Terakawa Ayako, Natsume Ayane, Okada Atsushi, Nishihata Shogo, Kuse Junko, Tanaka Kosei, Takenaka Shinji, Ishikawa Shu, Yoshida Ken-Ichi
Department of Agrobioscience, Kobe University, 1-1 Rokkodai, Nada, Kobe, Hyogo, 657-8501, Japan.
Organization of Advanced Science and Technology, Kobe University, 1-1 Rokkodai, Nada, Kobe, Hyogo, 657-8501, Japan.
BMC Microbiol. 2016 Oct 26;16(1):249. doi: 10.1186/s12866-016-0866-5.
In Escherichia coli, nagD, yrfG, yjjG, yieH, yigL, surE, and yfbR encode 5'-nucleotidases that hydrolyze the phosphate group of 5'-nucleotides. In Bacillus subtilis, genes encoding 5'-nucleotidase have remained to be identified.
We found that B. subtilis ycsE, araL, yutF, ysaA, and yqeG show suggestive similarities to nagD. Here, we expressed them in E. coli to purify the respective His-tagged proteins. YcsE exhibited significant 5'-nucleotidase activity with a broader specificity, whereas the other four enzymes had rather weak but suggestive activities with various capacities and substrate specificities. In contrast, B. subtilis yktC shares high similarity with E. coli suhB encoding an inositol monophosphatase. YktC exhibited inositol monophosphatase activity as well as 5'-nucleotidase activity preferential for GMP and IMP. The ycsE, yktC, and yqeG genes are induced by oxidative stress and were dispensable, although yqeG was required to maintain normal growth on solid medium. In the presence of diamide, only mutants lacking yktC exhibited enhanced growth defects, whereas the other mutants without ycsE or yqeG did not.
Accordingly, in B. subtilis, at least YcsE and YktC acted as major 5'-nucleotidases and the four minor enzymes might function when the intracellular concentrations of substrates are sufficiently high. In addition, YktC is involved in resistance to oxidative stress caused by diamide, while YqeG is necessary for normal colony formation on solid medium.
在大肠杆菌中，nagD、yrfG、yjjG、yieH、yigL、surE和yfbR编码5'-核苷酸酶，可水解5'-核苷酸的磷酸基团。在枯草芽孢杆菌中，编码5'-核苷酸酶的基因尚未被鉴定。
我们发现枯草芽孢杆菌的ycsE、araL、yutF、ysaA和yqeG与nagD有相似之处。在此，我们在大肠杆菌中表达它们以纯化各自带有His标签的蛋白质。YcsE表现出显著的5'-核苷酸酶活性，特异性较广，而其他四种酶的活性较弱，但具有不同的能力和底物特异性。相比之下，枯草芽孢杆菌的yktC与大肠杆菌中编码肌醇单磷酸酶的suhB高度相似。YktC表现出肌醇单磷酸酶活性以及对GMP和IMP优先的5'-核苷酸酶活性。ycsE、yktC和yqeG基因受氧化应激诱导，且是可有可无的，尽管yqeG对于在固体培养基上维持正常生长是必需的。在存在二酰胺的情况下，只有缺乏yktC的突变体表现出增强的生长缺陷，而其他没有ycsE或yqeG的突变体则没有。
因此，在枯草芽孢杆菌中，至少YcsE和YktC作为主要的5'-核苷酸酶，而当底物的细胞内浓度足够高时，这四种次要酶可能发挥作用。此外，YktC参与对由二酰胺引起的氧化应激的抗性，而YqeG对于在固体培养基上正常菌落形成是必需的。
