Abstract
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 (Hapo and Hlip) of the shuttle protein H, the latter (Hlip) is essential for the function of H protein and determines the activity of GCS. Effects of temperature, concentrations of lipoic acid and Hapo and the expression of H protein on its lipoylation were studied. It is found that Hlip 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, Hapo 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 Hlip 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.
Highlights
Glycine cleavage system (GCS) is the major degradation pathway of glycine widely distributed in animals, plants and bacteria (Kikuchi et al 2008)
Effect of Hapo protein on GCS activity In the GCS system, H protein can only function after lipoylation
This greatly facilitates the study of lipoylation of H protein in the GCS system
Summary
Glycine cleavage system (GCS) is the major degradation pathway of glycine widely distributed in animals, plants and bacteria (Kikuchi et al 2008). The reversed GCS reactions have been successfully used to construct novel C1 assimilation pathways in Escherichia coli for the use of formate and CO2 [5,6,7,8,9,10,11]. To this end, endogenous GCS and exogenous formyl-methenyl-methylenetetrahydrofolate synthetase were overexpressed in engineered E. coli to convert formate into glycine and serine, and channeled into the central metabolism pathway. It is essential to better understand and reengineer GCS for a truly formatotrophic growth in both C1 utilization and CO2 fixation
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