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accessGlucose-6-phosphate dehydrogenase and its 3D structures from crystallography and electron cryo-microscopy
aDepartment of Neuroscience and Rehabilitation, University of Ferrara, Ferrara, Italy, and bDepartment of Chemistry, University of Manchester, Manchester M13 9PL, United Kingdom
*Correspondence e-mail: hns@unife.it, john.helliwell@manchester.ac.uk
SH dedicates this article to her deceased husband Arben Caushi.
Glucose-6-phosphate dehydrogenase (G6PD) is the first enzyme in the pentose phosphate pathway. It has been extensively studied by biochemical and structural techniques. 13 X-ray crystal structures and five electron cryo-microscopy structures in the PDB are focused on in this topical review. Two F420-dependent glucose-6-phosphate dehydrogenase (FGD) structures are also reported. The significant differences between human and parasite G6PDs can be exploited to find selective drugs against infections such as malaria and leishmaniasis. Furthermore, G6PD is a prognostic marker in several cancer types and is also considered to be a tumour target. On the other hand, FGD is considered to be a target against Mycobacterium tuberculosis and possesses a high biotechnological potential in biocatalysis and bioremediation.
Keywords: glucose-6-phosphate dehydrogenase; X-ray crystallography; electron cryo-microscopy; fused glucose-6-phosphate dehydrogenase–6-phosphogluconolactonase; F420-dependent glucose-6-phosphate dehydrogenase; human and trypanosomatid species.
1. Introduction
1.1. Different types of glucose-6-phosphate dehydrogenase
It is possible to recognize different types of glucose-6-phosphate dehydrogenase, namely:
Group 1: glucose-6-phosphate dehydrogenase (G6PD; EC 1.1.1.49), which catalyses the NAD+- or NADP+-dependent dehydrogenation of β-D-glucose 6-phosphate (G6P) to 6-phosphoglucono-δ-lactone.
Group 1a: this consists of G6PD fused in a bifunctional enzyme with the second enzyme of the pentose phosphate pathway (PPP), 6-phosphogluconolactonase (6PGL), and includes endoplasmic reticulum (ER) hexose-6-phosphate dehydrogenase (H6PD) and G6PDs from the parasites Giardia and Plasmodium.
Group 2: bacterial cofactor F420-dependent glucose-6-phosphate dehydrogenase (FGD). F420 is a 5-deazariboflavin that has previously been described in methanogenic archaea and claimed to be essential for antioxidant defence in mycobacteria (Eirich et al., 1979![[Eirich, L. D., Vogels, G. D. & Wolfe, R. S. (1979). J. Bacteriol. 140, 20-27.]](../../../../../../logos/arrows/f_arr.gif "Eirich, L. D., Vogels, G. D. & Wolfe, R. S. (1979). J. Bacteriol. 140, 20-27."){kind=small}
; Hasan et al., 2010).
1.2. G6PD is the first enzyme of the pentose phosphate pathway
G6PD is the first enzyme of the PPP (Fig. 1) and catalyses its rate-limiting step. It is a cytosolic enzyme that is active as a homodimer and a homotetramer (Cohen & Rosemeyer, 1969; Garcia et al., 2022). In trypanosomatids it is also present in glycosomes (Gupta et al., 2011).
![[Figure 1]](va5059fig1thm.gif){kind=small} | Figure 1 The pentose phosphate pathway (PPP). Metabolites common to glycolysis are in red. |
The PPP is also named the hexose monophosphate shunt since it diverts the use of G6P in the cytosol from glycolysis to the production of both NADPH and ribose 5-phosphate (Fig. 1). The metabolic pathway divides into two phases, the oxidative PPP (OPPP) branch and the non-oxidative branch, the first of which is absent in some lower organisms such as Cryptosporidium (Stover et al., 2011). The non-oxidative branch shares the intermediates fructose 6-phosphate and glyceraldehyde 3-phosphate with glycolysis, so that either glycolysis (or gluconeogenesis in some tissues, in particular the liver) or ribose production can be furnished according to the needs of the cell (Stincone et al., 2015; García-Domínguez et al., 2022). Two NADPH molecules are produced per G6P molecule in the OPPP: one is produced in the reaction catalyzed by G6PD; 6-phosphoglucono-δ-lactone is then hydrolysed by 6PGL to 6-phosphogluconate, which is then oxidatively decarboxylated to ribulose 5-phosphate by 6-phosphogluconate dehydrogenase (6PGDH), producing the second NADPH (Hanau & Helliwell, 2022).
