Zhang, XinyiXinyiZhangLi, MeiMeiLiXu, YingyingYingyingXuRen, JieJieRenZeng, An-PingAn-PingZeng2019-05-082019-05-082019-04-23Journal of Biological Engineering 1 (13): 32 (2019-04-23)http://hdl.handle.net/11420/2638Glycine 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 apo and H lip ) of the shuttle protein H, the latter (H lip ) is essential for the function of H protein and determines the activity of GCS. Effects of temperature, concentrations of lipoic acid and H apo and the expression of H protein on its lipoylation were studied. It is found that H lip 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 apo 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 lip 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.en1754-16112019132Springerhttps://creativecommons.org/licenses/by/4.0/FormateGlycine cleavage systemH proteinLipoylationLplAIngenieurwissenschaftenJournal Articleurn:nbn:de:gbv:830-882.03447510.15480/882.224410.1186/s13036-019-0164-510.15480/882.2244Journal Article