Kti12 (Kluyveromyces lactis toxin insensitive 12) is an evolutionary highly conserved ATPase, crucial for the tRNA-modification activity of the eukaryotic Elongator complex. The protein consists of an N-terminal ATPase and a C-terminal tRNA-binding domain, which are connected by a flexible linker. The precise role of the linker region and its involvement in the communication between the two domains and their activities remain elusive. Here, we analyzed all available Kti12 protein sequences and report the discovery of a subset of Kti12 proteins with abnormally long linker regions. These Kti12 proteins are characterized by a co-occurring lysine to leucine substitution in their Walker A motif, previously thought to be invariable. We show that the K14L substitution lowers the affinity to ATP, but does not affect the catalytic activity of Kti12 at high ATP concentrations. We compare the activity of mutated variants of Kti12 in vitro with complementation assays in vivo in yeast. Ultimately, we compared Kti12 to other known p-loop ATPase family members known to carry a similar deviant Walker A motif. Our data establish Kti12 of Eurotiomycetes as an example of eukaryotic ATPase harboring a significantly deviating but still functional Walker A motif.

Kti12（乳酸克鲁维酵母毒素不敏感12）是进化上高度保守的ATPase，对于真核Elongator复合物的tRNA修饰活性至关重要。该蛋白质由N末端ATPase和C末端tRNA结合结构域组成，它们通过柔性接头连接。链接区域的确切作用及其在两个领域之间的交流及其活动中的参与仍然难以捉摸。在这里，我们分析了所有可用的Kti12蛋白序列，并报告了具有异常长接头区域的Kti12蛋白子集的发现。这些Kti12蛋白的特征是，在以前认为是不变的Walker A母题中，赖氨酸同时发生亮氨酸取代。我们表明，K14L取代降低了对ATP的亲和力，但在高ATP浓度下不影响Kti12的催化活性。我们用酵母体内的互补分析比较了体外Kti12突变体的活性。最终，我们将Kti12与其他已知带有类似Walker A异常基序的已知p环ATPase家族成员进行了比较。我们的数据建立了Kti12Eurotiomycetes作为真核ATPase的一个例子，它具有明显偏离但仍具有功能的Walker A基序。