Identification of formyl kynurenine formamidase and kynurenine aminotransferase from Saccharomyces cerevisiae using crystallographic, bioinformatic and biochemical evidence.
Identification of formyl kynurenine formamidase and kynurenine aminotransferase from Saccharomyces cerevisiae using crystallographic, bioinformatic and biochemical evidence.
复制标题
使用晶体学、生物信息学和生化证据鉴定酿酒酵母中的甲酰犬尿氨酸甲酰胺酶和犬尿氨酸转氨酶。
DOI：
10.1021/bi701172v
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发表时间：
2008
期刊：
影响因子：
2.9
通讯作者：
David K Wilson
中科院分区：
文献类型：
--
作者：
M. Wogulis;E. Chew;Paul D. Donohoue;David K Wilson
许多真核生物，包括酿酒酵母菌和哺乳动物，都可以用色氨酸作为原料合成必需的酶辅因子NAD+。通路上或分支上的代谢物具有重要的生物学功能。例如，犬尿酸可以作为NMDA拮抗剂，从而在广泛的病理状态下发挥神经保护剂的作用。n -甲酰基犬尿氨酸甲酰胺酶（N-Formyl kynurenine formamidase， FKF）通过水解n -甲酰基犬尿氨酸生成犬尿氨酸和甲酸酯，催化NAD+生物合成途径的第二步。据报道，酿酒酵母FKF是一种由BNA3编码的吡哆醛磷酸依赖酶。我们使用晶体学、生物信息学和生化方法联合证明Bna3p不是一种FKF，而很可能是酵母犬尿氨酸转氨酶，将犬尿氨酸转化为犬尿酸。此外，我们鉴定了YDR428C，一个编码α / β水解酶的酵母ORF，没有先前指定的功能，作为FKF。我们根据对先前结构基因组学结果的解释以及它与已知FKFs的序列同源性预测了它的功能。生化、生物信息学、遗传和体内代谢物的LC-MS数据表明，YDR428C（我们命名为BNA7）是酵母的FKF。
The essential enzymatic cofactor NAD+ can be synthesized in many eukaryotes, including Saccharomyces cerevisiae and mammals, using tryptophan as a starting material. Metabolites along the pathway or on branches have important biological functions. For example, kynurenic acid can act as an NMDA antagonist, thereby functioning as a neuroprotectant in a wide range of pathological states. N-Formyl kynurenine formamidase (FKF) catalyzes the second step of the NAD+ biosynthetic pathway by hydrolyzing N-formyl kynurenine to produce kynurenine and formate. The S. cerevisiae FKF had been reported to be a pyridoxal phosphate-dependent enzyme encoded by BNA3. We used combined crystallographic, bioinformatic and biochemical methods to demonstrate that Bna3p is not an FKF but rather is most likely the yeast kynurenine aminotransferase, which converts kynurenine to kynurenic acid. Additionally, we identify YDR428C, a yeast ORF coding for an alpha/beta hydrolase with no previously assigned function, as the FKF. We predicted its function based on our interpretation of prior structural genomics results and on its sequence homology to known FKFs. Biochemical, bioinformatics, genetic and in vivo metabolite data derived from LC-MS demonstrate that YDR428C, which we have designated BNA7, is the yeast FKF.