A novel Nudix hydrolase for oxidized purine nucleoside triphosphates encoded by ORFYLR151c (PCD1 gene) in Saccharomyces cerevisiae.
A novel Nudix hydrolase for oxidized purine nucleoside triphosphates encoded by ORFYLR151c (PCD1 gene) in Saccharomyces cerevisiae.
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DOI：
10.1093/nar/gkh868
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发表时间：
2004
影响因子：
14.9
通讯作者：
T. Nunoshiba;Rikiya Ishida;Michi Sasaki;S. Iwai;Y. Nakabeppu;Kazuo Yamamoto
中科院分区：
文献类型：
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作者：
T. Nunoshiba;Rikiya Ishida;Michi Sasaki;S. Iwai;Y. Nakabeppu;Kazuo Yamamoto
在NCBI-BLAST数据库中使用Nutrition box（大肠杆菌MutT、普通假单胞菌MutT以及人、大鼠和小鼠MTH 1之间保守的短氨基酸序列）搜索酵母酿酒酵母中大肠杆菌MutT的功能同源物的候选物。在五个候选者中，我们关注开放阅读框架YLR 151 c，因为它具有与MutT的N-末端一半（包括NuVibe盒）具有约76%相似性的区域。因此，我们评估了YLR 151 c作为大肠杆菌MutT在链球菌中的功能同源物的能力。YLR 151 c的表达能够抑制由大肠杆菌mutT缺陷菌株中氧化核苷酸8-oxo-dGTP的错误掺入引起的从A：T到C：G的颠换。与野生型相比，酵母菌株中YLR 151 c的破坏导致自发突变频率增加约14倍。此外，生化分析表明，GST-YLR 151 c融合蛋白对7，8-二氢-8-氧代-2 ′-脱氧鸟苷三磷酸（8-oxo-dGTP）和1，2-二氢-2-羟基-2 ′-脱氧腺苷三磷酸（2-OH-dATP）都具有焦磷酸酶活性。GST-YLR 151 c对8-oxo-dGTP的比活性为5.6 x 10（-3）microM（-1）s（-1），与大肠杆菌中MutT的备用酶RibA相似，但比hMTH 1低150倍。从这些结果中，我们得出结论，YLR 151 c具有通过氧化核苷酸的消毒来防止自发突变的能力，并且它可能是大肠杆菌MutT在S.ae中的功能同源物。
A search for candidates for a functional homologue of Escherichia coli MutT in yeast Saccharomyces cerevisiae was made in the NCBI-BLAST database using the Nudix box, a short amino acid sequence conserved among E.coli MutT, Pseudomonoas vulgaris MutT, and human, rat and mouse MTH1. Among five candidates, we focused on the open reading frame YLR151c, because it had a region with approximately 76% similarity to the N-terminal half of MutT including the Nudix box. We thus evaluated the ability of YLR151c as a functional homologue of E.coli MutT in S.cerevisiae. Expression of YLR151c was able to suppress the transversion from A:T to C:G caused by misincorporation of the oxidized nucleotide 8-oxo-dGTP in the E.coli mutT-deficient strain. The disruption of the YLR151c in yeast strain caused approximately 14-fold increase in the frequency of spontaneous mutation compared to the wild type. Additionally, biochemical analysis indicated that GST-YLR151c fusion protein possessed pyrophosphatase activity for both 7,8-dihydro-8-oxo-2'-deoxyguanosine triphosphate (8-oxo-dGTP) and 1,2-dihydro-2-hydroxy-2'-deoxyadenosine triphosphate (2-OH-dATP). The specific activity of GST-YLR151c for 8-oxo-dGTP was 5.6 x 10(-3) microM(-1) s(-1), which was similar to that of RibA, a backup enzyme for MutT in E.coli, but was 150-fold lower than that of hMTH1. From these results, we conclude that YLR151c has an ability to prevent spontaneous mutagenesis via sanitization of oxidized nucleotides, and that it may be the functional homologue of E.coli MutT in S.cerevisiae.