非磷酸化甘油醛-3-磷酸脱氢酶的计算机辅助工程以实现无细胞生物催化,ACS Catalysis

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非磷酸化甘油醛-3-磷酸脱氢酶的计算机辅助工程以实现无细胞生物催化
ACS Catalysis ( IF 11.3 ) Pub Date : 2023-08-22 , DOI: 10.1021/acscatal.3c01452
Sam J. B. Mallinson ₁ , Delphine Dessaux ₂ , Sophie Barbe ₂ , Yannick J. Bomble ₁

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氧化还原辅助因子的利用是实现高效且具有成本竞争力的无细胞生物催化的主要障碍之一，特别是在涉及多个氧化还原步骤的情况下。针对糖酵解等典型代谢途径的多功能、辅因子平衡模块的设计是克服此类障碍的一种途径。在这里，我们建立了一个计算机辅助设计框架来设计变形链球菌的非磷酸化甘油醛-3-磷酸脱氢酶（GapN）用于实现 NADH 连接的高效无细胞糖酵解途径，且 ATP 使用量为零。这种合理的设计方法将分子动力学模拟与多态计算设计方法相结合，使我们能够考虑 GapN 酶催化循环中遇到的不同构象状态。特别是，辅因子翻转（该酶家族的特征并在产物水解之前发生）被考虑在内，以重新设计辅因子结合袋以利用 NAD ⁺。虽然 GapN 仅对 NAD ⁺表现出微量活性，但该活性增强了约 10,000 倍，相当于野生型酶对 NADP + 的催化效率恢复了约 72 ^%，GapN 酶仅包含 5 个突变。

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Computer-Aided Engineering of a Non-Phosphorylating Glyceraldehyde-3-Phosphate Dehydrogenase to Enable Cell-Free Biocatalysis

Redox cofactor utilization is one of the major barriers to the realization of efficient and cost-competitive cell-free biocatalysis, especially where multiple redox steps are concerned. The design of versatile, cofactor balanced modules for canonical metabolic pathways, such as glycolysis, is one route to overcoming such barriers. Here, we set up a computer-aided design framework to engineer the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GapN) from Streptococcus mutans for enabling an NADH linked efficient cell-free glycolytic pathway with a net zero ATP usage. This rational design approach combines molecular dynamics simulations with a multistate computational design method that allowed us to consider different conformational states encountered along the GapN enzyme catalytic cycle. In particular, the cofactor flip, characteristic of this enzyme family and occurring before product hydrolysis, was taken into account to redesign the cofactor binding pocket for NAD⁺ utilization. While GapN exhibits only trace activity with NAD⁺, a ∼10,000-fold enhancement of this activity was achieved, corresponding to a recovery of ∼72% of the catalytic efficiency of the wild-type enzyme on NADP⁺, with a GapN enzyme harboring only 5 mutations.

更新日期：2023-08-22

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