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

Zhang et al. Journal of Biological Engineering https://doi.org/10.1186/s13036-019-0164-5 (2019) 13:32 RESEARCH Open Access Quantitative study of H protein lipoylation of the glycine cleavage system and a strategy to increase its activity by coexpression of LplA Xinyi Zhang1, Mei Li1, Yingying Xu1, Jie Ren1* and An-Ping Zeng1,2* 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 coexpressing 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. Keywords: Glycine cleavage system, H protein, Lipoylation, LplA, Formate Introduction Glycine cleavage system (GCS) is the major degradation pathway of glycine widely distributed in animals, plants and bacteria (Kikuchi et al. 2008). In GCS glycine is enzymatically cleaved into CO2, NH4+, and a methylene group (Fig. 1). The methylene group is accepted by tetrahydrofolate (THF), forming 5,10-methylene-THF as the one-carbon (C1) source for purine synthesis and cell growth, and yielding one molecule of NADH as reducing power [1]. GCS also catalyzes the reversible reaction of glycine synthesis from CO2, ammonium, 5,10-methylene-THF and NADH, especially in anaerobic bacteria such as Clostridium acidiurici [2, 3]. * Correspondence: ; 1 Beijing Advanced Innovation Center for Soft Matter Science and Engineering, Beijing University of Chemical Technology, North Third Ring Road 15, Beijing 100029, China Full list of author information is available at the end of the article Bar-Even et al. (2013) proposed the use of reversed GCS reactions as a central part of the so-called reductive glycine pathway as the most promising pathway for developing a synthetic formatotropic microorganism for the use of formate and CO2 [4]. Recently, the reversed GCS reactions have been successfully used to construct novel C1 assimilation pathways in Escherichia coli for the use of formate and CO2 [5–11]. 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 then channeled into the central metabolism pathway. However, the reaction rate or flux of glycerin synthesis is still quite low and only about 10% of the carbon for cell growth can be supplied by the synthetic pathway. It is essential to better understand and reengineer GCS for a truly formatotrophic growth in both C1 utilization and CO2 fixation. © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Zhang et al. Journal of Biological Engineering (2019) 13:32 Page 2 of 9 Fig. 1 Glycine cleavage system (GCS) with H protein as a shuttle among its components, also shown are the lipoylation of H protein and the roles of GCS in the utilization of formate and purine biosynthesis GCS consists of four enzymes: glycine decarboxylase (P protein), aminomethyl-transferase (T protein), dihydrolipoyl dehydrogenase (L protein) and a carrier protein (H protein) (Fig. 1) [12–14]. The H protein plays a pivotal role and interacts with the other three proteins through a lipoic acid arm bound to a lysine residue [15]. The lipoyl group is the “true” shuttle which carries the aminomethyl group between the P protein and the T protein, and regenerates through the L protein yielding NADH at the same time. It may therefore play a key role in determine the overall reaction rate. Two mechanisms are known to perform lipoylation reaction in nature: one is to transfer the lipoyl group from lipoylated E2 protein of keto-acid dehydrogenase catalyzed by lipoyl (octanoyl) transferase (EC 2.3.1.181LipB) [16], and the other is lipoylation with exogenous lipoic acid under the involvement of ATP and lipoate-protein ligase A (EC 6.3.1.20, LplA) [17]. Fujiwara and Motokawa (1990) developed a method to quantify the rate of H protein lipoylation via mapping digestion peptides of the apo-form of H protein (Hapo) and the lipolated H protein (Hlip) using HPLC and mass spectroscopy [18]. They proved that only a trace amount of the H protein was lipoylated when H protein was overexpressed in E. coli cultured without addition of lipoic acid. When the cells were cultured in medium supplemented with 30 μM lipoic acid, about 10% of the recombinant protein expressed had the correctly lipoylated active form, the other 10% were in an inactive aberrantly modified form, presumably with an octanoyl group [19], and the remaining 80% were the apo-form. However, Macherel et. al. (2010) reached different results: with the same expression vector (PET system) they obtained more than 90% of a recombinant pea H protein in the lipoylated form with 100 μM lipoic acid added. [20] They assumed that the difference might be due to the different induction time. In engineered E. coli overexpressing GCS, the lipoylation rate of H protein is an important factor that may limit the C1 assimilation pathway. Despite intensive studies of GCS in the past, quantitative data and information are still scare regarding the interactions of the GCS components and potential limiting steps in both the forward and reversed reaction directions of GCS. In particular, uncertainties exist in literature regarding a potential inhibiting role of Hapo and the extent of H protein lipoylatio (...truncated)