PRECORRIN-6X REDUCTASE FROM PSEUDOMONAS-DENITRIFICANS - PURIFICATION AND CHARACTERIZATION OF THE ENZYME AND IDENTIFICATION OF THE STRUCTURAL GENE
PRECORRIN-6X REDUCTASE FROM PSEUDOMONAS-DENITRIFICANS - PURIFICATION AND CHARACTERIZATION OF THE ENZYME AND IDENTIFICATION OF THE STRUCTURAL GENE
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DOI：
10.1128/jb.174.3.1036-1042.1992
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发表时间：
1992-02-01
影响因子：
3.2
通讯作者：
CROUZET, J
中科院分区：
文献类型：
--
作者：
BLANCHE, F;THIBAUT, D;CROUZET, J
前咕啉-6x还原酶催化前咕啉-6x还原成二氢衍生物前咕啉-6y，从重组假单胞菌的提取物中纯化14，300倍至均一，产率为8%。 用快原子轰击-质谱法鉴定前受体蛋白-6 γ。 它被转化为高产率（90%）的无细胞蛋白制剂从P. acetificans的氢硼酸。 为了纯化和表征前受体蛋白-6x还原酶，开发了偶联酶放射酶测定法，其中前受体蛋白-6y被cobL基因产物原位甲基化（F. Blanche，A. Famechon，D.蒂博湖德彪西，B。卡梅隆，J. Crouzet，细菌学杂志。174：1050-1052，1992）。 通过凝胶过滤法（M（r）= 27，000）和分析型十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法（M（r）= 31，000）获得的前咕啉-6x还原酶的分子量与作为单体的酶一致。 在pH 7.7下，获得前啡蛋白-6x的K（m）值为3.6 +/- 0.2-μ-M，NADPH的K（m）值为23.5 +/- 3.5-μ-M，V（max）值为17，000 U mg-1。 通过微测序确定酶的胰蛋白酶消化后获得的N-末端序列（6个氨基酸）和3个内部序列，并确定cobK基因编码的前乳清蛋白-6x还原酶，该基因位于先前描述的8.7-kb EcoRI片段上（J. Crouzet，B.卡梅隆湖Cauchois，S. Rigault，M. C. Rouyez，F.布兰奇，D。Thibaut和L. Debussche，J. Bacteriol. 172：5980-5990，1990）。 然而，编码序列显示在与先前提出的编码链互补的链上。
Precorrin-6x reductase, which catalyzes the NADPH-dependent reduction of precorrin-6x to a dihydro derivative named precorrin-6y, was purified 14,300-fold to homogeneity with an 8% yield from extracts of a recombinant strain of Pseudomonas denitrificans. Precorrin-6y was identified by fast atom bombardment-mass spectrometry. It was converted in high yield (90%) to hydrogenobyrinic acid by cell-free protein preparations from P. denitrificans. For the purification and characterization of precorrin-6x reductase, a coupled-enzyme radioenzymatic assay was developed in which precorrin-6y was methylated in situ by the cobL gene product (F. Blanche, A. Famechon, D. Thibaut, L. Debussche, B. Cameron, J. Crouzet, J. Bacteriol. 174:1050-1052, 1992) in the presence of [methyl-H-3]S-adenosyl-L-methionine. Molecular weights of precorrin-6x reductase obtained by gel filtration (M(r) congruent-to 27,000) and by analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis (M(r) congruent-to 31,000) were consistent with the enzyme being a monomer. K(m) values of 3.6 +/- 0.2-mu-M for precorrin-6x and 23.5 +/- 3.5-mu-M for NADPH and a V(max) value of 17,000 U mg-1 were obtained at pH 7.7. The N-terminal sequence (six amino acids) and three internal sequences obtained after tryptic digestion of the enzyme were determined by microsequencing and established that precorrin-6x reductase is encoded by the cobK gene, located on a previously described 8.7-kb EcoRI fragment (J. Crouzet, B. Cameron, L. Cauchois, S. Rigault, M.-C. Rouyez, F. Blanche, D. Thibaut, and L. Debussche, J. Bacteriol. 172:5980-5990, 1990). However, the coding sequence was shown to be on the strand complementary to the one previously proposed as the coding strand.