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首页> 美国卫生研究院文献>Journal of Bacteriology >Reduction of Cob(III)alamin to Cob(II)alamin in Salmonella enterica Serovar Typhimurium LT2
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Reduction of Cob(III)alamin to Cob(II)alamin in Salmonella enterica Serovar Typhimurium LT2

机译：肠炎沙门氏菌鼠伤寒沙门氏菌LT2中的Cob（III）阿拉明还原为Cob（II）阿拉明

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Reduction of the cobalt ion of cobalamin from the Co(III) to the Co(I) oxidation state is essential for the synthesis of adenosylcobalamin, the coenzymic form of this cofactor. A cob(II)alamin reductase activity in Salmonella enterica serovar Typhimurium LT2 was isolated to homogeneity. N-terminal analysis of the homogeneous protein identified NAD(P)H:flavin oxidoreductase (Fre) (EC 1.6.8.1) as the enzyme responsible for this activity. The fre gene was cloned, and the overexpressed protein, with a histidine tag at its N terminus, was purified to homogeneity by nickel affinity chromatography. His-tagged Fre reduced flavins (flavin mononucleotide [FMN] and flavin adenine dinucleotide [FAD]) and cob(III)alamin to cob(II)alamin very efficiently. Photochemically reduced FMN substituted for Fre in the reduction of cob(III)alamin to cob(II)alamin, indicating that the observed cobalamin reduction activity was not Fre dependent but FMNH2 dependent. Enzyme-independent reduction of cob(III)alamin to cob(II)alamin by FMNH2 occurred at a rate too fast to be measured. The thermodynamically unfavorable reduction of cob(II)alamin to cob(I)alamin was detectable by alkylation of the cob(I)alamin nucleophile with iodoacetate. Detection of the product, caboxymethylcob(III)alamin, depended on the presence of FMNH2 in the reaction mixture. FMNH2 failed to substitute for potassium borohydride in in vitro assays for corrinoid adenosylation catalyzed by the ATP:co(I)rrinoid adenosyltransferase (CobA) enzyme, even under conditions where Fre and NADH were present in the reaction mixture to ensure that FMN was always reduced. These results were interpreted to mean that Fre was not responsible for the generation of cob(I)alamin in vivo. Consistent with this idea, a fre mutant displayed wild-type cobalamin biosynthetic phenotypes. It is proposed that S. enterica serovar Typhimurium LT2 may not have a cob(III)alamin reductase enzyme and that, in vivo, nonadenosylated cobalamin and other corrinoids are maintained as co(II)rrinoids by reduced flavin nucleotides generated by Fre and other flavin oxidoreductases.
机译：将钴胺素的钴离子从Co（III）还原为Co（I）氧化态对于合成腺苷钴胺素（该辅因子的辅酶形式）至关重要。肠炎沙门氏菌血清鼠伤寒沙门氏菌LT2中的cob（II）阿拉明还原酶活性被分离到同质。均质蛋白的N端分析确定NAD（P）H：黄素氧化还原酶（Fre）（EC 1.6.8.1）是负责此活性的酶。克隆了fre基因，并通过镍亲和色谱将过表达的蛋白在其N端带有一个组氨酸标签，纯化至同质。带有组氨酸标签的Fre将黄素（黄素单核苷酸[FMN]和黄素腺嘌呤二核苷酸[FAD]）和cob（III）alamin高效地还原为cob（II）alamin。光化学还原的FMN在将Cob（III）alamin还原为Cob（II）alamin中代替了Fre，表明观察到的Cobalamin还原活性不依赖Fre，而是依赖FMNH2。 FMNH2以酶非依赖性的方式将Cob（III）阿拉明还原为Cob（II）阿拉明的速度太快，无法测量。通过用碘乙酸酯将钴（I）阿拉明亲核体烷基化，可以检测到钴（II）丙氨酸的热力学不利还原为钴（I）阿拉明。产物羧甲基甲基钴（III）丙氨酸的检测取决于反应混合物中FMNH 2的存在。 FMNH2在由ATP：co（I）类rinrinoid腺苷基转移酶（CobA）酶催化的皮质类固醇腺苷化的体外分析中无法替代硼氢化钾，即使在反应混合物中存在Fre和NADH以确保始终减少FMN的条件下。这些结果被解释为意味着Fre不负责体内Cob（I）alamin的产生。与此想法一致，fre突变体显示出野生型钴胺素的生物合成表型。有人提出，肠炎链球菌血清型鼠伤寒沙门氏菌LT2可能没有钴（III）丙氨酸还原酶，并且在体内，通过由Fre和其他黄素产生的黄素核苷酸减少，将腺苷化的钴胺素和其他类海藻素维持为co（II）类海藻素。氧化还原酶。

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