5'-Nucleotidases (EC 3.1.3.5) are enzymes that catalyze the hydrolytic dephosphorylation of 5'-ribonucleotides and 5'-deoxyribonucleotides to their corresponding nucleosides plus phosphate. In the present study, to search for new genes encoding 5'-nucleotidases specific for purine nucleotides in industrially important Bacillus species, "shotgun" cloning and the direct selection of recombinant clones grown in purine nucleosides at inhibitory concentrations were performed in the Escherichia coli GS72 strain, which is sensitive to these compounds. As a result, orthologous yitU genes from Bacillus subtilis and Bacillus amyloliquefaciens, whose products belong to the ubiquitous haloacid dehalogenase superfamily (HADSF), were selected and found to have a high sequence similarity of 87%. B. subtilis YitU was produced in E. coli as an N-terminal hexahistidine-tagged protein, purified and biochemically characterized as a soluble 5'-nucleotidase with broad substrate specificity with respect to various deoxyribo- and ribonucleoside monophosphates: dAMP, GMP, dGMP, CMP, AMP, XMP, IMP and 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranosyl 5'-monophosphate (AICAR-P). However, the preferred substrate for recombinant YitU was shown to be flavin mononucleotide (FMN). B. subtilis and B. amyloliquefaciens yitU overexpression increased riboflavin (RF) and 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) accumulation and can be applied to breed highly performing RF- and AICAR-producing strains.

中文翻译：

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从芽孢杆菌属物种中表达和纯化5'-核苷酸酶YitU：其酶学性质及其在生物技术中的应用。

5'-核糖核酸酶（EC 3.1.3.5）是催化5'-核糖核苷酸和5'-脱氧核糖核苷酸水解为相应的核苷加磷酸的酶。在本研究中，为了在工业上重要的芽孢杆菌属物种中寻找编码嘌呤核苷酸特异的5'-核苷酸酶的新基因，在大肠杆菌GS72中进行了“ shot弹”克隆以及在嘌呤核苷中以抑制浓度生长的重组克隆的直接选择。对这些化合物敏感的菌株。结果，选择了来自枯草芽孢杆菌和解淀粉芽孢杆菌的直系同源yitU基因，其产物属于遍在的卤酸脱卤素酶超家族（HADSF），并具有87％的高序列相似性。枯草芽孢杆菌YitU在E. 大肠杆菌是一种N-末端六组氨酸标签蛋白，经过纯化和生化鉴定为可溶性5'-核苷酸酶，对各种脱氧核糖和核糖核苷一磷酸具有广泛的底物特异性：dAMP，GMP，dGMP，CMP，AMP，XMP，IMP和5-氨基咪唑-4-羧酰胺-1-β-D-呋喃呋喃糖基5'-单磷酸酯（AICAR-P）。然而，重组YitU的优选底物显示为黄素单核苷酸（FMN）。枯草芽孢杆菌和解淀粉芽孢杆菌过表达会增加核黄素（RF）和5-氨基咪唑-4-羧酰胺核糖核苷（AICAR）的积累，可用于繁殖高性能RF和AICAR产生菌株。-对各种脱氧核糖核糖核苷和核糖核苷单磷酸具有广泛的底物特异性的核苷酸酶：dAMP，GMP，dGMP，CMP，AMP，XMP，IMP和5-氨基咪唑-4-甲酰胺-1-β-D-呋喃呋喃糖基5'-单磷酸（ AICAR-P）。然而，重组YitU的优选底物显示为黄素单核苷酸（FMN）。枯草芽孢杆菌和解淀粉芽孢杆菌过表达会增加核黄素（RF）和5-氨基咪唑-4-羧酰胺核糖核苷（AICAR）的积累，可用于繁殖高性能RF和AICAR产生菌株。-核苷酸酶对多种脱氧核糖核糖核糖核糖核苷具有广泛的底物特异性：dAMP，GMP，dGMP，CMP，AMP，XMP，IMP和5-氨基咪唑-4-甲酰胺-1-β-D-呋喃呋喃糖基5'-单磷酸（ AICAR-P）。然而，重组YitU的优选底物显示为黄素单核苷酸（FMN）。枯草芽孢杆菌和解淀粉芽孢杆菌过表达会增加核黄素（RF）和5-氨基咪唑-4-羧酰胺核糖核苷（AICAR）的积累，可用于繁殖高性能RF和AICAR产生菌株。