Metal-free class Ie ribonucleotide reductase from pathogens initiates catalysis with a tyrosine-derived dihydroxyphenylalanine radical.
Metal-free class Ie ribonucleotide reductase from pathogens initiates catalysis with a tyrosine-derived dihydroxyphenylalanine radical.
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DOI：
10.1073/pnas.1811993115
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发表时间：
2018-10-02
影响因子：
11.1
通讯作者：
Boal AK
中科院分区：
文献类型：
--
作者：
Blaesi EJ;Palowitch GM;Hu K;Kim AJ;Rose HR;Alapati R;Lougee MG;Kim HJ;Taguchi AT;Tan KO;Laremore TN;Griffin RG;Krebs C;Matthews ML;Silakov A;Bollinger JM Jr;Allen BD;Boal AK
核糖核苷酸转化为DNA生物合成所需的2′-脱氧核糖核苷酸是由核糖核苷酸还原酶（RNR）通过自由基机制催化的。已知类型的RNR都依赖于氧化还原活性过渡金属-锰、铁或钴-用于自由基引发。病原菌受到过渡金属螯合和由其宿主施加氧化应激的挑战，并且具有不同金属需求和自由基引发氧化剂的多个RNR的部署是已知的细菌对策。来自两种细菌病原体的I类RNR在其活性状态下完全缺乏过渡金属，并使用酪氨酸衍生的二羟基苯丙氨酸自由基作为其引发剂，体现了对抗过渡金属和氧化剂介导的先天免疫的新策略，并加强细菌RNR作为潜在的抗生素靶标。所有细胞通过核糖核苷酸还原酶（RNR）的活性获得用于DNA合成的2′-脱氧核糖核苷酸。在人类和病原菌中发现的I类RNR的不同之处在于（i）使用Fe（II）、Mn（II）或两者来活化双核金属辅因子亚基β;（ii）还原的双金属中心与分子氧或超氧化物的反应来进行这种活化;（iii）需要Fe（II）、Mn（II）或两者来活化双核金属辅因子亚基β。（或缺乏）黄素蛋白活化酶NrdI从O2提供超氧化物;和（iv）使用稳定的酪氨酰基自由基或高价双金属簇，通过将α亚基中的半胱氨酸残基氧化为自由基（Cys·）来启动每次转换。细菌I类，亚类b-d RNR使用锰，这与真核Ia酶专门使用铁形成对比，似乎是某些病原体对抗其宿主所施加的铁剥夺的对策。在这里，我们报告了一个无金属类型的I类RNR（e亚类）从两个人类病原体。α亚基中的Cys·由β亚基中稳定的酪氨酸衍生的二羟基苯丙氨酸自由基（DOPA·）产生。只有当β与病原体基因组中相邻编码的NrdI活化酶共表达时，才能在大肠杆菌中发生三电子氧化产生DOPA·。这种新的RNR与过渡金属的独立性，或对单个金属离子的需要仅短暂激活，可能为病原体提供针对过渡金属导向的先天免疫的更有效的对策。
Conversion of ribonucleotides to the 2′-deoxyribonucleotides required for DNA biosynthesis is catalyzed by ribonucleotide reductases (RNRs) via a free-radical mechanism. Known types of RNRs all depend on redox-active transition metals—manganese, iron, or cobalt—for radical initiation. Pathogenic bacteria are challenged by transition metal sequestration and infliction of oxidative stress by their hosts, and the deployment of multiple RNRs with different metal requirements and radical-initiating oxidants is a known bacterial countermeasure. A class I RNR from two bacterial pathogens completely lacks transition metals in its active state and uses a tyrosine-derived dihydroxyphenylalanine radical as its initiator, embodying a novel tactic to combat transition metal- and oxidant-mediated innate immunity and reinforcing bacterial RNRs as potential antibiotic targets. All cells obtain 2′-deoxyribonucleotides for DNA synthesis through the activity of a ribonucleotide reductase (RNR). The class I RNRs found in humans and pathogenic bacteria differ in (i) use of Fe(II), Mn(II), or both for activation of the dinuclear-metallocofactor subunit, β; (ii) reaction of the reduced dimetal center with dioxygen or superoxide for this activation; (iii) requirement (or lack thereof) for a flavoprotein activase, NrdI, to provide the superoxide from O2; and (iv) use of either a stable tyrosyl radical or a high-valent dimetal cluster to initiate each turnover by oxidizing a cysteine residue in the α subunit to a radical (Cys•). The use of manganese by bacterial class I, subclass b-d RNRs, which contrasts with the exclusive use of iron by the eukaryotic Ia enzymes, appears to be a countermeasure of certain pathogens against iron deprivation imposed by their hosts. Here, we report a metal-free type of class I RNR (subclass e) from two human pathogens. The Cys• in its α subunit is generated by a stable, tyrosine-derived dihydroxyphenylalanine radical (DOPA•) in β. The three-electron oxidation producing DOPA• occurs in Escherichia coli only if the β is coexpressed with the NrdI activase encoded adjacently in the pathogen genome. The independence of this new RNR from transition metals, or the requirement for a single metal ion only transiently for activation, may afford the pathogens an even more potent countermeasure against transition metal-directed innate immunity.
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