Quantitative study of H protein lipoylation of the glycine cleavage system and a strategy to increase its activity by co-expression of LplA.

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.