YtsJ has the major physiological role of the four paralogous malic enzyme isoforms in Bacillus subtilis
YtsJ has the major physiological role of the four paralogous malic enzyme isoforms in Bacillus subtilis
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DOI：
10.1128/jb.00167-06
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发表时间：
2006-07-01
影响因子：
3.2
通讯作者：
Aymerich, Stephane
中科院分区：
文献类型：
--
作者：
Lerondel, Guillaume;Doan, Thierry;Aymerich, Stephane
枯草芽孢杆菌基因组含有几组旁系同源物。一个极端的例子是四个假定的苹果酸酶基因maeA、malS、ytsJ和mleA。已证明maeA编码苹果酸酶活性，可被苹果酸诱导，但也可在苹果酸上生长。我们报告了系统的实验，以测试这四个基因是否确保备份或覆盖不同的功能。单突变株和多突变株的分析表明，ytsJ具有重要的生理作用，在苹果酸的利用，没有其他三个基因可以补偿。相比之下，maeA，malS和mleA在苹果酸利用中具有不同的作用，它们可以相互补偿。这四种蛋白质表现出苹果酸酶活性;迈斯、MieA和MaeA在NAD（+）下的活性比NADP（+）高4 - 90倍。相比之下，NADP（+）的YtsJ活性比NAD（+）高70倍，K-m值分别为0.055和2.8 mM。lacZ融合显示了ytsJ的强转录，在苹果酸中比在葡萄糖培养基中高两倍，但malS和mleA的弱转录。相反，mleA在复杂培养基中强烈转录。代谢通量分析证实了YtsJ在苹果酸-丙酮酸互变中的主要作用。虽然过表达依赖NADP的大肠杆菌苹果酸酶MaeB不能抑制苹果酸对ytsJ突变体的生长缺陷，但过表达来自大肠杆菌的转氢酶UdhA。这些结果表明YtsJ除了苹果酸转化为丙酮酸之外还有另外的生理作用。
The Bacillus subtilis genome contains several sets of paralogs. An extreme case is the four putative malic enzyme genes maeA, malS, ytsJ, and mleA. maeA was demonstrated to encode malic enzyme activity, to be inducible by malate, but also to be dispensable for growth on malate. We report systematic experiments to test whether these four genes ensure backup or cover different functions. Analysis of single- and multiple-mutant strains demonstrated that ytsJ has a major physiological role in malate utilization for which none of the other three genes could compensate. In contrast, maeA, malS, and mleA had distinct roles in malate utilization for which they could compensate one another. The four proteins exhibited malic enzyme activity; MaIS, MIeA, and MaeA exhibited 4- to 90-fold higher activities with NAD(+) than with NADP(+). YtsJ activity, in contrast, was 70-fold higher with NADP(+) than with NAD(+), with K-m values of 0.055 and 2.8 mM, respectively. lacZ fusions revealed strong transcription of ytsJ, twofold higher in malate than in glucose medium, but weak transcription of malS and mleA. In contrast, mleA was strongly transcribed in complex medium. Metabolic flux analysis confirmed the major role of YtsJ in malate-to-pyruvate interconversion. While overexpression of the NADP-dependent Escherichia coli malic enzyme MaeB did not suppress the growth defect of a ytsJ mutant on malate, overexpression of the transhydrogenase UdhA from E. coli partially suppressed it. These results suggest an additional physiological role of YtsJ beyond that of malate-to-pyruvate conversion.