NADPH is necessary for reductive biosynthesis, protection against oxidative stress and the function of the numerous NADPH-dependent enzymes, such as NADPH oxidases in immune cells. At the same time, it is also crucial for cancer cell metabolism and chemoresistance (Zhang et al., 2021; Song et al., 2022). Thus, overall, G6PD activity is regulated at different levels: firstly in the processes of transcription and RNA processing, and then at the level of the protein, such as stability, localization, post-translational modification, polymerization and allostery (Stanton, 2012; Meng et al., 2022). Among the transcription factors that directly bind to a G6PD promoter, c-Myc is involved in hepatocellular carcinoma growth (Yin et al., 2017), where G6PD represents a marker of poor prognosis. This is also the case for several other tumours, including lung cancer, both clear cell and papillary renal cell carcinomas, ovarian and prostate cancer, glioma, gastric and colorectal cancer (Tsouko et al., 2014; Zhang et al., 2020; Song et al., 2022; Zeng et al., 2023; Thakor et al., 2024). G6PD has also been found to be related to angiogenesis and distant metastasis (Zeng et al., 2023). In breast cancer Nrf2 was shown to be the transcription factor that is involved (Zhang et al., 2019), while in glioblastoma and renal cell carcinoma it is STAT3 (Song et al., 2022). The G6PD gene also contains vitamin D and sterol regulatory element-binding protein response elements and p53 family protein response elements. Furthermore, several other transcription factors have been reported to bind to the G6PD promoter (Meng et al., 2022).
G6PD deficiency is the most common human enzymopathy, affecting 500 million people worldwide. This arises due to mutation of the gene, which is located in the X chromosome. The mutation affects above all the red blood cells, causing hemolytic anaemia of different grades, which can be triggered by exposure to oxidative stress and was classified in 1989 by the World Health Organization (WHO) into classes I–IV, with the first being the most severe (WHO Working Group, 1989). This increased sensitivity to oxidative damage can manifest after the consumption of fava beans (favism), other foods, medications, especially antimalarial drugs, during infections and under particular environmental conditions (https://www.g6pd.org/en/G6PDDeficiency/SafeUnsafe/DaEvitare_ISS-it). A new WHO classification foresees four classes, A, B, C and U, where variants formerly in classes II and III are merged into the single class B (Luzzatto et al., 2024). On the other hand, deficient individuals and female carriers of deficient alleles are provided with beneficial effects against malaria, which is the reason why the frequency of G6PD deficiency is so high in those geographical regions with a high incidence of malaria (Mason et al., 2007; Nkhoma et al., 2009). G6PD deficiency has also been related to a longevity pattern (Schwartz & Pashko, 2004).
2. Crystallographic and electron cryo-microscopy (cryoEM) structures
In Table 1 we cite many of the available crystallographic structures and their PDB codes and report various details. We selected these structures based on their representativeness. The table includes a column in which peaks that are unmodelled in the PDB entry have been scrutinized using the Coot molecular-graphics visualization system (Emsley et al., 2010). Table 1 also includes a column in which the PDB validation reports are assessed by their clashscore. Specific comments of interest based on the PDB reports and an evaluation of the structure factors are also provided in this column. In Table 2 we cite cryo-EM structures and include a similar validation evaluation.
). ‡The validation report from the PDB concerns the derived model and not unmodelled peaks. The Fo − Fc map was inspected in the Coot visualization system to describe the unmodelled peaks (Emsley et al., 2010 ). §The difference-map evaluation in this table used the PDB-REDO entry for PDB entry 1qki as the deposited file of structure factors did not have map coefficients. The PDB-REDO file has the eight chains displayed in the Draw Sequence View tool in Coot (Emsley et al., 2010 ), whereas PDB entry 1qki shows chains A, B, C and D even though the PDB file contains all eight chains and Coot displays them, i.e. chains A–H. This is presumably a format-interconversion problem. ¶The difference-map evaluation in this table used the PDB-REDO entry. ∥Primary citation of related structure 7seh. #Primary citation of related structure 6e07. ††Primary citation of related structures 7zht, 7zhu, 7zhw, 7zhx, 7zhy and 7zhz. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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2.1. The G6PD subunit
The molecular weight of the human G6PD monomer is 59 kDa (Au et al., 2000). The first G6PD crystal structures, that from Leuconostoc mesenteroides (LM; PDB entry 1dpg) and the human form (PDB entry 1qki), both showed an N-terminal β–α–β `Rossmann-fold' coenzyme-binding domain and a β+α domain dominated by a curved nine-stranded antiparallel β-sheet (Fig. 2; Rowland et al., 1994; Au et al., 2000). In the amino-acid numbering system used in this paper the N-terminal methionine is designated `1'. The first of human G6PD was that of the class II mutant Canton R459L, which is one of the most common Chinese variants; it has only 18% of normal G6PD activity due to its decreased stability and has a higher Km for G6P in particular (Fig. 3; Au et al., 2000; Hwang et al., 2018). While no cysteine is present in the LM enzyme, there are seven cysteines in the human enzyme, with a disulfide bridge between Cys13 and Cys446, which most probably orders the mobile N-terminal segment (Au et al., 2000). After these structures, crystal structures of a deletion mutant ΔG6PD (i.e. without 25 N-terminal residues which were poorly ordered) were solved as binary complexes with G6P (PDB entry 2bhl) and NADP+ (PDB entry 2bh9) (Kotaka et al., 2005).
