Department of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109-0600.
Department of Chemistry, University of Michigan, Ann Arbor, MI 48109-0600.
Proc Natl Acad Sci U S A. 2020 Dec 1;117(48):30412-30422. doi: 10.1073/pnas.2007332117. Epub 2020 Nov 16.
Cobalamin is a complex organometallic cofactor that is processed and targeted via a network of chaperones to its dependent enzymes. AdoCbl (5'-deoxyadenosylcobalamin) is synthesized from cob(II)alamin in a reductive adenosylation reaction catalyzed by adenosyltransferase (ATR), which also serves as an escort, delivering AdoCbl to methylmalonyl-CoA mutase (MCM). The mechanism by which ATR signals that its cofactor cargo is ready (AdoCbl) or not [cob(II)alamin] for transfer to MCM, is not known. In this study, we have obtained crystallographic snapshots that reveal ligand-induced ordering of the N terminus of ATR, which organizes a dynamic cobalamin binding site and exerts exquisite control over coordination geometry, reactivity, and solvent accessibility. Cob(II)alamin binds with its dimethylbenzimidazole tail splayed into a side pocket and its corrin ring buried. The cosubstrate, ATP, enforces a four-coordinate cob(II)alamin geometry, facilitating the unfavorable reduction to cob(I)alamin. The binding mode for AdoCbl is notably different from that of cob(II)alamin, with the dimethylbenzimidazole tail tucked under the corrin ring, displacing the N terminus of ATR, which is disordered. In this solvent-exposed conformation, AdoCbl undergoes facile transfer to MCM. The importance of the tail in cofactor handover from ATR to MCM is revealed by the failure of 5'-deoxyadenosylcobinamide, lacking the tail, to transfer. In the absence of MCM, ATR induces a sacrificial cobalt-carbon bond homolysis reaction in an unusual reversal of the heterolytic chemistry that was deployed to make the same bond. The data support an important role for the dimethylbenzimidazole tail in moving the cobalamin cofactor between active sites.
钴胺素是一种复杂的有机金属辅因子，通过伴侣蛋白网络进行加工和靶向，以到达其依赖的酶。AdoCbl（5'-脱氧腺苷钴胺素）是由 cob(II)alamin 在腺苷转移酶（ATR）催化的还原腺苷化反应中合成的，ATR 还作为伴侣蛋白，将 AdoCbl 递送给甲基丙二酰辅酶 A 变位酶（MCM）。ATR 如何发出信号，表明其辅因子货物（AdoCbl）已准备好（cob(II)alamin）或未准备好（cob(II)alamin）转移到 MCM，目前尚不清楚。在这项研究中，我们获得了晶体学快照，揭示了配体诱导的 ATR N 端的有序排列，这组织了一个动态的钴胺素结合位点，并对配位几何形状、反应性和溶剂可及性进行了精细控制。cob(II)alamin 与二甲基苯并咪唑尾巴展开成侧袋，并将其 corrin 环埋藏，结合。共底物 ATP 强制 cob(II)alamin 形成四配位几何形状，促进不利的还原为 cob(I)alamin。AdoCbl 的结合模式与 cob(II)alamin 显著不同，二甲基苯并咪唑尾巴被塞进 corrin 环下，取代了 ATR 的 N 端，该 N 端无序。在这种溶剂暴露的构象中，AdoCbl 易于转移到 MCM。ATR 向 MCM 转移辅因子时，尾巴的重要性通过缺乏尾巴的 5'-脱氧腺苷钴胺素无法转移得到揭示。在没有 MCM 的情况下，ATR 诱导一个牺牲的钴-碳键均裂反应，这是一种异常的逆转，该反应用于形成相同的键。数据支持二甲基苯并咪唑尾巴在将钴胺素辅因子在活性位点之间移动中发挥重要作用。
