苯甲酸脱羧酶通过逆反应-羧化作用为CO2固定提供了一种优雅的替代品,被命名为bio-Kolbe-Schmitt反应,但它们不利于羧化。增强可逆苯甲酸脱羧酶的羧化效率受到无法解释的羧化机制的限制。可逆酶催化反应的方向取决于酶活性中心的催化残基是否质子化,这是由pH值。因此,在不同的pH值下可以分离正向和反向反应。可逆的2,3-二羟基苯甲酸脱羧酶在pH5.0下进行脱羧,在pH8.6下进行羧化。然而,目前尚不清楚酶在正向和反向反应中与底物和产物的相互作用是否可以在不利的方向上提高可逆酶的催化活性。这里,我们从米曲霉(2,3-DHBD_Ao)中鉴定了一个V形的2,3-二羟基苯甲酸脱羧酶隧道,底物通过该隧道在酶中行进,并证明酪氨酸残基的侧链构象控制可逆反应过程中底物/产物的进出。连同突变体的动力学研究,阐明了在2,3-DHBD_Ao中通过酶隧道行进的底物/产物之间的相互作用是方向依赖性的。这些结果丰富了对底物/产物与大分子可逆酶在不同反应方向上的相互作用的理解。从而证明了具有更高羧化效率的工程脱羧酶的可能途径。关键点:•2,3-DHBD_Ao的残基Trp23充当开关以控制邻苯二酚的进出•确定了用于脱羧和羧化反应的2,3-DHBD_Ao的V形通道•结果为工程脱羧酶提供了有希望的策略,该脱羧酶的底物/产物行进通道内部具有方向依赖性残基。
Benzoic acid decarboxylases offer an elegant alternative to CO2 fixation by reverse reaction-
carboxylation, which is named the bio-Kolbe-Schmitt reaction, but they are unfavorable to
carboxylation. Enhancing the
carboxylation efficiency of reversible benzoic acid decarboxylases is restricted by the unexplained
carboxylation mechanisms. The direction of reversible enzyme catalytic reactions depends on whether catalytic residues at the active center of the enzyme are protonated, which is subjected by the pH. Therefore, the forward and reverse reactions could be separated at different pH values. Reversible 2,3-dihydroxybenzoate acid decarboxylase undergoes decarboxylation at pH 5.0 and
carboxylation at pH 8.6. However, it is unknown whether the interaction of enzymes with substrates and products in the forward and reverse reactions can be exploited to improve the catalytic activity of reversible enzymes in the unfavorable direction. Here, we identify a V-shaped tunnel of 2,3-dihydroxybenzoic acid decarboxylase from Aspergillus oryzae (2,3-DHBD_Ao) through which the substrate travels in the enzyme, and demonstrate that the side chain conformation of a tyrosine residue controls the entry and exit of substrate/product during reversible reactions. Together with the kinetic studies of the mutants, it is clarified that interactions between substrate/product traveling through the enzyme tunnel in 2,3-DHBD_Ao are direction-dependent. These results enrich the understanding of the interactions of substrates/products with macromolecular reversible enzymes in different reaction directions, thereby demonstrating a possible path for engineering decarboxylases with higher carboxylation efficiency. KEY POINTS: • The residue Trp23 of 2,3-DHBD_Ao served as a switch to control the entry and exit of catechol • A V-shaped tunnel of 2,3-DHBD_Ao for decarboxylation and carboxylation reactions was identified • The results provide a promising strategy for engineering decarboxylases with direction-dependent residues inside the substrate/product traveling tunnel of the enzyme.