...
  • 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>Journal of bacteriology >Purification and characterization of S-adenosyl-L-methionine: uroporphyrinogen III methyltransferase from Pseudomonas denitrificans.
【24h】

Purification and characterization of S-adenosyl-L-methionine: uroporphyrinogen III methyltransferase from Pseudomonas denitrificans.

机译：纯化和表征S-腺苷-L-蛋氨酸：来自反硝化假单胞菌的尿卟啉原III甲基转移酶。

获取原文
           
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

S-Adenosyl-L-methionine:uroporphyrinogen III methyltransferase (SUMT), the enzyme of the cobalamin biosynthetic pathway which catalyzes C methylation of uroporphyrinogen III, was purified about 150-fold to homogeneity from extracts of a recombinant strain of Pseudomonas denitrificans derived from a cobalamin-overproducing strain by ammonium sulfate fractionation, anion-exchange chromatography, and hydroxyapatite chromatography. The purified protein has an isoelectric point of 6.4 and molecular weights of 56,500 as estimated by gel filtration and 30,000 as estimated by gel electrophoresis under denaturing conditions, suggesting that the active enzyme is a homodimer. It does not contain a chromophoric prosthetic group and does not seem to require metal ions or cofactors for activity. SUMT catalyzes the two successive C-2 and C-7 methylation reactions involved in the conversion of uroporphyrinogen III to precorrin-2 via the intermediate formation of precorrin-1. In vitro studies suggest that the intermediate monomethylated product (precorrin-1) is released from the protein and then added back to the enzyme for the second C-methylation reaction. The pH optimum was 7.7, the Km values for S-adenosyl-L-methionine and uroporphyrinogen III were 6.3 and 1.0 microM, respectively, and the turnover number was 38 h-1. The enzyme activity was shown to be completely insensitive to feedback inhibition by cobalamin and corrinoid intermediates tested at physiological concentration. At uroporphyrinogen III concentrations above 2 microM, SUMT exhibited a substrate inhibition phenomenon. It is suggested that this property might play a regulatory role in cobalamin biosynthesis in the cobalamin-overproducing strain studied.
机译：S-腺苷-L-蛋氨酸：尿卟啉原III甲基转移酶（SUMT）是钴胺素生物合成途径的酶，催化尿卟啉原III的C甲基化，从重组脱氮假单胞菌假单胞菌的重组菌株提取物中纯化至均质约150倍。硫酸铵分级分离，阴离子交换色谱法和羟基磷灰石色谱法纯化钴胺素过剩菌株。纯化的蛋白质的等电点为6.4，通过凝胶过滤估计的分子量为56,500，通过变性条件下的凝胶电泳估计的分子量为30,000，表明该活性酶是同型二聚体。它不含发色的修复基团，似乎不需要金属离子或辅助因子来发挥作用。 SUMT催化两个连续的C-2和C-7甲基化反应，涉及尿卟啉原III通过中间体precorrin-1的转化而转变为precorrin-2。体外研究表明，中间体单甲基化产物（precorrin-1）从蛋白质中释放出来，然后加回到酶中进行第二次C-甲基化反应。最适pH为7.7，S-腺苷-L-蛋氨酸和尿卟啉原III的Km值分别为6.3和1.0 microM，周转数为38 h-1。该酶活性显示出对在生理浓度下测试的钴胺素和类corrinoid中间体的反馈抑制完全不敏感。在尿卟啉原III浓度高于2 microM时，SUMT表现出底物抑制现象。建议该特性可能在所研究的钴胺素过量生产菌株中的钴胺素生物合成中起调节作用。

著录项

相似文献

  • 外文文献
  • 中文文献
  • 专利
获取原文

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号