![[Figure 2]](va5059fig2thm.gif) | Figure 2 Ribbon diagram of the human G6PD subunit in complex with structural NADP (NADP+s), catalytic NADP (NADP+c) and G6P (PDB entry 7sni; reproduced from Wei et al., 2022 ). |
![[Figure 3]](va5059fig3thm.gif) | Figure 3 The human G6PDCanton (R459L) dimer with structural NADP+ bound (PDB entry 1qki; reproduced from Kotaka et al., 2005 ). |
In all higher organisms an additional allosteric binding site for NADP+ between the β-sheet and the C-terminus, proximal to the dimer interface, supports the binding of a second NADP+, called the `structural' NADP+ (NADP+s), which is not present in the LM enzyme (Fig. 2). NADP+s structurally stabilizes the enzyme, promotes oligomer assembly and regulates G6P binding and catalysis (De Flora et al., 1974; Au et al., 2000; Wang et al., 2008; Garcia et al., 2022; Wei et al., 2022). Thus, when the cytosolic ratio [NADPH]/[NADP+] decreases, NADP+ binding to the regulatory site of G6PD should allow the activation of G6PD, which is thought to work at low efficiency under normal cell conditions while being necessary during oxidative stress (Filosa et al., 2003). Although a second dinucleotide fingerprint is present in G6PDH sequences, this sequence does not appear to be involved in NADP+s binding in the structure of the human enzyme, and contacts are all made to side-chain atoms of one subunit (Fig. 3; PDB entries 1qki and 6e08; Au et al., 2000). NADP+s is 77% buried in the protein at a positively charged binding site with many arginine and lysine residues, at a distance of 7 Å from residues of the second subunit (Fig. 4). Its nicotinamide amide binds to Asp421, which is at the centre of the dimer interface. The aromatic rings of the adenine and nicotinamide form π–π interactions, with the adenine being between Tyr503 and Arg487, while the nicotinamide is between Trp509 and Tyr401 (Fig. 4; Au et al., 2000; Kotaka et al., 2005).
![[Figure 4]](va5059fig4thm.gif){kind=small} | Figure 4 Structural NADP binding site in human G6PDCanton (R459L) (PDB entry 1qki). This figure was prepared in UCSF ChimeraX (Meng et al., 2023 ; https://www.rbvi.ucsf.edu/chimerax). |
of the last residue by the Src-family kinase Fyn in red blood cells has been shown to activate G6PD (Mattè et al., 2020). Glu416, Glu417 and Glu419 can attract the positive charge on the oxidized nicotinamide ring. Hence, NADPH is not well bound at this site (Au et al., 2000; Kotaka et al., 2005). NADP+s is present in crystals of the human enzyme (containing tetramers) even if the coenzyme is not present in the protein solution prior to crystallization (Kotaka et al., 2005; Hwang et al., 2018). The Kd for NADP+s is 37 nM, which is 200-fold lower than that for the `catalytic' NADP+ (NADP+c; Wang et al., 2008). However, it is not present in the binary complex with G6P, in which the C-terminus is disordered (Kotaka et al., 2005). It is not present in cryo-electron microscopy (cryo-EM) structures of G6PD (tetramer; PDB entry 7sng), in which the C-terminus is also disordered (Wei et al., 2022). It was in fact shown that dimeric G6PD without NADP+s is active, although it is much less stable (Wang et al., 2008). Also, NADP+s can be reduced in the presence of G6P after migrating to the catalytic site (Kotaka et al., 2005; Wang et al., 2008).
