Myrach Till, Zhu Anting, Witte Claus-Peter
From the Freie Universität Berlin, Dahlem Centre of Plant Sciences, Department of Plant Biochemistry, Königin-Luise-Strasse 12-16, 14195 Berlin, Germany and.
Leibniz Universität Hannover, Institute of Plant Nutrition, Molecular Nutrition and Biochemistry of Plants, Herrenhäuser Strasse 2, 30419 Hannover, Germany.
J Biol Chem. 2017 Sep 1;292(35):14556-14565. doi: 10.1074/jbc.M117.780403. Epub 2017 Jul 14.
Urease is a ubiquitous nickel metalloenzyme. In plants, its activation requires three urease accessory proteins (UAPs), UreD, UreF, and UreG. In bacteria, the UAPs interact with urease and facilitate activation, which involves the channeling of two nickel ions into the active site. So far this process has not been investigated in eukaryotes. Using affinity pulldowns of Strep-tagged UAPs from and rice transiently expressed , we demonstrate that a urease-UreD-UreF-UreG complex exists in plants and show its stepwise assembly. UreG is crucial for nickel delivery because UreG-dependent urease activation was observed only with UreG obtained from nickel-sufficient plants. This activation competence could not be generated by incubation of UreG with nickel, bicarbonate, and GTP. Compared with their bacterial orthologs, plant UreGs possess an N-terminal extension containing a His- and Asp/Glu-rich hypervariable region followed by a highly conserved sequence comprising two potential HH metal-binding sites. Complementing the mutant of with N-terminal deletion variants of UreG demonstrated that the hypervariable region has a minor impact on activation efficiency, whereas the conserved region up to the first HH motif is highly beneficial and up to the second HH motif strictly required for activation. We also show that urease reaches its full activity several days after nickel becomes available in the leaves, indicating that urease activation is limited by nickel accessibility Our data uncover the crucial role of UreG for nickel delivery during eukaryotic urease activation, inciting further investigations of the details of this process.
脲酶是一种广泛存在的镍金属酶。在植物中，其激活需要三种脲酶辅助蛋白（UAPs），即脲酶D（UreD）、脲酶F（UreF）和脲酶G（UreG）。在细菌中，UAPs与脲酶相互作用并促进其激活，这涉及到将两个镍离子导入活性位点。到目前为止，这一过程在真核生物中尚未得到研究。通过对来自烟草和水稻瞬时表达的带有链霉亲和标签的UAPs进行亲和下拉实验，我们证明了植物中存在脲酶-UreD-UreF-UreG复合物，并展示了其逐步组装过程。UreG对于镍的传递至关重要，因为只有从镍充足的植物中获得的UreG才能观察到依赖UreG的脲酶激活。将UreG与镍、碳酸氢盐和鸟苷三磷酸（GTP）一起孵育并不能产生这种激活能力。与它们的细菌直系同源物相比，植物UreG具有一个N端延伸，其中包含一个富含组氨酸和天冬氨酸/谷氨酸的高变区，随后是一个高度保守的序列，该序列包含两个潜在的HH金属结合位点。用UreG的N端缺失变体对烟草的突变体进行互补实验表明，高变区对激活效率的影响较小，而直到第一个HH基序的保守区对激活非常有利，直到第二个HH基序则是激活严格必需的。我们还表明，在叶片中镍可用几天后，脲酶才达到其完全活性，这表明脲酶的激活受到镍可及性的限制。我们的数据揭示了UreG在真核生物脲酶激活过程中对镍传递的关键作用，促使对这一过程的细节进行进一步研究。
