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.