Cheng Zhongjun, Song Feng, Shan Xiaoyue, Wei Zhiyi, Wang Yanli, Dunaway-Mariano Debra, Gong Weimin
School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230026, PR China.
Biochem Biophys Res Commun. 2006 Oct 13;349(1):172-7. doi: 10.1016/j.bbrc.2006.08.025. Epub 2006 Aug 14.
Hotdog-fold has been identified in more than 1000 proteins, yet many of which in eukaryotes are less studied. No structural or functional studies of human thioesterase superfamily member 2 (hTHEM2) have been reported before. Since hTHEM2 exhibits about 20% sequence identity to Escherichia coli PaaI protein, it was proposed to be a thioesterase with a hotdog-fold. Here, we report the crystallographic structure of recombinant hTHEM2, determined by the single-wavelength anomalous dispersion method at 2.3A resolution. This structure demonstrates that hTHEM2 indeed contains a hotdog-fold and forms a back-to-back tetramer as other hotdog proteins. Based on structural and sequence conservation, the thioesterase active site in hTHEM2 is predicted. The structure and substrate specificity are most similar to those of the bacterial phenylacetyl-CoA hydrolase. Asp65, located on the central alpha-helix of subunit B, was shown by site-directed mutagenesis to be essential to catalysis.
热狗折叠结构已在1000多种蛋白质中被鉴定出来，但其中许多真核生物中的蛋白质研究较少。此前尚未有关于人类硫酯酶超家族成员2（hTHEM2）的结构或功能研究报道。由于hTHEM2与大肠杆菌PaaI蛋白具有约20%的序列同一性，因此它被认为是一种具有热狗折叠结构的硫酯酶。在此，我们报告了重组hTHEM2的晶体结构，该结构通过单波长反常散射法在2.3埃分辨率下测定。该结构表明hTHEM2确实包含一个热狗折叠结构，并像其他热狗蛋白一样形成背靠背的四聚体。基于结构和序列保守性，预测了hTHEM2中的硫酯酶活性位点。其结构和底物特异性与细菌苯乙酰辅酶A水解酶最为相似。位于亚基B中央α螺旋上的Asp65经定点诱变显示对催化作用至关重要。
