Zhang Xinyi, Li Mei, Xu Yingying, Ren Jie, Zeng An-Ping
1Beijing Advanced Innovation Center for Soft Matter Science and Engineering, Beijing University of Chemical Technology, North Third Ring Road 15, Beijing, 100029 China.
2Institute of Bioprocess and Biosystems Engineering, Hamburg University of Technology, Denickestrasse 15, D-21073 Hamburg, Germany.
J Biol Eng. 2019 Apr 23;13:32. doi: 10.1186/s13036-019-0164-5. eCollection 2019.
Glycine cleavage system (GCS) plays a key role in one-carbon (C1) metabolism related to the biosynthesis of a number of key intermediates with significance in both biomedicine and biotechnology. Despite extensive studies of the proteins (H, T, P and L) involved and the reaction mechanisms of this important enzyme complex little quantitative data are available. In this work, we have developed a simple HPLC method for direct analysis and quantification of the apo- and lipoylated forms (H and H) of the shuttle protein H, the latter (H) is essential for the function of H protein and determines the activity of GCS. Effects of temperature, concentrations of lipoic acid and H and the expression of H protein on its lipoylation were studied. It is found that H is as low as only 20-30% of the total H protein with lipoic acid concentration in the range of 10-20 μM and at a favorable temperature of 30 °C. Furthermore, H seems to inhibit the overall activity of GCS. We proposed a strategy of co-expressing LplA to improve the lipoylation of H protein and GCS activity. With this strategy the fraction of H was increased, for example, from 30 to 90% at a lipoic acid concentration of 20 μM and GCS activity was increased by more than 2.5 fold. This work lays a quantitative foundation for better understanding and reengineering the GCS system.
甘氨酸裂解系统（GCS）在一碳（C1）代谢中起着关键作用，该代谢与许多关键中间体的生物合成相关，这些中间体在生物医学和生物技术领域都具有重要意义。尽管对该重要酶复合物所涉及的蛋白质（H、T、P和L）以及反应机制进行了广泛研究，但可用的定量数据很少。在这项工作中，我们开发了一种简单的高效液相色谱法，用于直接分析和定量穿梭蛋白H的脱辅基形式和硫辛酰化形式（H和H），后者（H）对H蛋白的功能至关重要，并决定了GCS的活性。研究了温度、硫辛酸浓度、H浓度以及H蛋白的表达对其硫辛酰化的影响。结果发现，在硫辛酸浓度为10 - 20μM且温度为30°C的适宜条件下，H仅占总H蛋白的20 - 30%。此外，H似乎会抑制GCS的整体活性。我们提出了一种共表达LplA的策略来提高H蛋白的硫辛酰化水平和GCS活性。采用该策略，例如在硫辛酸浓度为20μM时，H的比例从30%增加到90%，GCS活性提高了2.5倍以上。这项工作为更好地理解和改造GCS系统奠定了定量基础。
