Hoffmann B, Konrat R, Bothe H, Buckel W, Kräutler B
Institut of Organic Chemistry, University of Innsbruck, Innsbruck, Austria.
Eur J Biochem. 1999 Jul;263(1):178-88. doi: 10.1046/j.1432-1327.1999.00482.x.
Glutamate mutase (Glm) is an adenosylcobamide-dependent enzyme that catalyzes the reversible rearrangement of (2S)-glutamate to (2S, 3S)-3-methylaspartate. The active enzyme from Clostridium cochlearium consists of two subunits (of 53.6 and 14.8 kDa) as an alpha2beta2 tetramer, whose assembly is mediated by coenzyme B12. The smaller of the protein components, GlmS, has been suggested to be the B12-binding subunit. Here we report the solution structure of GlmS, determined from a heteronuclear NMR-study, and the analysis of important dynamical aspects of this apoenzyme subunit. The global fold and dynamic behavior of GlmS in solution are similar to those of the corresponding subunit MutS from C. tetanomorphum, which has previously been investigated using NMR-spectroscopy. Both solution structures of the two Glm B12-binding subunits share striking similarities with that determined by crystallography for the B12-binding domain of methylmalonyl CoA mutase (Mcm) from Propionibacterium shermanii, which is B12 bound. In the crystal structure a conserved histidine residue was found to be coordinated to cobalt, displacing the endogenous axial ligand of the cobamide. However, in GlmS and MutS the sequence motif, Asp-x-His-x-x-Gly, which includes the cobalt-coordinating histidine residue, and a predicted alpha-helical region following the motif, are present as an unstructured and highly mobile loop. In the absence of coenzyme, the B12-binding site apparently is only partially formed. By comparing the crystal structure of Mcm with the solution structures of B12-free GlmS and MutS, a consistent picture on the mechanism of B12 binding has been obtained. Important elements of the binding site only become structured upon binding B12; these include the cobalt-coordinating histidine residue, and an alpha helix that forms one side of the cleft accommodating the nucleotide 'tail' of the coenzyme.
谷氨酸变位酶（Glm）是一种依赖腺苷钴胺素的酶，可催化（2S）-谷氨酸可逆重排为（2S，3S）-3-甲基天冬氨酸。来自耳蜗梭菌的活性酶由两个亚基（53.6 kDa和14.8 kDa）组成，为α2β2四聚体，其组装由辅酶B12介导。较小的蛋白质组分GlmS被认为是B12结合亚基。在此，我们报道了通过异核核磁共振研究确定的GlmS的溶液结构，以及对该脱辅酶亚基重要动力学方面的分析。GlmS在溶液中的整体折叠和动力学行为与先前使用核磁共振光谱研究过的来自破伤风梭菌的相应亚基MutS相似。两个Glm B12结合亚基的溶液结构与由晶体学确定的来自谢氏丙酸杆菌的甲基丙二酰辅酶A变位酶（Mcm）的B12结合结构域（结合有B12）都有显著相似性。在晶体结构中，发现一个保守的组氨酸残基与钴配位，取代了钴胺素的内源性轴向配体。然而，在GlmS和MutS中，包含钴配位组氨酸残基的序列基序Asp-x-His-x-x-Gly以及该基序后的预测α螺旋区域，以无结构且高度可移动的环形式存在。在没有辅酶的情况下，B12结合位点显然仅部分形成。通过将Mcm的晶体结构与不含B12的GlmS和MutS的溶液结构进行比较，获得了关于B12结合机制的一致图景。结合位点的重要元件仅在结合B12时才形成结构；这些元件包括钴配位组氨酸残基以及形成容纳辅酶核苷酸“尾巴”的裂隙一侧的α螺旋。
