From cyclohydrolase to oxidoreductase:: Discovery of nitrile reductase activity in a common fold
From cyclohydrolase to oxidoreductase:: Discovery of nitrile reductase activity in a common fold
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DOI：
10.1073/pnas.0408056102
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发表时间：
2005-03-22
影响因子：
11.1
通讯作者：
Iwata-Reuyl, D
中科院分区：
文献类型：
--
作者：
Van Lanen, SG;Reader, JS;Iwata-Reuyl, D
来自枯草芽孢杆菌的酶YkvM先前沿着与其他三种酶（YkvJKL）一起在生物信息学搜索中鉴定，所述酶涉及肌苷的生物合成，肌苷是在细菌和真核生物的tRNA（GUN）中发现的7-脱氮鸟嘌呤修饰的核苷。对不动杆菌中ykvJKLM突变体的遗传分析证实，每一个突变体都是肌苷生物合成所必需的，因此将这些基因重命名为queCDEF。QueF与FolE所表征的I型GTP环水解酶具有显著的同源性。鉴于GTP是肌苷的前体，并且假定环水解酶样反应是肌苷生物合成的初始步骤，因此提出QueF是负责该反应的假定环水解酶样酶。我们已经从B中克隆了queF基因。枯草芽孢杆菌和大肠杆菌的重组酶和表征。与基于序列分析的预测相反，我们发现这些酶实际上催化一个机械上无关的反应，7-氰基-7-脱氮鸟嘌呤还原为7-氨甲基-7-脱氮鸟嘌呤的NADPH依赖性还原，这是肌苷生物合成的后期步骤。我们在这里报告，在体外和体内的研究，证明这种催化活性，以及初步的生化和生物信息学分析，提供深入了解这个家庭的酶的结构。
The enzyme YkvM from Bacillus subtilis was identified previously along with three other enzymes (YkvJKL) in a bioinformatics search for enzymes involved in the biosynthesis of queuosine, a 7-deazaguanine modified nucleoside found in tRNA(GUN) of Bacteria and Eukarya. Genetic analysis of ykvJKLM mutants in Acinetobacter confirmed that each was essential for queuosine biosynthesis, and the genes were renamed queCDEF. QueF exhibits significant homology to the type I GTP cyclohydrolases characterized by FolE. Given that GTP is the precursor to queuosine and that a cyclohydrolase-like reaction was postulated as the initial step in queuosine biosynthesis, QueF was proposed to be the putative cyclohydrolase-like enzyme responsible for this reaction. We have cloned the queF genes from B. subtilis and Escherichia coli and characterized the recombinant enzymes. Contrary to the predictions based on sequence analysis, we discovered that the enzymes, in fact, catalyze a mechanistically unrelated reaction, the NADPH-dependent reduction of 7-cyano-7-deazaguanine to7-aminomethyl-7-deazaguanine, a late step in the biosynthesis of queuosine. We report here in vitro and in vivo studies that demonstrate this catalytic activity, as well as preliminary biochemical and bioinformatics analysis that provide insight into the structure of this family of enzymes.