KEYWORDS:
H protein, lipoate-protein ligase A, lipoylation, hydrolysis, protein interaction
INTRODUCTION
Lipoic acid is an essential cofactor that is covalently bound to its cognate enzyme proteins, a process known as lipoylation, and conveys activated reaction intermediates between different active sites of several multienzyme complexes involved in the central metabolism of organisms (Cronan, 2016). These enzyme systems include pyruvate dehydrogenase complex (PDHC) and 2-oxoglutarate dehydrogenase complex (KDHC) for the entry of carbon into the tricarboxylic acid (TCA) cycle and the progression of carbon through the TCA cycle, respectively, the branched-chain alpha-ketoacid dehydrogenase complex (BCKDH) in amino acid metabolism and the glycine cleavage system (GCS) in one-carbon (C1) metabolism(Cronan, 2018; Perham, 2000). Recently, lipoylation of the GCS H protein has gained interest in the engineering synthetic C1 metabolism. In the so-called reductive glycine pathway, the reverse GCS reaction serves in the assimilation of the C1 carbon source CO2 into biomass (Bang and Lee, 2018; Bar-Even, 2016; Bar-Even et al., 2010; Bar-Even et al., 2012; Doring et al., 2018; Tashiro et al., 2018; Yishai et al., 2018). However, our recent studies showed that the degree of the intracellular H protein lipoylation is in general quite low and can represent a limiting step for C1-based biosynthesis (Zhang et al., 2019).
Lipoylation of the H protein has received much interest in the past (Cronan, 2016; Fujiwara et al., 1990; Fujiwara et al., 1991; Fujiwara et al., 1992; Macherel et al., 1996; Solmonson and DeBerardinis, 2018). It is now clear that in E. coli , lipoate-protein ligase A (LplA) catalyzes the lipoylation of the apo-H protein (Hapo) in the presence of lipoic acid and ATP (Fig. 1a). The reaction is divided into two steps: (1) the lipoate adenylation reaction, where lipoic acid is activated by ATP, forming the intermediate lipoyl-AMP (Lip-AMP); (2) the lipoate transfer reaction, where the lipoyl part is transferred onto a specific lysine residue of Hapo (Fujiwara et al., 2005). Recent years, the functionality of LplA as a connection tool was further explored (Fernández-Suárez et al., 2007; Uttamapinant et al., 2010). Uttamapinant, C. et al. first engineered LplA as a fluorophore ligase. The new protein labeling method, called probe incorporation mediated by enzymes (PRIME) will provide a much-needed alternative to GFP, and provide life scientists with a way to label proteins of interest in a minimally invasive and extremely specific manner.
The crystal structures of E. coli LplA and its complexes with lipoic acid (lipoate), Lip-AMP and H protein have been determined by Fujiwara et al. (Fujiwara et al., 2010; Fujiwara et al., 2005). LplA contains a large N-terminal domain and a small C-terminal domain. Based on information from the structural analysis and some kinetic studies of potential key residues involved in the binding of Lip-AMP and the lipoate transfer, Fujiwaraet al. (Fujiwara et al., 2010) proposed a reaction mechanism model of the H protein lipoylation catalyzed by E. coli LplA. It involves large conformational changes of LplA as summarized in Fig.1b. At the beginning of the lipoylation process the C-terminal domain of unliganded LplA adopts a bending conformation. Lipoic acid attaches to the hydrophobic cavity in the N-terminal domain by hydrophobic interactions without changing the bending conformation of LplA first (Fujiwara et al., 2005). After the adenylation of the lipoic acid (Step 1 in Fig.1a), LplA undergoes a dramatic conformational change: the adenylate-binding and lipoate-binding loops take certain movements to bind the reaction intermediate Lip-AMP, while the C-terminal domain rotates by about 180° and adopts a stretched conformation. The large conformational change is suggested to be a prerequisite for LplA to accommodate Hapo for the second reaction step (Fujiwara et al., 2010). However, little information is available about the quantitative kinetics of the lipoylation reaction, i.e. the kinetics of the individual reaction steps as well as the influence of structural factors affecting the reaction rates are basically unknown. This is probably among others due to lack of suitable analytic methods (Hong et al., 2020). While the product of the first adenylation step, Lip-AMP, can be measured relatively easily using HPLC (Fujiwara et al., 2010), assays available for the second lipoate transfer reaction are complicated to perform and less suitable for quantitative kinetic studies. They are relied on radioactive isotope labeling either directly by using [35S] lipoic acid (Morris et al., 1994), or indirectly by determining the activity of the glycine-14CO2 exchange reaction catalyzed by P protein of GCS (Fujiwara et al., 1992). The latter can only be carried out under low concentrations of Hapo, because the glycine-CO2 exchange reaction is inhibited when the concentration of Hapo is higher than 5μM (Fujiwara et al., 2005).
Recently, we developed an efficient direct assay method for Hapo and Hlip (the lipoylated form of H protein) using HPLC (Zhang et al., 2019). In this work, we have further developed an efficient HPLC method for a fast and reliable quantification of lipoic acid, Lip-AMP, AMP, ADP and ATP, enabling us thereby quantitative studies of the overall reaction and the individual steps of the LplA-catalyzed lipoylation of H protein. The quantitative and differentiated studies of the overall reaction and the individual steps revealed unusual the kinetic behaviors of H protein lipoylation. In particular, we discovered autocatalytic and feedback activations of lipoylation by the different forms of H protein and unexpected hydrolysis of Lip-AMP. The latter is a major phenomenon associated with the second step of the lipoylation. Based on these findings, a revised mechanistic model of H protein lipoylation is proposed.