Bui Soi, Gil-Guerrero Sara, van der Linden Peter, Carpentier Philippe, Ceccarelli Matteo, Jambrina Pablo G, Steiner Roberto A
Randall Centre for Cell and Molecular Biophysics, King's College London London SE1 1UL UK
Departamento de Química Física, University of Salamanca Salamanca 37008 Spain
Chem Sci. 2023 Sep 18;14(38):10547-10560. doi: 10.1039/d3sc03044j. eCollection 2023 Oct 4.
Protein fold adaptation to novel enzymatic reactions is a fundamental evolutionary process. Cofactor-independent oxygenases degrading -heteroaromatic substrates belong to the α/β-hydrolase (ABH) fold superfamily that typically does not catalyze oxygenation reactions. Here, we have integrated crystallographic analyses under normoxic and hyperoxic conditions with molecular dynamics and quantum mechanical calculations to investigate its prototypic 1--3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (HOD) member. O localization to the "oxyanion hole", where catalysis occurs, is an unfavorable event and the direct competition between dioxygen and water for this site is modulated by the "nucleophilic elbow" residue. A hydrophobic pocket that overlaps with the organic substrate binding site can act as a proximal dioxygen reservoir. Freeze-trap pressurization allowed the structure of the ternary complex with a substrate analogue and O bound at the oxyanion hole to be determined. Theoretical calculations reveal that O orientation is coupled to the charge of the bound organic ligand. When 1--3-hydroxy-4-oxoquinaldine is uncharged, O binds with its molecular axis along the ligand's C2-C4 direction in full agreement with the crystal structure. Substrate activation triggered by deprotonation of its 3-OH group by the His-Asp dyad, rotates O by approximately 60°. This geometry maximizes the charge transfer between the substrate and O, thus weakening the double bond of the latter. Electron density transfer to the O(π*) orbital promotes the formation of the peroxide intermediate intersystem crossing that is rate-determining. Our work provides a detailed picture of how evolution has repurposed the ABH-fold architecture and its simple catalytic machinery to accomplish metal-independent oxygenation.
蛋白质折叠对新型酶促反应的适应是一个基本的进化过程。降解杂芳族底物的非辅因子依赖性加氧酶属于α/β-水解酶（ABH）折叠超家族，该超家族通常不催化氧化反应。在这里，我们结合了常氧和高氧条件下的晶体学分析与分子动力学和量子力学计算，以研究其典型的1-3-羟基-4-氧代喹哪啶2,4-双加氧酶（HOD）成员。氧定位到发生催化作用的“氧阴离子洞”是一个不利事件，并且双氧和水在该位点的直接竞争由“亲核肘”残基调节。与有机底物结合位点重叠的疏水口袋可作为近端双氧储存库。冷冻捕集加压使得能够确定与底物类似物和结合在氧阴离子洞处的氧形成的三元复合物的结构。理论计算表明，氧的取向与结合的有机配体的电荷相关。当1-3-羟基-4-氧代喹哪啶不带电时，氧以其分子轴沿配体的C2-C4方向结合，这与晶体结构完全一致。由His-Asp二元组使其3-OH基团去质子化引发的底物活化，使氧旋转约60°。这种几何结构使底物与氧之间的电荷转移最大化，从而削弱了后者的双键。电子密度转移到氧的O(π*)轨道促进了过氧化物中间体系间窜越的形成，这是速率决定步骤。我们的工作详细描绘了进化如何重新利用ABH折叠结构及其简单的催化机制来完成非金属依赖性氧化作用。
