Properties of a thermostable nitrate reductase from the hyperthermophilic archaeon Pyrobaculum aerophilum
Properties of a thermostable nitrate reductase from the hyperthermophilic archaeon Pyrobaculum aerophilum
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DOI：
10.1128/jb.183.19.5491-5495.2001
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发表时间：
2001-10-01
影响因子：
3.2
通讯作者：
Schröder, I
中科院分区：
文献类型：
--
作者：
Afshar, S;Johnson, E;Schröder, I
从细胞膜中分离纯化了嗜热嗜热古细菌硝酸还原酶137倍。根据十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析，该酶复合体由三个亚基组成，表观分子量分别为130,000，52,000和32,000。该酶以钼(0.8-摩尔/摩尔络合物)、铁(15.4-摩尔/摩尔络合物)和细胞色素b(0.49-摩尔/摩尔络合物)为辅因子。嗜氧假单胞菌硝酸还原酶以还原的苯基紫精为电子供体，比活力极高，有别于中温细菌和古生菌的硝酸还原酶(硝酸盐的V-max为1,162 S(-1)(326U/mg)；氯酸盐的V-max为1,348 S(-1)(378U/mg)[在75℃下测定])。硝酸盐和氯酸盐的K-m值分别为58um和140um。叠氮是硝酸还原酶活性的竞争性抑制物，氰化物是非竞争性抑制物。酶活的最适温度为-95℃。在100℃孵育时，纯化的硝酸还原酶的半衰期为1.5h。这项研究首次描述了一种来自高温古生菌的硝酸还原酶。
The nitrate reductase of the hyperthermophilic archaeon Pyrobaculum aerophilum was purified 137-fold from the cytoplasmic membrane. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, the enzyme complex consists of three subunits with apparent molecular weights of 130,000, 52,000, and 32,000. The enzyme contained molybdenum (0.8-mol/mol complex), iron (15.4-mol/mol complex) and cytochrome b (0.49-mol/mol complex) as cofactors. The P. aerophilum nitrate reductase distinguishes itself from nitrate reductases of mesophilic bacteria and archaea by its very high specific activity using reduced benzyl viologen as the electron donor (V-max with nitrate, 1,162 s(-1) (326 U/mg); V-max with chlorate, 1,348 s(-1) (378 U/mg) [assayed at 75 degreesC]). The K-m values for nitrate and chlorate were 58 and 140 muM, respectively. Azide was a competitive inhibitor and cyanide was a noncompetitive inhibitor of the nitrate reductase activity. The temperature optimum for activity was > 95 degreesC. When incubated at 100 degreesC, the purified nitrate reductase had a half-life of 1.5 h. This study constitutes the first description of a nitrate reductase from a hyperthermophilic archaeon.