Department of Biological Sciences, Louisiana State University; Baton Rouge, LA 70803, USA.
Department of Biochemistry and Molecular Biology, Michigan State University; East Lansing, MI 48824, USA.
Biochim Biophys Acta Proteins Proteom. 2024 Sep 1;1872(5):141033. doi: 10.1016/j.bbapap.2024.141033. Epub 2024 Jul 15.
Malonyl-CoA reductase utilizes two equivalents of NADPH to catalyze the reduction of malonyl-CoA to 3-hydroxypropionic acid (3HP). This reaction is part of the carbon fixation pathway in the phototrophic bacterium Chloroflexus aurantiacus. The enzyme is composed of two domains. The C-terminal domain catalyzes the reduction of malonyl-CoA to malonic semialdehyde, while the N-terminal domain catalyzes the reduction of the aldehyde to 3HP. The two domains can be produced independently and retain their enzymatic activity. This report focuses on the kinetic characterization of the C-terminal domain. Initial velocity patterns and inhibition studies showed the kinetic mechanism is ordered with NADPH binding first followed by malonyl-CoA. Malonic semialdehyde is released first, while CoA and NADP are released randomly. Analogs of malonyl-CoA showed that the thioester carbon is reduced, while the carboxyl group is needed for proper positioning. The enzyme transfers the pro-S hydrogen of NADPH to malonyl-CoA and pH rate profiles revealed that a residue with a pK value of about 8.8 must be protonated for activity. Kinetic isotope effects indicated that NADPH is not sticky (that is, NADPH dissociates from the enzyme faster than the rate of product formation) and product release is partially rate-limiting. Moreover, the mechanism is stepwise with the pH dependent step occurring before or after hydride transfer. The findings from this study will aid in the development of an eco-friendly biosynthesis of 3HP which is an industrial chemical used in the production of plastics and adhesives.
丙二酰辅酶 A 还原酶利用两个当量的 NADPH 将丙二酰辅酶 A 还原为 3-羟基丙酸（3HP）。该反应是光养细菌黄杆菌中碳固定途径的一部分。该酶由两个结构域组成。C 端结构域催化丙二酰辅酶 A 还原为丙二醛半醛，而 N 端结构域催化醛还原为 3HP。两个结构域可以独立产生并保留其酶活性。本报告重点介绍 C 端结构域的动力学特征。初始速度模式和抑制研究表明，动力学机制是有序的，首先结合 NADPH，然后结合丙二酰辅酶 A。首先释放丙二醛半醛，而 CoA 和 NADP 随机释放。丙二酰辅酶 A 的类似物表明硫酯碳被还原，而羧基对于正确定位是必需的。该酶将 NADPH 的 pro-S 氢转移到丙二酰辅酶 A 上，并且 pH 速率曲线表明，活性需要一个 pK 值约为 8.8 的残基质子化。动力学同位素效应表明 NADPH 不粘滞（即，NADPH 从酶上解离的速度快于产物形成的速度），并且产物释放部分限速。此外，该机制是分步的，依赖 pH 的步骤发生在氢化物转移之前或之后。这项研究的结果将有助于开发一种环保的 3HP 生物合成方法，3HP 是一种用于生产塑料和粘合剂的工业化学品。
