Role of Metal Ions on the Activity of Mycobacterium tuberculosis Pyrazinamidase
Role of Metal Ions on the Activity of Mycobacterium tuberculosis Pyrazinamidase
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DOI：
10.4269/ajtmh.2012.10-0565
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发表时间：
2012-07-01
影响因子：
3.3
通讯作者：
Zimic, Mirko
中科院分区：
文献类型：
--
作者：
Sheen, Patricia;Ferrer, Patricia;Zimic, Mirko
结核分枝杆菌的吡嗪酰胺酶催化吡嗪酰胺转化为活性分子吡嗪酸。吡嗪酰胺酶活性的降低导致一定水平的吡嗪酰胺抗性。以前的研究表明，吡嗪酰胺酶有一个金属结合位点，二价金属辅因子是活性所必需的。为了确定二价金属对吡嗪酰胺酶的影响，在具有和不具有羧基末端的大肠杆菌中表达对应于H37 Rv吡嗪酰胺敏感参考菌株的重组野生型吡嗪酰胺酶。His标记的吡嗪酰胺酶通过金属耗尽失活，并通过用二价金属滴定重新活化。虽然Co 2+，Mn 2+，和Zn 2+恢复吡嗪酰胺酶的活性，只有Co 2+增强酶活性的水平高于野生型吡嗪酰胺酶。Cu ~（2+）、Fe ~（2+）、Fe ~（3+）和Mg ~（2+）在试验条件下不能恢复活性。具有适当折叠但不同酶活性的各种重组突变吡嗪酰胺酶显示出恢复活性的差异模式。X射线荧光和原子吸收光谱分析表明，重组野生型吡嗪酰胺酶在大肠杆菌中得到了表达。大肠杆菌很可能含有锌。总之，本研究表明，M。结核吡嗪酰胺酶是一种能够与多种离子配位的金属酶，但在体内，它更可能与Zn 2+配位。然而，在体外，金属耗尽的酶可以重新激活的几个二价金属具有更高的效率比锌。
Pyrazinamidase of Mycobacterium tuberculosis catalyzes the conversion of pyrazinamide to the active molecule pyrazinoic acid. Reduction of pyrazinamidase activity results in a level of pyrazinamide resistance. Previous studies have suggested that pyrazinamidase has a metal-binding site and that a divalent metal cofactor is required for activity. To determine the effect of divalent metals on the pyrazinamidase, the recombinant wild-type pyrazinamidase corresponding to the H37Rv pyrazinamide-susceptible reference strain was expressed in Escherichia coli with and without a carboxy terminal. His-tagged pyrazinamidase was inactivated by metal depletion and reactivated by titration with divalent metals. Although Co2+, Mn2+, and Zn2+ restored pyrazinamidase activity, only Co2+ enhanced the enzymatic activity to levels higher than the wild-type pyrazinamidase. Cu2+, Fe2+, Fe3+, and Mg2+ did not restore the activity under the conditions tested. Various recombinant mutated pyrazinamidases with appropriate folding but different enzymatic activities showed a differential pattern of recovered activity. X-ray fluorescence and atomic absorbance spectroscopy showed that recombinant wild-type pyrazinamidase expressed in E. coli most likely contained Zn. In conclusion, this study suggests that M. tuberculosis pyrazinamidase is a metalloenzyme that is able to coordinate several ions, but in vivo, it is more likely to coordinate Zn2+. However, in vitro, the metal-depleted enzyme could be reactivated by several divalent metals with higher efficiency than Zn.