Spectroscopic and Electrochemical Characterization of the Iron Sulfur and Cobalamin Cofactors of TsrM, an Unusual Radical S-Adenosylmethionine Methylase
Spectroscopic and Electrochemical Characterization of the Iron Sulfur and Cobalamin Cofactors of TsrM, an Unusual Radical S-Adenosylmethionine Methylase
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DOI：
10.1021/jacs.5b12592
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发表时间：
2016-03-16
影响因子：
15
通讯作者：
Booker, Squire J.
中科院分区：
文献类型：
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作者：
Blaszczyk, Anthony J.;Silakov, Alexey;Booker, Squire J.
TsrM是一种注释的自由基S-腺苷甲硫氨酸（SAM）酶，催化L-色氨酸吲哚环的碳2的甲基化。它的反应是硫链丝菌素A（一种硫肽抗生素）独特的喹哪酸部分生物合成的第一步。附加的甲基来自SAM;然而，该酶还需要钴胺素和铁硫簇辅因子进行周转。在这项工作中，我们报告的过量生产和纯化的TsrM和其金属辅因子的表征紫外可见，电子顺磁共振，超精细次能级相关（HYSCORE），穆斯堡尔谱以及蛋白膜电化学（PFE）。该酶在其分离状态下含有1当量的钴胺素辅因子，并且可以用铁和硫化物重构以含有一个具有位点分化的Fe 2 +/Fe 3+对的[4Fe-4S]簇。我们的光谱研究表明，TsrM结合钴胺素在一个不典型的五坐标碱基关闭/组氨酸关闭构象，其中二甲基苯并咪唑基团被取代的非含氮配体，这可能是一个水分子。通过PFE对蛋白质的电化学分析表明，具有中点电位为-550 mV的单电子氧化还原特征，其归属于[4Fe-4S]（2+）/[4Fe-4S]（+）-氧化还原对。TsrM的穆斯堡尔和HYSCORE光谱分析表明，SAM不绑定到唯一的铁网站的集群在相同的方式在其他自由基SAM（RS）酶，但其结合仍然扰乱了电子构型的Fe/S集群和钴（II）的丙氨酸辅因子。这些生物物理学研究表明，TsrM是一种非典型的RS酶，与其报道的不能催化5 '-脱氧腺苷5'-自由基的形成一致。
TsrM, an annotated radical S-adenosylmethionine (SAM) enzyme, catalyzes the methylation of carbon 2 of the indole ring of L-tryptophan. Its reaction is the first step in the biosynthesis of the unique quinaldic acid moiety of thiostrepton A, a thiopeptide antibiotic. The appended methyl group derives from SAM; however, the enzyme also requires cobalamin and iron-sulfur cluster cofactors for turnover. In this work we report the overproduction and purification of TsrM and the characterization of its metallocofactors by UV-visible, electron paramagnetic resonance, hyperfine sublevel correlation (HYSCORE), and Mossbauer spectroscopies as well as protein-film electrochemistry (PFE). The enzyme contains 1 equiv of its cobalamin cofactor in its as-isolated state and can be reconstituted with iron and sulfide to contain one [4Fe-4S] cluster with a site-differentiated Fe2+/Fe3+ pair. Our spectroscopic studies suggest that TsrM binds cobalamin in an uncharacteristic five-coordinate base-off/His-off conformation, whereby the dimethylbenzimidazole group is replaced by a non-nitrogenous ligand, which is likely a water molecule. Electrochemical analysis of the protein by PFE indicates a one-electron redox feature with a midpoint potential of -550 mV, which is assigned to a [4Fe-4S](2+)/[4Fe-4S](+)- redox couple. Analysis of TsrM by Mossbauer and HYSCORE spectroscopies suggests that SAM does not bind to the unique iron site of the cluster in the same manner as in other radical SAM (RS) enzymes, yet its binding still perturbs the electronic configuration of both the Fe/S cluster and the cob(II)alamin cofactors. These biophysical studies suggest that TsrM is an atypical RS enzyme, consistent with its reported inability to catalyze formation of a 5'-deoxyadenosyl 5'-radical.