Purification, Characterization, and Gene Cloning of Purine Nucleosidase from Ochrobactrum anthropi
Purification, Characterization, and Gene Cloning of Purine Nucleosidase from Ochrobactrum anthropi
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人苍白杆菌嘌呤核苷酶的纯化、表征和基因克隆
DOI：
10.1128/aem.67.4.1783-1787.2001
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发表时间：
2001
影响因子：
4.4
通讯作者：
S. Shimizu
中科院分区：
文献类型：
--
作者：
J. Ogawa;S. Takeda;S. Xie;H. Hatanaka;T. Ashikari;T. Amachi;S. Shimizu
摘要：一种名为人苍白杆菌（Ochrobactrum anthropi）的细菌在与嘌呤核苷一起培养时会产生大量的核苷酶。将核苷酶纯化至同质。该酶的分子量约为170,000，由四个相同的亚基组成。它特异性催化嘌呤核苷的不可逆N-核苷水解，Km值为11.8至56.3μM。最适活性温度和pH分别为50℃和pH 4.5至6.5。嘧啶核苷、嘌呤和嘧啶核苷酸、NAD、NADP 和烟酰胺单核苷酸不被酶水解。该酶的嘌呤核苷水解活性受到嘧啶核苷的抑制（混合抑制），Ki和Ki'值为0.455～11.2 μM。金属离子螯合剂抑制活性，添加Zn2+或Co2+可恢复活性。一个 1.5 kb DNA 片段包含编码核苷酶的开放阅读框，已在大肠杆菌中进行克隆、测序和表达。推导的 363 个氨基酸序列（包括 22 个残基的前导肽）与酶分子量以及 NH2 末端和内部肽的氨基酸序列一致，并且该酶与已知的原生动物寄生虫核苷酶同源。形成催化位点并参与与金属离子结合的氨基酸残基在这些核苷酶中得到很好的保守。
ABSTRACT A bacterium, Ochrobactrum anthropi, produced a large amount of a nucleosidase when cultivated with purine nucleosides. The nucleosidase was purified to homogeneity. The enzyme has a molecular weight of about 170,000 and consists of four identical subunits. It specifically catalyzes the irreversibleN-riboside hydrolysis of purine nucleosides, theKm values being 11.8 to 56.3 μM. The optimal activity temperature and pH were 50°C and pH 4.5 to 6.5, respectively. Pyrimidine nucleosides, purine and pyrimidine nucleotides, NAD, NADP, and nicotinamide mononucleotide are not hydrolyzed by the enzyme. The purine nucleoside hydrolyzing activity of the enzyme was inhibited (mixed inhibition) by pyrimidine nucleosides, with Ki and Ki′ values of 0.455 to 11.2 μM. Metal ion chelators inhibited activity, and the addition of Zn2+ or Co2+ restored activity. A 1.5-kb DNA fragment, which contains the open reading frame encoding the nucleosidase, was cloned, sequenced, and expressed inEscherichia coli. The deduced 363-amino-acid sequence including a 22-residue leader peptide is in agreement with the enzyme molecular mass and the amino acid sequences of NH2-terminal and internal peptides, and the enzyme is homologous to known nucleosidases from protozoan parasites. The amino acid residues forming the catalytic site and involved in binding with metal ions are well conserved in these nucleosidases.
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影响因子：
2.9
作者：
Gopaul, DN;Meyer, SL;Schramm, VL
通讯作者：
Schramm, VL
底物的结合模式和原生动物核苷水解酶的拟议过渡态类似物。
影响因子：
2.9
作者：
Parkin,DW;Schramm,VL
通讯作者：
Schramm,VL
与嘌呤和嘧啶代谢相关的疾病。
DOI：
--
发表时间：
1984
期刊：
Special topics in endocrinology and metabolism
影响因子：
--
作者：
Edwards,NL;Fox,IH
通讯作者：
Fox,IH
DOI：
10.1016/s0021-9258(18)54759-1
发表时间：
1991-11
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
D. Parkin;B. Horenstein;D. Abdulah;B. Estupiñán;V. Schramm
通讯作者：
D. Parkin;B. Horenstein;D. Abdulah;B. Estupiñán;V. Schramm