O'Brien Jessica Rae, Raynaud Celine, Croux Christian, Girbal Laurence, Soucaille Philippe, Lanzilotta William N
Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602, USA.
Biochemistry. 2004 Apr 27;43(16):4635-45. doi: 10.1021/bi035930k.
The molecular characterization of a B12-independent glycerol dehydratase from Clostridium butyricum has recently been reported [Raynaud, C., et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 5010-5015]. In this work, we have further characterized this system by biochemical and crystallographic methods. Both the glycerol dehydratase (GD) and the GD-activating enzyme (GD-AE) could be purified to homogeneity under aerobic conditions. In this form, both the GD and GD-AE were inactive. A reconstitution procedure, similar to what has been reported for pyruvate formate lyase activating enzyme (PFL-AE), was employed to reconstitute the activity of the GD-AE. Subsequently, the reconstituted GD-AE could be used to reactivate the GD under strictly anaerobic conditions. We also report here the crystal structure of the inactive GD in the native (2.5 A resolution, Rcryst = 17%, Rfree = 20%), glycerol-bound (1.8 A resolution, Rcryst = 21%, Rfree = 24%), and 1,2-propanediol-bound (2.4 A resolution, Rcryst = 20%, Rfree = 24%) forms. The overall fold of the GD monomer was similar to what has been observed for pyruvate formate lyase (PFL) and anaerobic ribonucleotide reductase (ARNR), consisting of a 10-stranded beta/alpha barrel motif. Clear density was observed for both substrates, and a mechanism for the dehydration reaction is presented. This mechanism clearly supports a concerted pathway for migration of the OH group through a cyclic transition state that is stabilized by partial protonation of the migrating OH group. Finally, despite poor alignment (rmsd approximately 6.8 A) of the 10 core strands that comprise the barrel structure of the GD and PFL, the C-terminal domains of both proteins align well (rmsd approximately 0.7 A) and have structural properties consistent with this being the docking site for the activating enzyme. A single point mutation within this domain, at a strictly conserved arginine residue (R782K) in the GD, resulted in formation of a tight protein-protein complex between the GD and the GD-AE in vivo, thereby supporting this hypothesis.
最近有报道称对丁酸梭菌中一种不依赖维生素B12的甘油脱水酶进行了分子特征分析[雷诺，C.等人（2003年）《美国国家科学院院刊》100，5010 - 5015]。在这项工作中，我们通过生化和晶体学方法对该系统进行了进一步表征。甘油脱水酶（GD）和GD激活酶（GD - AE）在有氧条件下均可纯化至均一状态。在此形式下，GD和GD - AE均无活性。采用了一种类似于丙酮酸甲酸裂解酶激活酶（PFL - AE）所报道的重构程序来重构GD - AE的活性。随后，重构后的GD - AE可用于在严格厌氧条件下重新激活GD。我们在此还报道了无活性GD在天然状态（分辨率为2.5 Å，Rcryst = 17%，Rfree = 20%）、甘油结合状态（分辨率为1.8 Å，Rcryst = 21%，Rfree = 24%）和1,2 - 丙二醇结合状态（分辨率为2.4 Å，Rcryst = 20%，Rfree = 24%）下的晶体结构。GD单体的整体折叠结构与丙酮酸甲酸裂解酶（PFL）和厌氧核糖核苷酸还原酶（ARNR）所观察到的相似，由一个10链的β/α桶状基序组成。两种底物均观察到清晰的电子密度，并提出了脱水反应的机制。该机制明确支持通过一个环状过渡态使OH基团迁移的协同途径，该过渡态通过迁移的OH基团的部分质子化得以稳定。最后，尽管构成GD和PFL桶状结构的10条核心链的比对效果较差（均方根偏差约为6.8 Å），但两种蛋白质的C末端结构域比对良好（均方根偏差约为0.7 Å），并且具有与作为激活酶对接位点相一致的结构特性。GD中该结构域内一个严格保守的精氨酸残基（R782K）处的单点突变，导致在体内GD与GD - AE之间形成紧密的蛋白质 - 蛋白质复合物，从而支持了这一假说。
