Enzymatic production of ß-glucose 1,6-bisphosphate through manipulation of catalytic magnesium coordination
Enzymatic production of ß-glucose 1,6-bisphosphate through manipulation of catalytic magnesium coordination
复制标题
通过操纵催化镁配位酶法生产β-葡萄糖1,6-二磷酸
DOI：
10.1039/d0gc03290e
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发表时间：
2021
期刊：
影响因子：
9.8
通讯作者：
Wood H
中科院分区：
文献类型：
--
作者：
Wood H
操纵酶的行为是一种可持续的技术，可用于提高有价值的代谢物和化学前体的生产。β-葡萄糖-1，6-二磷酸(βG16BP)是β-磷酸葡萄糖变位酶(β-PGM)催化循环中的天然反应中间体，已被提出用于治疗涉及磷酸甘露糖变位酶2的人类先天性糖基化障碍。迄今为止，合成βG16BP的策略存在产率低或使用化学品和对环境有重大影响的步骤。在这里，我们报道了使用βPGM的D170N变体(βPGMD170N)高效地酶法合成异构体特异性的βG16BP，其中第170位天冬氨酸到天冬酰胺的取代扰乱了催化镁离子的配位。核磁共振谱和动力学分析相结合的结果表明，β对βG16BP的亲和力和反应活性减弱，导致两步催化循环中的第二步明显滞后，导致βG16BP的显著积累，特别是在较高的MgCl2浓度下。采用简单的环保沉淀法进行纯化，只需要标准的生化工具箱，即可获得高纯度和高产量的βG16BP产品。总而言之，这一合成策略说明了如何利用酶的催化镁配位来产生大量有价值的代谢物。
Manipulation of enzyme behaviour represents a sustainable technology that can be harnessed to enhance the production of valuable metabolites and chemical precursors. β-Glucose 1,6-bisphosphate (βG16BP) is a native reaction intermediate in the catalytic cycle of β-phosphoglucomutase (βPGM) that has been proposed as a treatment for human congenital disorder of glycosylation involving phosphomannomutase 2. Strategies to date for the synthesis of βG16BP suffer from low yields or use chemicals and procedures with significant environmental impacts. Herein, we report the efficient enzymatic synthesis of anomer-specific βG16BP using the D170N variant of βPGM (βPGMD170N), where the aspartate to asparagine substitution at residue 170 perturbs the coordination of a catalytic magnesium ion. Through combined use of NMR spectroscopy and kinetic assays, it is shown that the weakened affinity and reactivity of βPGMD170N towards βG16BP contributes to the pronounced retardation of the second step in the two-step catalytic cycle, which causes a marked accumulation of βG16BP, especially at elevated MgCl2 concentrations. Purification, employing a simple environmentally considerate precipitation procedure requiring only a standard biochemical toolset, results in a βG16BP product with high purity and yield. Overall, this synthesis strategy illustrates how manipulation of the catalytic magnesium coordination of an enzyme can be utilised to generate large quantities of a valuable metabolite.