Rigden Daniel J, Littlejohn James E, Henderson Keith, Jedrzejas Mark J
National Centre of Genetic Resources and Biotechnology, Cenargen/Embrapa, SAIN Parque Rural, Final W5, Asa Norte, 70770-900 Brasília, Brazil.
J Mol Biol. 2003 Jan 17;325(3):411-20. doi: 10.1016/s0022-2836(02)01229-9.
Bacillus stearothermophilus phosphatase PhoE is a member of the cofactor-dependent phosphoglycerate mutase superfamily possessing broad specificity phosphatase activity. Its previous structural determination in complex with glycerol revealed probable bases for its efficient hydrolysis of both large, hydrophobic, and smaller, hydrophilic substrates. Here we report two further structures of PhoE complexes, to higher resolution of diffraction, which yield a better and thorough understanding of its catalytic mechanism. The environment of the phosphate ion in the catalytic site of the first complex strongly suggests an acid-base catalytic function for Glu83. It also reveals how the C-terminal tail ordering is linked to enzyme activation on phosphate binding by a different mechanism to that seen in Escherichia coli phosphoglycerate mutase. The second complex structure with an unusual doubly covalently bound trivanadate shows how covalent modification of the phosphorylable His10 is accompanied by small structural changes, presumably to catalytic advantage. When compared with structures of related proteins in the cofactor-dependent phosphoglycerate mutase superfamily, an additional phosphate ligand, Gln22, is observed in PhoE. Functional constraints lead to the corresponding residue being conserved as Gly in fructose-2,6-bisphosphatases and Thr/Ser/Cys in phosphoglycerate mutases. A number of sequence annotation errors in databases are highlighted by this analysis. B. stearothermophilus PhoE is evolutionarily related to a group of enzymes primarily present in Gram-positive bacilli. Even within this group substrate specificity is clearly variable highlighting the difficulties of computational functional annotation in the cofactor-dependent phosphoglycerate mutase superfamily.
嗜热栖热放线菌磷酸酶PhoE是辅因子依赖性磷酸甘油酸变位酶超家族的成员，具有广泛特异性的磷酸酶活性。其先前与甘油形成复合物的结构测定揭示了其有效水解大型疏水性和亲水性较小底物的可能基础。在此，我们报告了PhoE复合物的另外两种结构，其衍射分辨率更高，从而能更好、更全面地理解其催化机制。第一个复合物催化位点中磷酸根离子的环境强烈表明Glu83具有酸碱催化功能。它还揭示了C末端尾巴的有序排列如何通过与大肠杆菌磷酸甘油酸变位酶不同的机制与磷酸结合时的酶激活相联系。第二个复合物结构与一种不寻常的双共价结合的三钒酸盐显示了可磷酸化的His10的共价修饰如何伴随着小的结构变化，推测这具有催化优势。与辅因子依赖性磷酸甘油酸变位酶超家族中相关蛋白质的结构相比，在PhoE中观察到一个额外的磷酸配体Gln22。功能限制导致相应残基在果糖-2,6-双磷酸酶中保守为甘氨酸，在磷酸甘油酸变位酶中保守为苏氨酸/丝氨酸/半胱氨酸。该分析突出了数据库中一些序列注释错误。嗜热栖热放线菌PhoE在进化上与一组主要存在于革兰氏阳性杆菌中的酶相关。即使在这一组中，底物特异性也明显不同，这突出了辅因子依赖性磷酸甘油酸变位酶超家族中计算功能注释的困难。