G6P binds in the pocket between the N- and the C-terminal subdomains proximal to NADP+c (Fig. 2) at a highly conserved nine-residue region in helix αf′, with Asp200 involved in catalysis and His201, Tyr202 and Lys205 involved in substrate phosphate binding (Fig. 5, left) (Au et al., 2000; Cosgrove et al., 2000; Kotaka et al., 2005; Mason et al., 2007; Wei et al., 2022). His263 is the general base, which is hydrogen-bonded to Asp200, which together form a catalytic dyad as found in other enzymes. The Asp200 carboxylate stabilizes the positive charge of the histidine after proton abstraction from the C1 hydroxyl of G6P, allowing transfer of the C1 hydride to the nicotinamide ring of the coenzyme (Fig. 6; Cosgrove et al., 2000). Some differences between the LM and human G6PDs are present in the region of the binding of 6PG to elements near the mixed β-sheet, such as the positions of Asp258 from helix αi, Lys360 from a loop of the β-sheet and of human Gln395 binding to the phosphate (Fig. 5, left; Kotaka et al., 2005; Wei et al., 2022). This Gln is near Arg393 in the β-sheet, interacting with the O atom of the amide of NADP+s (Fig. 4).
![[Figure 5]](va5059fig5thm.gif) | Figure 5 The human G6PD–G6P complex (left; PDB entry 2bhl) and G6PD–catalytic NADP+ complex (right; PDB entry 2bh9). Reproduced from Kotaka et al. (2005 ). |
![[Figure 6]](va5059fig6thm.gif) | Figure 6 The His–Asp catalytic dyad of human G6PD. |
In the polypeptide loop between the βJ and βK strands, Arg365 binds the phosphate of G6P, while Lys366 at the beginning of the βK strand interacts with the 2′-phosphate of NADP+s (Figs. 2, 3 and 5, left). Also, other residues in this region interact by means of bound water molecules, allowing communication between NADP+s and the substrate-binding sites (for instance Asn363). Many class I variants only have mutations at these positions (Kotaka et al., 2005; Mason et al., 2007; Hernández-Ochoa et al., 2023).
The dinucleotide-binding fingerprint GxxGDLx encompasses residues 38–44 in the βA–αA turn, with the main-chain amino groups of Gly41 and Asp42 hydrogen-bonded to the O atoms of the adenine ribose 3′-hydroxyl and the bisphosphate (Fig. 5, right). Arg72 (Arg46 in the LM enzyme) is proximal to the 2′-phosphate (Fig. 5, right); however, there are differences in the catalytic coenzyme binding site between the human and LM enzymes that are related to the potential of the LM enzyme, but not the human enzyme, to use both NAD+ and NADP+. Among these, LM G6PD has a more hydrophobic environment for the adenine ring (Rowland et al., 1994; Levy, 1979; Kotaka et al., 2005). The hinge angle between the two domains of LM G6PD can vary, allowing three different shapes: a closed structure binding G6P, a half-open structure with NAD+ bound and an open form that tightly binds NADP+ (Naylor et al., 2001). The NADP+ reaction is an ordered sequential reaction, with G6P binding after NADP+. Fluorescence studies indicate that upon G6P binding there is an open-to-closed conformational change (Naylor et al., 2001; Haghighi & Levy, 1982). In both NADP+- and NAD+-bound structures the nicotinamide ribose makes a hydrogen bond to Lys148 (Lys171 in the human enzyme; Fig. 5, right) of the conserved EKPxG sequence. The cis–trans isomerization of the proline in this region in helix αe is critical in allowing the lysine to interact with both O1 of G6P and the 3′-hydroxyl of the nicotinamide ribose for the correct positioning of nicotinamide (Fig. 5; Bautista et al., 1995; Kotaka et al., 2005). A P172S mutation is found in the class I Volendam G6PD, which leads to chronic nonspherocytic hemolytic anaemia (CNSHA) and also seriously affects G6PD activity in leucocytes (Roos et al., 1999). In mammalian cells the conserved Ser84 was identified to be O-GlcNAcylated in a small percentage of G6PD; glycosylation increased under hypoxia or other conditions and has been found to significantly increase in lung and oesophageal cancer tissue compared with adjacent normal tissue (Rao et al., 2015; Su et al., 2021). When induced by means of the overexpression of O-GlcNAc transferase, glycosylation resulted in a higher NADP+ binding affinity (Rao et al., 2015). Ionizing radiation-induced Thr145 by casein kinase 2 in human epithelial cells also increases NADP+ binding affinity; hence, the G6PD activity increases (Hao et al., 2023).
