In vitro biosynthesis of diphthamide, studied with mutant Chinese hamster ovary cells resistant to diphtheria toxin.

In vitro biosynthesis of diphthamide, studied with mutant Chinese hamster ovary cells resistant to diphtheria toxin.
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用抗白喉毒素的突变型中国仓鼠卵巢细胞研究了白喉酰胺的体外生物合成。

DOI:
10.1128/mcb.4.4.642-650.1984
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发表时间:
1984
影响因子:
5.3
通讯作者:
Moehring,JM
Moehring,JM
中科院分区:
生物学2区
文献类型:
--
作者:
Moehring,TJ;Danley,DE;Moehring,JM

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敌草胺是一种独特的氨基酸,是组氨酸的翻译后衍生物,存在于蛋白质合成延伸因子2的白喉毒素催化的ADP-核糖化延伸因子2的位置。我们研究了在这种复杂的翻译后修饰的不同步骤中,中国仓鼠卵巢细胞的突变体生物合成敌草胺的步骤。生化证据表明,这种修饰至少需要三个步骤,其中两个步骤是在体外完成的。我们鉴定了一种甲基转移酶活性,它能将甲基基团从腺苷甲硫氨酸转移到去甲基化形式的敌草胺(去甲胺化的形式),并初步鉴定了一种依赖于ATP的合成酶活性,该合成酶参与了敌草胺的生物合成。我们的结果与建议的敌草胺的结构是一致的(B.G.VanNess等,J.Biol。化学255:10710-10716,1980年)。
Diphthamide, a unique amino acid, is a post-translational derivative of histidine that exists in protein synthesis elongation factor 2 at the site of diphtheria toxin-catalyzed ADP-ribosylation of elongation factor 2. We investigated steps in the biosynthesis of diphthamide with mutants of Chinese hamster ovary cells that were altered in different steps of this complex post-translational modification. Biochemical evidence indicates that this modification requires a minimum of three steps, two of which we accomplished in vitro. We identified a methyltransferase activity that transfers methyl groups fromS-adenosyl methionine to an unmethylated form of diphthine (the deamidated form of diphthamide), and we tentatively identified an ATP-dependent synthetase activity involved in the biosynthesis of diphthamide from diphthine. Our results are in accord with the proposed structure of diphthamide (B. G. VanNess, et al., J. Biol. Chem.255:10710–10716, 1980).
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