Graduate School of Biological Sciences, Nara Institute of Science and Technology-NAIST, Ikoma, Nara, Japan.
PLoS One. 2013 Jul 1;8(7):e67385. doi: 10.1371/journal.pone.0067385. Print 2013.
To recycle reduced sulfur to methionine in the methionine salvage pathway (MSP), 5-methylthioribulose-1-phosphate is converted to 2-keto-4-methylthiobutyrate, the methionine precursor, by four steps; dehydratase, enolase, phosphatase, and dioxygenase reactions (catalyzed by MtnB, MtnW, MtnX and MtnD, respectively, in Bacillus subtilis). It has been proposed that the MtnBD fusion enzyme in Tetrahymena thermophila catalyzes four sequential reactions from the dehydratase to dioxygenase steps, based on the results of molecular biological analyses of mutant yeast strains with knocked-out MSP genes, suggesting that new catalytic function can be acquired by fusion of enzymes. This result raises the question of how the MtnBD fusion enzyme can catalyze four very different reactions, especially since there are no homologous domains for enolase and phosphatase (MtnW and MtnX, respectively, in B. subtilis) in the peptide. Here, we tried to identify the domains responsible for catalyzing the four reactions using recombinant proteins of full-length MtnBD and each domain alone. UV-visible and ¹H-NMR spectral analyses of reaction products revealed that the MtnB domain catalyzes dehydration and enolization and the MtnD domain catalyzes dioxygenation. Contrary to a previous report, conversion of 5-methylthioribulose-1-phosphate to 2-keto-4-methylthiobutyrate was dependent on addition of an exogenous phosphatase from B. subtilis. This was observed for both the MtnB domain and full-length MtnBD, suggesting that MtnBD does not catalyze the phosphatase reaction. Our results suggest that the MtnB domain of T. thermophila MtnBD acquired the new function to catalyze both the dehydratase and enolase reactions through evolutionary gene mutations, rather than fusion of MSP genes.
为了在蛋氨酸补救途径（MSP）中将还原态硫回收为蛋氨酸，5-甲基硫代核糖-1-磷酸通过四个步骤转化为蛋氨酸前体 2-酮-4-甲基硫代丁酸；脱水酶、烯醇酶、磷酸酶和双加氧酶反应（分别由枯草芽孢杆菌中的 MtnB、MtnW、MtnX 和 MtnD 催化）。基于对敲除 MSP 基因的突变酵母菌株的分子生物学分析结果，提出了四膜虫中的 MtnBD 融合酶催化从脱水酶到双加氧酶步骤的四个连续反应，这表明酶的融合可以获得新的催化功能。这一结果提出了一个问题，即 MtnBD 融合酶如何能够催化四个非常不同的反应，特别是在肽中没有与烯醇酶和磷酸酶（枯草芽孢杆菌中的 MtnW 和 MtnX）相对应的同源结构域。在这里，我们试图使用全长 MtnBD 和每个单独的结构域的重组蛋白来鉴定负责催化这四个反应的结构域。反应产物的紫外可见和 1H-NMR 光谱分析表明，MtnB 结构域催化脱水和烯醇化，而 MtnD 结构域催化双加氧化。与之前的报告相反，5-甲基硫代核糖-1-磷酸向 2-酮-4-甲基硫代丁酸的转化依赖于枯草芽孢杆菌外源磷酸酶的添加。这在 MtnB 结构域和全长 MtnBD 中都观察到，这表明 MtnBD 不催化磷酸酶反应。我们的结果表明，四膜虫 MtnBD 的 MtnB 结构域通过进化基因突变获得了催化脱水酶和烯醇酶反应的新功能，而不是 MSP 基因的融合。
