Biosynthesis of thiamin thiazole in eukaryotes: Conversion of NAD to an advanced intermediate
Biosynthesis of thiamin thiazole in eukaryotes: Conversion of NAD to an advanced intermediate
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DOI：
10.1021/ja067606t
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发表时间：
2007-03-14
影响因子：
15
通讯作者：
Begley, Tadhg P.
中科院分区：
文献类型：
--
作者：
Chatterjee, Abhishek;Jurgenson, Christopher T.;Begley, Tadhg P.
噻唑合酶催化焦磷酸硫胺素的噻唑部分的形成。来自酿酒酵母的酶（THI 4）与一组强结合的腺苷酸化代谢物共纯化。其中一种化合物的结构为5-（2-羟乙基）-4-甲基噻唑-2-羧酸的ADP加合物。迄今为止，通过释放蛋白质结合的代谢物来产生活性野生型THI 4的尝试失败了。在这里，我们描述了THI 4的两个部分活性突变体（C204 A和H200 N）的鉴定和表征。两种突变体都催化烟酰胺部分从NAD释放以产生ADP-核糖，ADP-核糖进一步转化为ADP-核酮糖。在甘氨酸的存在下，这两种突变体都催化形成高级中间体。该中间体被困与邻苯二胺，产生一个稳定的喹喔啉衍生物，其特征在于通过NMR光谱和ESI-MS。这些观察证实NAD作为底物THI 4和阐明这种独特的生物合成的噻唑部分硫胺素在真核生物中的早期步骤。
Thiazole synthase catalyzes the formation of the thiazole moiety of thiamin pyrophosphate. The enzyme from Saccharomyces cerevisiae (THI4) copurifies with a set of strongly bound adenylated metabolites. One of them has been characterized as the ADP adduct of 5-(2-hydroxyethyl)-4-methylthiazole-2- carboxylic acid. Attempts toward yielding active wild-type THI4 by releasing protein-bound metabolites have failed so far. Here, we describe the identification and characterization of two partially active mutants (C204A and H200N) of THI4. Both mutants catalyzed the release of the nicotinamide moiety from NAD to produce ADP-ribose, which was further converted to ADP-ribulose. In the presence of glycine, both the mutants catalyzed the formation of an advanced intermediate. The intermediate was trapped with ortho-phenylenediamine, yielding a stable quinoxaline derivative, which was characterized by NMR spectroscopy and ESI-MS. These observations confirm NAD as the substrate for THI4 and elucidate the early steps of this unique biosynthesis of the thiazole moiety of thiamin in eukaryotes.