. These metalloform selective inhibitors were used to assign the metalloform of the cellular MtMetAP1c. The fact that only the Fe(II)-form selective inhibitors inhibited the cellular MtMetAP1c activity and inhibited the MtMetAP1c-complemented cell growth suggests that Fe(II) is the native metal used by MtMetAP1c in an E. coli cellular environment. Finally, X-ray structures of MtMetAP1c in complex with three metalloform-selective inhibitors were analyzed, which showed different binding modes and different interactions with metal ions and active site residues.