PURIFICATION, CHARACTERIZATION, AND GENETIC-ANALYSIS OF MYCOBACTERIUM-TUBERCULOSIS UREASE, A POTENTIALLY CRITICAL DETERMINANT OF HOST-PATHOGEN INTERACTION
PURIFICATION, CHARACTERIZATION, AND GENETIC-ANALYSIS OF MYCOBACTERIUM-TUBERCULOSIS UREASE, A POTENTIALLY CRITICAL DETERMINANT OF HOST-PATHOGEN INTERACTION
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DOI：
10.1128/jb.177.19.5644-5652.1995
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发表时间：
1995-10-01
影响因子：
3.2
通讯作者：
HORWITZ, MA
中科院分区：
文献类型：
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作者：
CLEMENS, DL;LEE, BY;HORWITZ, MA
纯化的结核分枝杆菌尿素酶(尿素氨基水解酶[EC 3.5.1.5])含有三个亚基：两个小亚基，每个亚基约11,000 Da，一个大亚基62,000 Da。这三个亚基的N末端序列分别与其他细菌尿素酶的A、B和C亚基的序列同源。结核分枝杆菌尿素酶对尿素有专一性，K-m为0.3 mM，不对硫脲、羟基脲、精氨酸和天冬酰胺进行水解。该酶在较宽的pH范围内具有较高的活力(最适pH为7.2)，对60℃的温度非常稳定，不被尿素变性，1 mM EDTA不抑制该酶的活性，但可被N-乙基马来酰亚胺、羟基脲、乙酰羟基氨基甲酸酯和苯基磷二酸二酯抑制。在富氮肉汤中生长的结核分枝杆菌中很容易检测到尿素酶的活性，但在缺氮条件下表达增加了10倍，这与该酶在细菌吸收氮素中的作用是一致的。编码尿素酶的基因簇与其他细菌的尿素酶基因簇在组织上具有相似性。结核分枝杆菌尿素酶基因簇的核苷酸序列显示出与尿素酶A、B和C亚基以及尿素酶辅助分子F和G相对应的开放阅读框。
Mycobacterium tuberculosis urease (urea amidohydrolase [EC 3.5.1.5]) was purified and shown to contain three subunits: two small subunits, each approximately 11,000 Da, and a large subunit of 62,000 Da. The N-terminal sequences of the three subunits were homologous to those of the A, B, and C subunits, respectively, of other bacterial ureases. M. tuberculosis urease was specific for urea, with a K-m of 0.3 mM, and did not hydrolyze thiourea, hydroxyurea, arginine, or asparagine. The enzyme was active over a broad pH range (optimal activity at pH 7.2) and was remarkably stable against heating to 60 degrees C and resistant to denaturation with urea, The enzyme was not inhibited by 1 mM EDTA but was inhibited by N-ethylmaleimide, hydroxyurea, acetohydroxamate, and phenylphosphorodiamidate. Urease activity was readily detectable in M. tuberculosis growing in nitrogen-rich broth, but expression increased 10-fold upon nitrogen deprivation, which is consistent with a role for the enzyme in nitrogen acquisition by the bacterium, The gene cluster encoding urease was shown to have organizational similarities to urease gene clusters of other bacteria, The nucleotide sequence of the M. tuberculosis urease gene cluster revealed open reading frames corresponding to the urease A, B, and C subunits, as well as to the urease accessory molecules F and G.