Comparisons of the cryo-EM structure of the D200N G6PD mutant bound to all of the ligands (PDB entry 7sni), the with NADPs bound (PDB entry 1qki) and the cryo-EM structure of wild-type G6PD (G6PD WT; PDB entry 7sng) are informative. These suggest that a structural ordering of the subunit C-terminus in the presence of allosteric NADP+s involves movement of Phe237 and Phe241 from the β-sheet and flips of His201, Tyr202 and Lys205, which would be in a position that is incompatible with G6P binding in the apoenzyme. At the NADP+s binding site the loop 201–204 would form helix αf′, and His263 moves from a catalytically incompetent location to a competent location (Wei et al., 2022; Fig. 7). Tyr112 has been reported to be phosphorylated by tyrosine kinase c-Src in colorectal cancer, causing increases in 6PG affinity and (Ma et al., 2021). In contrast, in breast cancer cells (MCF7) the Src protein was found to decrease G6PD activity by phosphorylating Tyr249 and Tyr322, which are spatially close to the G6P binding pocket, and this is reverted in the presence of 20 mM lactic acid, mimicking the metabolic microenvironment of the solid tumour (Sun et al., 2023).
![[Figure 7]](va5059fig7thm.gif) | Figure 7 Superposition of G6PD WT and G6PD D200N–NADP+s–G6P suggesting NADP+s-mediated reorientation of Phe237 and Phe241, repositioning His201, Tyr202 and Lys205 (forming helix αf′), and of the catalytic His263 (reproduced from Wei et al., 2022 ). |
2.2. The active forms of G6PD are both as a dimer and as a tetramer
While LM G6PD is a dimer, the human enzyme in solution is in a monomer–dimer–tetramer equilibrium, where the monomer is not active and is induced by NADPH in a regulatory way (Bonsignore et al., 1971). The dimer–tetramer equilibrium is shifted towards the dimer by one of the following: a pH higher than 8, a high enzyme dilution or the presence of G6P (Cohen & Rosemeyer, 1969; Cancedda et al., 1973; Horikoshi et al., 2021). In contrast, NADP+ and certain metal ions favour the tetramer (Kirkman & Hendrickson, 1962; Wang et al., 2008), which has been shown to be more active and more stable than dimeric G6PD (Ranzani & Cordeiro, 2017; Garcia et al., 2022). The crystallized human G6PD is a tetramer consisting of a side-by-side dimer of dimers (Au et al., 2000; Fig. 8), while high-resolution cryo-electron microscopy structures show a mixture of dimers and tetramers, with an overall structure like that in the crystal (Wei et al., 2022).
![[Figure 8]](va5059fig8thm.gif)
57 amino-acid residues are involved in the dimer interface of human G6PD, while there are 48 in the LM enzyme, although in both approximatively half are hydrophobic and the gross structure of the interface is conserved. Hydrogen bonds and salt bridges are present, and the two βN strands of the β-sheets are associated in both cases (Fig. 3; Au et al., 2000; Rowland et al., 1994). In the human enzyme dimer, the longer C-terminal tail is flexible unless NADP+s is bound in the positively charged crevice between the β-sheet and the C-terminus (Kotaka et al., 2005). Lys407 is a completely conserved residue in the interface. Close by, Lys403 is an NADP+s-bound conserved residue that is found to be acetylated in immortalized cells and mouse embryonic fibroblasts. This acetylation hinders dimerization, causing enzyme inactivation, while NAD+-dependent SIRT2 can directly reactivate G6PD by deacetylation of Lys403 in response to oxidative stress (Wang et al., 2019). In agreement with this, most of the G6PD variants that cause deficiencies of classes I and II have mutations in the dimer interface region and at the NADP+s binding site, thereby showing decreased activity and stability (Au et al., 2000; Mason et al., 2007; Gomes-Manzo et al., 2017; Luzzatto et al., 2020; Chandran et al., 2024). In certain mutants, recovers on increasing the NADP+ concentration and stability increases when it is present (Beutler et al., 1991; Hernández-Ochoa et al., 2023). There is ongoing active research in finding molecules that activate and also stabilize G6PD mutants by promoting dimer formation (Hwang et al., 2018; Raub et al., 2019). G6PDA− (V68M and N126D), a class III double variant and is also the most common African variant (WHO Working Group, 1989; Nkhoma et al., 2009), has significantly improved activity when the dimers are stabilized (Garcia et al., 2022). Sirtuin 5 can activate G6PD by deglutarylating it, although the residue involved is not known to date (Zhou et al., 2016). Lactylation, specifically of Lys45, was also found to inhibit G6PD, and the oncoprotein E6 of human papilloma virus HPV16 causing cervical cancer can revert it, thus activating the PPP (Meng et al., 2024). Lys403, together with Lys366, was also shown to be the main ubiquitination site of G6PD (Wang et al., 2019).
