Institute of Plant Biology, National Taiwan University, Taipei 106, Taiwan, Republic of China.
Plant Physiol. 2011 Nov;157(3):1015-25. doi: 10.1104/pp.111.184739. Epub 2011 Sep 9.
The membrane protein AtTLP18.3 of Arabidopsis (Arabidopsis thaliana) contains a domain of unknown function, DUF477; it forms a polysome with photosynthetic apparatuses in the thylakoid lumen. To explore the molecular function of AtTLP18.3, we resolved its crystal structures with residues 83 to 260, the DUF477 only, and performed a series of biochemical analyses to discover its function. The gene expression of AtTLP18.3 followed a circadian rhythm. X-ray crystallography revealed the folding of AtTLP18.3 as a three-layer sandwich with three α-helices in the upper layer, four β-sheets in the middle layer, and two α-helices in the lower layer, which resembles a Rossmann fold. Structural comparison suggested that AtTLP18.3 might be a phosphatase. The enzymatic activity of AtTLP18.3 was further confirmed by phosphatase assay with various substrates (e.g. p-nitrophenyl phosphate, 6,8-difluoro-4-methylumbelliferyl phosphate, O-phospho-L-serine, and several synthetic phosphopeptides). Furthermore, we obtained the structure of AtTLP18.3 in complex with O-phospho-L-serine to identify the binding site of AtTLP18.3. Our structural and biochemical studies revealed that AtTLP18.3 has the molecular function of a novel acid phosphatase in the thylakoid lumen. DUF477 is accordingly renamed the thylakoid acid phosphatase domain.
拟南芥（Arabidopsis thaliana）膜蛋白 AtTLP18.3 含有一个未知功能结构域（DUF477）；它与类囊体腔中的光合器官一起形成多核糖体。为了探索 AtTLP18.3 的分子功能，我们解析了其残基 83 至 260 位、仅含 DUF477 的晶体结构，并进行了一系列生化分析以发现其功能。AtTLP18.3 的基因表达呈现出昼夜节律。X 射线晶体学揭示了 AtTLP18.3 的折叠方式为三层夹心结构，上层有三个α螺旋，中层有四个β折叠，下层有两个α螺旋，类似于 Rossmann 折叠。结构比较表明，AtTLP18.3 可能是一种磷酸酶。通过用各种底物（例如对硝基苯磷酸酯、6,8-二氟-4-甲基伞形酮磷酸酯、O-磷酸-L-丝氨酸和几种合成的磷酸肽）进行磷酸酶测定，进一步证实了 AtTLP18.3 的酶活性。此外，我们获得了 AtTLP18.3 与 O-磷酸-L-丝氨酸复合物的结构，以确定 AtTLP18.3 的结合位点。我们的结构和生化研究表明，AtTLP18.3 在类囊体腔中具有一种新型酸性磷酸酶的分子功能。因此，将 DUF477 重新命名为类囊体酸性磷酸酶结构域。
