The alpha subunit of nitrile hydratase is sufficient for catalytic activity and post-translational modification.
The alpha subunit of nitrile hydratase is sufficient for catalytic activity and post-translational modification.
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DOI：
10.1021/bi500260j
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发表时间：
2014-06-24
期刊：
影响因子：
2.9
通讯作者：
Bandarian V
中科院分区：
文献类型：
--
作者：
Nelp MT;Astashkin AV;Breci LA;McCarty RM;Bandarian V
腈水合酶（nase）具有单个核铁或钴辅因子，其配位环境包括罕见的翻译后氧化半胱氨酸亚砜和亚砜酸配体。该辅因子位于异二聚体酶界面活性位点的α-亚基上。与典型的nase不同，来自链霉菌的toyocamycin nitrile hydratase （TNHase）是该家族中独特的三个亚基成员，参与吡咯嘧啶类抗生素的生物合成。TNHase的亚基与典型nase的α-亚基和β-亚基同源。在此，我们报道了TNHase α-亚基的表达、纯化和表征。α-亚基TNHase的紫外可见光谱、EPR和质谱证明，该亚基能够单独合成具有完整翻译后修饰的活性位点复合物。值得注意的是，分离的翻译后修饰的α-亚基对天然底物toyocamycin以及烟酸前体3-氰吡啶也具有催化活性。单亚基变体与异三聚体蛋白的稳态动力学参数比较清楚地表明，额外的亚基具有底物特异性和催化效率。我们得出结论，α-亚基是腈水化所需的最小序列，为研究这类重要催化剂的机理和翻译后修饰提供了一个简化的支架。
Nitrile hydratases (NHases) possess a mononuclear iron or cobalt cofactor whose coordination environment includes rare post-translationally oxidized cysteine sulfenic and sulfinic acid ligands. This cofactor is located in the α-subunit at the interfacial active site of the heterodimeric enzyme. Unlike canonical NHases, toyocamycin nitrile hydratase (TNHase) from Streptomyces rimosus is a unique three-subunit member of this family involved in the biosynthesis of pyrrolopyrimidine antibiotics. The subunits of TNHase are homologous to the α- and β-subunits of prototypical NHases. Herein we report the expression, purification, and characterization of the α-subunit of TNHase. The UV–visible, EPR, and mass spectra of the α-subunit TNHase provide evidence that this subunit alone is capable of synthesizing the active site complex with full post-translational modifications. Remarkably, the isolated post-translationally modified α-subunit is also catalytically active with the natural substrate, toyocamycin, as well as the niacin precursor 3-cyanopyridine. Comparisons of the steady state kinetic parameters of the single subunit variant to the heterotrimeric protein clearly show that the additional subunits impart substrate specificity and catalytic efficiency. We conclude that the α-subunit is the minimal sequence needed for nitrile hydration providing a simplified scaffold to study the mechanism and post-translational modification of this important class of catalysts.
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