Ueda M, Kinoshita H, Maeda S-I, Zou W, Tanaka A
Laboratory of Applied Biological Chemistry, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Yoshida, 606-8501 Kyoto, Sakyo-ku, Japan.
Appl Microbiol Biotechnol. 2003 Jun;61(5-6):488-94. doi: 10.1007/s00253-003-1251-5. Epub 2003 Feb 20.
Analysis of the protein structure of bovine liver catalase suggested that the N-terminal region containing two alpha-helices may function as a linker binding to another subunit. The number of amino-acid residues in catalase from the n-alkane-assimilating yeast Candida tropicalis (CTC) is the lowest of any eukaryotic catalase molecule hitherto investigated, and only one helix, corresponding to the helix alpha2 in bovine liver catalase, is estimated to be present in the same region. In the present study, N-terminal-deleted mutants of CTC were characterized to evaluate the role of the alpha-helix structure in the N-terminal region. CTCDelta1-4 and CTCDelta1-24, whose N-terminal regions were shortened by four and 24 amino-acid residues, respectively, showed an 80% decrease in specific activity compared to wild-type CTC in spite of containing the same amount of heme as in the wild-type. Polyacrylamide gel electrophoresis under nondenaturing conditions revealed that the mutants contained large amounts of oligomeric forms with molecular masses less than 220 kDa (tetramer assembly). Although the smaller oligomers were found to be bound with heme, only the tetramer exhibited catalase activity in activity staining on nondenaturing gel. CTCDelta1-49, a mutant with deletion of the N-terminal 49 amino-acid residues which contain the conserved helix alpha2, showed no catalase activity and no heme binding. However, the CD spectrum profiles of CTCDelta1-49, CTCDelta1-4, and CTCDelta1-24 indicated that these mutant subunits could attain secondary conformations similar to that of wild-type CTC, regardless of their binding with heme. From these results, it was concluded that the N-terminal stretch of catalase is significant for complete assembly into active tetramer and that the conserved helix alpha2, although it has little effect on the formation of the subunit secondary structure, is indispensable not only in assembling tetramer but also in binding heme.
牛肝过氧化氢酶的蛋白质结构分析表明，包含两个α-螺旋的N端区域可能作为与另一个亚基结合的连接体发挥作用。热带假丝酵母（CTC）这种同化正构烷烃的酵母中的过氧化氢酶所含氨基酸残基数量，是迄今为止所研究的任何真核过氧化氢酶分子中最少的，据估计在同一区域仅存在一个与牛肝过氧化氢酶中的α2螺旋相对应的螺旋。在本研究中，对CTC的N端缺失突变体进行了表征，以评估N端区域α-螺旋结构的作用。CTCDelta1-4和CTCDelta1-24的N端区域分别缩短了4个和24个氨基酸残基，尽管它们所含血红素的量与野生型相同，但与野生型CTC相比，其比活性降低了80%。非变性条件下的聚丙烯酰胺凝胶电泳显示，这些突变体含有大量分子量小于220 kDa的寡聚体形式（四聚体组装）。虽然发现较小的寡聚体与血红素结合，但在非变性凝胶上的活性染色中只有四聚体表现出过氧化氢酶活性。CTCDelta1-49是一个缺失包含保守α2螺旋的N端49个氨基酸残基的突变体，既没有过氧化氢酶活性也没有血红素结合。然而，CTCDelta1-49、CTCDelta1-4和CTCDelta1-24的圆二色光谱图谱表明，这些突变体亚基无论是否与血红素结合，都能获得与野生型CTC相似的二级构象。从这些结果可以得出结论，过氧化氢酶的N端延伸对于完全组装成活性四聚体很重要，并且保守的α2螺旋虽然对亚基二级结构的形成影响很小，但不仅在组装四聚体中不可或缺，而且在结合血红素中也不可或缺。
