Selective targeting of the conserved active site cysteine of Mycobacterium tuberculosis methionine aminopeptidase with electrophilic reagents
Selective targeting of the conserved active site cysteine of Mycobacterium tuberculosis methionine aminopeptidase with electrophilic reagents
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DOI：
10.1111/febs.12847
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发表时间：
2014-09-01
期刊：
影响因子：
5.4
通讯作者：
Addlagatta, Anthony
中科院分区：
文献类型：
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作者：
Reddi, Ravikumar;Arya, Tarun;Addlagatta, Anthony
蛋氨酸氨基肽酶（Methionine aminopeptidases, MetAPs）在每个活细胞中从大约70%的新合成蛋白质中切割引发物蛋氨酸，特异性抑制或敲除该功能是有害的。metap是一种金属酶，大致分为I型和II型两个亚型。细菌只含有I型metap，这些酶的活性位点含有一个保守的半胱氨酸。相比之下，在II型酶中，类似的位置由保守的甘氨酸占据。在这里，我们报道了结核分枝杆菌I型MetAP， MetAP1c （MtMetAP1c）的活性位点半胱氨酸对高选择性半胱氨酸特异性试剂的反应性。通过位点定向诱变和共价和非共价复合物的晶体结构测定，确定MtMetAP1c Cys105选择性修饰的真实性。在这些观察的基础上，我们提出活性位点的金属离子通过适当地定向试剂来帮助Cys105的共价修饰，从而使反应成功。这些研究首次证实了I型MetAPs的保守半胱氨酸可以作为选择性抑制的靶点，我们相信这种化学性质可以用于微生物MetAPs的进一步药物发现工作。
Methionine aminopeptidases (MetAPs) cleave initiator methionine from similar to 70% of the newly synthesized proteins in every living cell, and specific inhibition or knockdown of this function is detrimental. MetAPs are metalloenzymes, and are broadly classified into two subtypes, type I and type II. Bacteria contain only type I MetAPs, and the active site of these enzymes contains a conserved cysteine. By contrast, in type II enzymes the analogous position is occupied by a conserved glycine. Here, we report the reactivity of the active site cysteine in a type I MetAP, MetAP1c, of Mycobacterium tuberculosis (MtMetAP1c) towards highly selective cysteine-specific reagents. The authenticity of selective modification of Cys105 of MtMetAP1c was established by using site-directed mutagenesis and crystal structure determination of covalent and noncovalent complexes. On the basis of these observations, we propose that metal ions in the active site assist in the covalent modification of Cys105 by orienting the reagents appropriately for a successful reaction. These studies establish, for the first time, that the conserved cysteine of type I MetAPs can be targeted for selective inhibition, and we believe that this chemistry can be exploited for further drug discovery efforts regarding microbial MetAPs.