Brown K L, Cheng S, Zou X, Li J, Chen G, Valente E J, Zubkowski J D, Marques H M
Department of Chemistry, Ohio University, Athens 45701, USA.
Biochemistry. 1998 Jul 7;37(27):9704-15. doi: 10.1021/bi980707m.
A new analogue of coenzyme B12 (5'-deoxyadenosylcobalamin, AdoCbl), in which the configuration of the N-glycosidic bond in the Ado ligand is inverted [(alpha-ribo)AdoCbl], has been synthesized and its crystal structure determined by X-ray diffraction [MoKalpha, lambda = 0.71073 A, monoclinic P212121, a = 16.132(12) A, b = 21. 684(15) A, c = 27.30(3) A, 9611 independent reflections, R1 = 0. 0708]. As suggested by molecular mechanics modeling before the structure was known, the Ado ligand lies over the southern quadrant of the molecule, as is the case for AdoCbl. The most striking feature of the structure is disorder in the orientation of the adenine (Ade) moiety relative to the ribose of the Ado ligand. This was resolved with a two-state model in which in the major (0.57 occupancy) conformer the A16(O)-A11-A9(N)-A8 dihedral angle is 1.9 degrees and the Ade is virtually perpendicular to the corrin ring; in the minor conformer, the Ade is tilted down, and this dihedral is -48.7 degrees. The Co-C and axial Co-N bond lengths and the Co-C-C bond angle are quite similar to those in AdoCbl. The corrin ring is considerably flatter than that of AdoCbl, with a fold angle of 11.7 degrees. The molecule was successfully modeled by molecular mechanics (MM), and rotation of the Ado ligand relative to the corrin gave rise to four locally minimum structures with the Ado in the southern, eastern, northern, or western quadrant, with the southern conformation as the global minimum, as is the case with AdoCbl itself. Nuclear Overhauser effects (nOe's) observed by two-dimensional (2D) NMR were incorporated as restraints in molecular dynamics (MD) and simulated annealing (SA) calculations. A MD simulation at 300 K showed that only the southern conformation is populated with the Ado ligand confined to an arc from over C15 to over C12, while the Ade ring oscillates from perpendicular to parallel to the corrin ring. Twenty-seven structures were collected by MD-SA. Most of these annealed into the southern conformation, but examples of the other conformations were also found. The new analogue is a partially active coenzyme for the ribonucleotide reductase from Lactobacillus leichmanii with maximal activity that is 9.7% of that of AdoCbl itself, and a very high Km value (245 microM compared to 0.54 microM for AdoCbl). In addition, the rate constant for enzyme-induced carbon-cobalt bond cleavage of (alpha-ribo)AdoCbl is 160-fold smaller than that for AdoCbl, and only 1/3 as much cob(II)alamin is produced at the active site.
已合成了辅酶B12（5'-脱氧腺苷钴胺素，AdoCbl）的一种新类似物，其中Ado配体中N-糖苷键的构型发生了反转[（α-核糖）AdoCbl]，并通过X射线衍射确定了其晶体结构[MoKα，λ = 0.71073 Å，单斜晶系P212121，a = 16.132(12) Å，b = 21.684(15) Å，c = 27.30(3) Å，9611个独立反射，R1 = 0.0708]。正如在结构已知之前分子力学建模所表明的那样，Ado配体位于分子的南象限，AdoCbl也是如此。该结构最显著的特征是腺嘌呤（Ade）部分相对于Ado配体核糖的取向无序。这通过双态模型得以解决，在主要构象（占有率0.57）中，A16(O)-A11-A9(N)-A8二面角为1.9°，Ade实际上垂直于咕啉环；在次要构象中，Ade向下倾斜，该二面角为-48.7°。Co-C键和轴向Co-N键长度以及Co-C-C键角与AdoCbl中的非常相似。咕啉环比AdoCbl的要平得多，折叠角为11.7°。该分子通过分子力学（MM）成功建模，Ado配体相对于咕啉的旋转产生了四个局部最小结构，Ado分别位于南、东、北或西象限，其中南构象为全局最小值，AdoCbl本身也是如此。二维（2D）NMR观察到的核Overhauser效应（nOe）被用作分子动力学（MD）和模拟退火（SA）计算的约束条件。在300 K下的MD模拟表明，只有南构象存在，Ado配体局限于从C15上方到C12上方的一段弧线，而Ade环从垂直于咕啉环振荡到与咕啉环平行。通过MD-SA收集了27个结构。这些结构中的大多数退火后形成南构象，但也发现了其他构象的例子。这种新类似物是来自赖氏乳杆菌的核糖核苷酸还原酶的部分活性辅酶，最大活性为AdoCbl本身的9.7%，且Km值非常高（245 μM，而AdoCbl为0.54 μM）。此外，（α-核糖）AdoCbl的酶促碳-钴键断裂速率常数比AdoCbl小160倍，在活性位点产生的钴胺素（II）只有AdoCbl的1/3。