The residues involved in tetramerization are mostly charged, coming from the junction αi–αj, αj and the βI–βJ and βK–βL loops. They form few salt bridges, thus explaining the sensitivity of the dimer–tetramer equilibrium to pH or (Au et al., 2000; Kotaka et al., 2005). Only a quarter of the surface area (706 Å2 from the two dimers) is buried compared with the monomer surface covered in dimer formation (Fig. 8; Au et al., 2000; Kotaka et al., 2005). Nevertheless, a spatial scan statistic obtained by analysing pathogenic and benign G6PD variants suggested that disruption of tetramerization decreases the and is frequently found in class II variants (Cunningham & Mochly Rosen, 2017). Also, several class I mutants and the K403Q mutant were shown to be dysfunctional dimers that are less prone to dissociation and are unable to form tetramers (Garcia et al., 2022). A change in the NADPs binding site like that in the K403Q mutant (PDB entry 7sei) caused allosteric modification in the substrate-binding site, with helix αf moving in a way that hinders G6P binding (Garcia et al., 2022). At the same time, mutations at the dimer interface (F216L) or the tetramer interface (E274K and K275N) induce an allosteric loss of NADPs binding and a dysfunctional dimer. In both cases tetramerization is missing (Garcia et al., 2022). Induced tetramerization by the introduction of cysteines and disulfide bonds increases the activity and stability of both the WT and mutants such as G6PDCanton and G6PDMed (S188F) (Garcia et al., 2022). The latter is a class II variant that is most prevalent in the Middle East and is highly prevalent in India (Mason et al., 2007; Sukumar et al., 2004).
Horikoshi and coworkers have solved structures of class I mutants: V394L (a residue in the β–α interaction site), F381L (a residue in the dimer interface), P396L and R393H (residues in the NADPs site) (Fig. 4). These have impaired NADP+ binding and are not able to dissociate into monomers or to form tetramers (Garcia et al., 2022). In these structures the βM–βN strands are disordered, causing a shift of the αf helix into the G6P-binding site and its occlusion by the αf′ helix (Fig. 7), highlighting the long-distance communication between the NADPs and the G6P binding sites (Horikoshi et al., 2021).
In cancer cells, of Thr406 and Thr466 by Polo-like kinase 1 in the regulating cell cycle has been shown to affect G6PD dimerization and activity (Ma et al., 2017), while protein kinase A inhibits G6PD by serine (Xu et al., 2005). Conversely, of Ser40 (Fig. 5, right) by NF-κB-inducing kinase in CD8+ effector T cells activates the enzyme, the glycolytic enzyme hexokinase 2 and antitumor immunity (Gu et al., 2021).
3. Reported ligands of G6PD
As well as the detailed protein assembly described thus far, it is interesting to explore the ways in which G6PD can bind to each of the large number of proteins that have so far been reported to associate with it (Malhotra et al., 2021; He et al., 2022). Enzymes of the PPP, including G6PD, can form a supramolecular complex that can increase the shunt efficiency by substrate channelling (Huang et al., 2005). Furthermore, growth factors stimulate G6PD such as VEGF activating the enzyme by Tyr428 and Tyr507 Akt which allows translocation of G6PD to the cell membrane (Pan et al., 2009). Acetylation and other post-translational modifications have also been reported in the previous sections. Also, under glucose deprivation glutathione S-transferase P1 (GSTP1) has been shown to S-glutathionylate G6PD in MCF7 cells, while this modification is reduced in the presence of lactic supporting an increase in both G6PD activity and cell proliferation (Sun et al., 2023). Moreover, GSTP1 has been proposed as a lactic acid sensor since this metabolite would hamper the formation of a tripartite complex of GSTP1, G6PD and Src (Fig. 9; Sun et al., 2023).
