Chen L Y, Chen M Y, Leu W M, Tsai T Y, Lee Y H
Institute of Biochemistry, National Yang-Ming Medical College, Taipei, Taiwan, Republic of China.
J Biol Chem. 1993 Sep 5;268(25):18710-6.
The melanin operon (melC) of Streptomyces antibioticus contains two genes, melC1 and melC2 (apotyrosinase). Our previous studies indicated that MelC1 forms a transient binary complex with the downstream apotyrosinase MelC2 to facilitate the incorporation of copper ion and the secretion of tyrosinase. In this study, we investigated the role of histidine residues in the function of MelC1 by examining a series of substitution or deletion mutants. Of eight mutants only the substitution of His-117 with Asp in the mutant M-117D rendered the complete abolishment of the intracellular tyrosinase activity in both Streptomyces and Escherichia coli. Replacement of His-102 by Leu in the mutant M-102L also caused a 64-70% reduction of tyrosinase activity in Streptomyces and E. coli. These two mutations also affected the secretion of both MelC1 and MelC2 proteins. In vitro copper activation of the purified MelC1.MelC2 binary complex from these two mutants regained only 20-30% tyrosinase activity of the wild type. Biochemical characterization of the tyrosinases from these two mutants revealed that they were different in several aspects. The intracellular tyrosinase activity in M-117D, but not in M-102L, could be partially reactivated by copper ion or by the cell extract containing MelC1. The copper content and the specific activity of the tyrosinase purified from the culture supernatant from M-117D were only 40% of those in wild type and M-102L. Additionally, fast protein liquid chromatography analysis indicated that in these two mutants the copper activation process was defective, very likely due to the incompetent MelC1.MelC2 binary complex formed: reduced association in M-117D and elevated association in M-102L. Furthermore, the conformation of MelC2 in the binary complex or in the mature enzyme form in wild type could be differentiated by the proteinase K digestion pattern, and so did the conformation of MelC2 found in those of M-102L, but not in M-117D mutant. Taken together, our results demonstrate that MelC1 is indispensable in the incorporation of copper ion into MelC2 apotyrosinase via a transient, competent binary complex formation, during which a conformational transition of MelC2 has occurred. This strongly suggests that MelC1 is a chaperone for the apotyrosinase MelC2.
抗生链霉菌的黑色素操纵子（melC）包含两个基因，melC1和melC2（脱辅基酪氨酸酶）。我们之前的研究表明，MelC1与下游的脱辅基酪氨酸酶MelC2形成瞬时二元复合物，以促进铜离子的掺入和酪氨酸酶的分泌。在本研究中，我们通过检测一系列取代或缺失突变体，研究了组氨酸残基在MelC1功能中的作用。在八个突变体中，只有突变体M-117D中His-117被Asp取代，导致链霉菌和大肠杆菌中的细胞内酪氨酸酶活性完全丧失。突变体M-102L中His-102被Leu取代，也导致链霉菌和大肠杆菌中酪氨酸酶活性降低64 - 70%。这两个突变也影响了MelC1和MelC2蛋白的分泌。从这两个突变体纯化的MelC1.MelC2二元复合物的体外铜激活仅恢复了野生型酪氨酸酶活性的20 - 30%。对这两个突变体的酪氨酸酶进行生化特性分析表明，它们在几个方面存在差异。M-117D中的细胞内酪氨酸酶活性，但不是M-102L中的，可以被铜离子或含有MelC1的细胞提取物部分重新激活。从M-117D培养上清液中纯化的酪氨酸酶的铜含量和比活性仅为野生型和M-102L中的40%。此外，快速蛋白质液相色谱分析表明，在这两个突变体中，铜激活过程存在缺陷，很可能是由于形成的MelC1.MelC2二元复合物功能不全：M-117D中结合减少，M-102L中结合增加。此外，野生型二元复合物或成熟酶形式中MelC2的构象可以通过蛋白酶K消化模式来区分，M-102L中的MelC2构象也是如此，但M-117D突变体中的不是。综上所述，我们的结果表明，MelC1通过形成瞬时的、有功能的二元复合物，在将铜离子掺入MelC2脱辅基酪氨酸酶的过程中是不可或缺的，在此过程中MelC2发生了构象转变。这强烈表明MelC1是脱辅基酪氨酸酶MelC2的伴侣蛋白。
