Structural and enzymatic studies of a new analogue of coenzyme B12 with an alpha-adenosyl upper axial ligand.
Structural and enzymatic studies of a new analogue of coenzyme B12 with an alpha-adenosyl upper axial ligand.
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DOI：
10.1021/bi980707m
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发表时间：
1998-07
期刊：
影响因子：
2.9
通讯作者：
K. Brown;S. Cheng;X. Zou;J. Li;G. Chen;E. Valente;J. Zubkowski;H. Marques
中科院分区：
文献类型：
--
作者：
K. Brown;S. Cheng;X. Zou;J. Li;G. Chen;E. Valente;J. Zubkowski;H. Marques
本文合成了一种新的辅酶B_（12）类似物（5 ′-脱氧腺苷钴胺素，简称5 ′-Cbl），其中Ado配体中的N-糖苷键构型发生了反转[（α-ribo）Cbl]，并通过X射线衍射测定了其晶体结构[MoK α，λ = 0.71073 A，单斜晶系P212121，a = 16.132（12）A，B = 21. 684（15）A，c = 27.30（3）A，9611个独立反射，R1 = 0。0708]。正如在结构已知之前的分子力学模型所建议的那样，Ado配体位于分子的南部象限，就像Ado Cbl的情况一样。该结构最显著的特征是腺嘌呤（Ade）部分相对于Ado配体的核糖的取向紊乱。这可以用一个双态模型来解决，其中在主要构象（占有率为0.57）中，A16（O）-A11-A9（N）-A8二面角为1.9度，Ade几乎垂直于咕啉环;在次要构象中，Ade向下倾斜，二面角为-48.7度。Co-C键长和轴向Co-N键长以及Co-C-C键角与C_2Cbl中的那些非常相似。柯林环比柯林环平坦得多，折叠角为11.7度。该分子被成功地模拟了分子力学（MM），和旋转的Ado配体相对于咕啉产生了四个局部最小的结构与Ado在南部，东部，北方，或西部象限，与南部的构象作为全球最小值，是与cDNACbl本身的情况。二维（2D）NMR观察到的核Overhauser效应（nOe）被纳入分子动力学（MD）和模拟退火（SA）计算的约束。在300 K的MD模拟表明，只有南部的构象是人口与Ado配体局限于一个弧从C15以上到C12以上，而Ade环振荡从垂直到平行于咕啉环。通过MD-SA收集了27个结构。这种新的类似物是来自莱氏乳杆菌（Lactobacillus leichmanii）的核糖核苷酸还原酶的部分活性辅酶，其最大活性是Lactobacillus Cbl本身的9.7%，并且具有非常高的Km值（245 μ M，而Lactobacillus Cbl为0.54 μ M）。此外，酶诱导的碳-钴键裂解的速率常数（α-ribo）cobalt Cbl是160倍，小于cobalt Cbl，只有1/3的cob（II）alamin是在活性位点产生的。
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.