![[Figure 9]](va5059fig9thm.gif) | Figure 9 A modelled spatial arrangement of the GSTP1–SRC–G6PD complex. Green, GSTP1 (PDB entry 3gus); blue, SRC (PDB entry 2h8h); orange, G6PD (PDB entry 2bhl) [reproduced from Sun et al. (2023 ) according to https://creativecommons.org/licenses/by/4.0/]. |
Giving the low basal activity and the high cytosolic NADPH/NADP+ ratio, G6PD binding to activating proteins was suggested a long time ago (Eggleston & Krebs, 1974; Barcia-Vieitez & Ramos-Martinez, 2014). Binding to Hsp27 activates G6PD (Cosentino et al., 2011), and SUMOylation has also been shown to stabilize G6PD after its deacetylation by SIRT2 (Ni et al., 2021). Conversely, HSPA8/HSC70 binds to G6PD, eliminating it by a chaperone-mediated autophagy process (Deng et al., 2023), while monomeric cytoplasmic p53 binds to G6PD and prevents the formation of an active dimer (Jiang et al., 2011). The enzyme hepatic aldolase B (AldoB) potentiates p53 inhibition through an AldoB–G6PD–p53 protein complex (Li et al., 2020). The interaction of Leishmania donovani G6PD with trypanothione reductase has been shown to potentiate the peculiar and fundamental parasite thiol antioxidant machinery (Ghosh et al., 2017). Finally, G6PD has been shown to support metastasis by upregulating the redox-balance capacity, since its active dimer and tetramer can bind and activate NAD kinase I (Zhang et al., 2021, 2022).
In Fig. 10 only a few of the compounds that inhibit the enzyme are shown. The steroid dehydroepiandrosterone (DHEA) and its derivatives are strong uncompetitive inhibitors of mammalian and parasite G6PD with respect to both substrate and coenzyme, indicating that they only bind to the ternary complex (Marks & Banks, 1960; Gordon et al., 1995). Molecular docking has predicted binding in the active site (Zhao et al., 2014). Quinazolinone and thienopyrimidine derivatives have been shown to compete with steroid inhibitors and some were selective for the Trypanosoma cruzi enzyme compared with the human enzyme (Mercaldi et al., 2014).
![[Figure 10]](va5059fig10thm.gif) | Figure 10 Representative G6PD inhibitors. |
Wedeolactone, a noncompetitive reversible G6PD inhibitor with a Kd of 3.6 µM, was found by high-throughput screening (Luo et al., 2021). Polydatin is another natural compound (resveratrol glucoside) that has been shown to inhibit G6PD (Mele et al., 2018). Using in silico molecular docking, phytol and methotrexate have been predicted to have a good binding affinity (Thakor et al., 2017), while G6PDi-1, which was developed to inhibit G6PD in immune cells, showed a half-maximal G6PD inhibition of 100 nM in astrocyte cultures (Watermann et al., 2023).
4. G6PD in trypanosomatids
G6PD is considered to be a target to kill these parasites since it is highly important in defending them from the immune system of the host (Opperdoes & Michels, 2001; Gupta et al., 2011; Kovářová & Barrett, 2016). Compared with the human enzyme it possesses an additional N-terminal domain. This domain also differs among the three species T. cruzi, T. brucei and Leishmania (Igoillo-Esteve & Cazzulo, 2006; Gupta et al., 2011).
No structural NADP+ has been reported nor a dimer–tetramer equilibrium, even in the presence of ligands or Mg2+ or by varying the in either T. cruzi and L. donovani, and in the latter even over the pH range 4.8–7.8 (Ortíz et al., 2019; Berneburg, Rahlfs et al., 2022). The T. cruzi G6PD tetramer is particularly stable, having more salt bridges than the human tetramer at the dimer–dimer interface. PDB entries 6d23 and 6d24 are for the free enzyme and the G6P complex of a truncated form lacking the first 37 amino acids, respectively (Ortíz et al., 2019). Arg323 is critical, forming two salt bridges per subunit with Asp332 and Glu333, and also stabilizing the active enzyme conformation, being in a loop just after the structural elements of the active site (Ortíz et al., 2019). Only in the N-terminal domain of the T. cruzi G6PD are there two disulfide bonds joining subunits in the dimer. These are involved in the redox regulation of the enzyme, which is inactivated by reducing agents, similarly to cyanobacterial and chloroplast G6PDs (Wenderoth et al., 1997; Wendt et al., 1999; Igoillo-Esteve & Cazzulo, 2006; Gupta et al., 2011; Ortíz et al., 2019).
The structure of the ternary complex of a truncated T. cruzi G6PD, lacking 57 N-terminal and ten C-terminal residues (TcΔG6PDH), with G6P and NADPH (PDB entry 5aq1) revealed the presence of a cavity near the catalytic NADPH binding site which is not present in the human enzyme. This difference, which is exploitable in the search for parasite-specific inhibitors, arises because in T. cruzi G6PD a phenylalanine (Phe191) replaces a tyrosine (Tyr147) in human G6PD, allowing a completely different orientation far from the coenzyme binding site (Fig. 11; Mercaldi et al., 2016).
