Sun Li, Warncke Kurt
Department of Physics, N201 Mathematics and Science Center, 400 Dowman Drive, Emory University, Atlanta, Georgia 30322, USA.
Proteins. 2006 Aug 1;64(2):308-19. doi: 10.1002/prot.20997.
The structure of the EutB protein from Salmonella typhimurium, which contains the active site of the coenzyme B12 (adenosylcobalamin)-dependent enzyme, ethanolamine ammonia-lyase, has been predicted by using structural proteomics techniques of comparative modelling. The 453-residue EutB protein displays no significant sequence identity with proteins of known structure. Therefore, secondary structure prediction and fold recognition algorithms were used to identify templates. Multiple three-dimensional template matching (threading) servers identified predominantly beta8alpha8, TIM-barrel proteins, and in particular, the large subunits of diol dehydratase (PDB: 1eex:A, 1dio:A) and glycerol dehydratase (PDB: 1mmf:A), as templates. Consistent with this identification, the dehydratases are, like ethanolamine ammonia-lyase, Class II coenzyme B12-dependent enzymes. Model building was performed by using MODELLER. Models were evaluated by using different programs, including PROCHECK and VERIFY3D. The results identify a beta8alpha8, TIM-barrel fold for EutB. The beta8alpha8, TIM-barrel fold is consistent with a central role of the alpha/beta-barrel structures in radical catalysis conducted by the coenzyme B12- and S-adenosylmethionine-dependent (radical SAM) enzyme superfamilies. The EutB model and multiple sequence alignment among ethanolamine ammonia-lyase, diol dehydratase, and glycerol dehydratase from different species reveal the following protein structural features: (1) a "cap" loop segment that closes the N-terminal region of the barrel, (2) a common cobalamin cofactor binding topography at the C-terminal region of the barrel, and (3) a beta-barrel-internal guanidinium group from EutB R160 that overlaps the position of the active-site potassium ion found in the dehydratases. R160 is proposed to have a role in substrate binding and radical catalysis.
鼠伤寒沙门氏菌的EutB蛋白结构已通过比较建模的结构蛋白质组学技术进行了预测，该蛋白包含依赖辅酶B12（腺苷钴胺素）的乙醇胺氨裂解酶的活性位点。453个残基的EutB蛋白与已知结构的蛋白质没有显著的序列同一性。因此，使用二级结构预测和折叠识别算法来识别模板。多个三维模板匹配（穿线）服务器主要识别出β8α8、TIM桶状蛋白，特别是二醇脱水酶（PDB：1eex:A，1dio:A）和甘油脱水酶（PDB：1mmf:A）的大亚基作为模板。与该识别结果一致，脱水酶与乙醇胺氨裂解酶一样，是II类依赖辅酶B12的酶。使用MODELLER进行模型构建。通过使用包括PROCHECK和VERIFY3D在内的不同程序对模型进行评估。结果确定EutB具有β8α8、TIM桶状折叠。β8α8、TIM桶状折叠与α/β桶状结构在由辅酶B12和S-腺苷甲硫氨酸依赖（自由基SAM）酶超家族进行的自由基催化中的核心作用一致。来自不同物种的乙醇胺氨裂解酶、二醇脱水酶和甘油脱水酶之间的EutB模型和多序列比对揭示了以下蛋白质结构特征：（1）一个封闭桶状结构N端区域的“帽”环段，（2）在桶状结构C端区域的一个共同的钴胺素辅因子结合拓扑结构，以及（3）EutB R160的一个β桶状结构内部的胍基，其与脱水酶中活性位点钾离子的位置重叠。R160被认为在底物结合和自由基催化中起作用。