![[Figure 11]](va5059fig11thm.gif) | Figure 11 Comparison of TcΔG6PDH (a) and human G6PG (HsG6PDH) (b) shows the entry of a side cavity in the parasite enzyme, close to the nicotinamide riboside, that is not present in the human enzyme. (c) Stereo diagram showing a section of the cavity observed in the Rossmann-like domain of TcΔG6PDH. Residues defining the surface of this cavity are mainly hydrophobic. Probes (green) were added using KVFINDER and were used to estimate a volume of about 220 Å3 (PDB entry 5aq1) [reproduced from Mercaldi et al. (2016 ) with the permission of John Wiley & Sons]. |
While the T. cruzi and T. brucei G6PDs are inhibited by DHEA and epiandrosterone (EA), the Leishmania enzyme is insensitive to them (Cordeiro et al., 2009; Cordeiro & Thiemann, 2010; Gupta et al., 2011). In fact, the tetramer of L. donovani G6PD (PDB entry 7zht) is completely different, since the N-terminal domains form the dimer–dimer interface (face-to-face orientation; Fig. 12), whereas the other crystallized G6PDs, including those from human, LM and T. cruzi, present a back-to-back orientation of the two dimers (Berneburg, Rahlfs et al., 2022; Fig. 8). That is, in Leishmania G6PD the two helical N-terminal domains are revealed to be essential for tetramerization. Also, this domain rotates after substrate binding, leading to both a decreased angle between the `Rossmann-like' domain and the β+α domain and to an important shift of the active-site residues (PDB entry 7zhv; Berneburg, Rahlfs et al., 2022).
![[Figure 12]](va5059fig12thm.gif) | Figure 12 The L. donovani G6PD tetramer [PDB entry 7zht; reproduced from Berneburg, Ralhfs et al. (2022 ) according to https://creativecommons.org/licenses/by/4.0/]. |
5. Bifunctional G6PDs
In vertebrates, the microsomial H6PD is believed to have been derived from duplication of G6PD through sequence divergence and gene fusion (Stover et al., 2011). In fact, it is a bifunctional enzyme with 6PGL activity in the C-terminus that has been shown to be important in providing NADPH in the endoplasmic reticulum (ER), which is necessary for glucocorticoid reduction and neutralization of oxidative stress, and the fine tuning of the supply of which seems to be involved in control of ER activity (Marcolongo et al., 2011; Gansemer & Rutkowski, 2022). High-resolution cryo-EM recently showed a dimeric structure (Fig. 13; PDB entry 8em2) with an extended flexible loop from the β-domain of each subunit protruding into the β-domain of the other subunit (Su et al., 2022). The subunit encompasses an αβ domain with six α-helices and six β-strands, an all-α-helical domain with six helices and an all-β-stranded domain with 12 β-strands (Su et al., 2022).
![[Figure 13]](va5059fig13thm.gif) | Figure 13 The dimeric hexose-6-phosphate dehydrogenase (H6PD) model (Su et al., 2022 ; PDB entry 8em2). |
The other fusions between G6PD and 6PGL that occur are in the microorganisms Giardia lamblia, Trichomonas vaginalis and Plasmodium (Morales-Luna et al., 2024), although 6PGL is at the N-terminus in Plasmodium (Clarke et al., 2001, 2003). For G. lamblia and T. vaginalis it was shown that there is a high-affinity site for NADP+s, as in human G6PD (Morales-Luna et al., 2024). This site is between the C-terminal part of the G6PD subdomain and the 6PGL module, and coenzyme binding there increases the stability of the enzyme, especially the 6PGL part (Morales-Luna et al., 2024). The 6PGL module has a similar structure to other 6PGLs, with many helices, mainly of the α-type, surrounding several β-sheets (Delarue et al., 2007). Research to find selective inhibitors of parasite bifunctional G6PDs, compared with mammal G6PD, is very active, and potential antimalarial drugs with nanomolar activity have been developed (Allen et al., 2015; Berneburg, Peddibhotla et al., 2022).
6. Bacterial G6PD and FGD
G6PD is an ubiquitous enzyme. The solved structures in this category, including the L. mesenteroides and Mycobacterium avium G6PDs, show a similar overall structure to the other two-domain G6PDs (Rowland et al., 1994; Vu et al., 2021). FGD was first identified in Mycobacterium in 1996, using the oxidized form of F420 to convert G6P to 6-phosphogluconolactone, thus producing the reduced F420H2 (Fig. 14). There was no significant homology to NADP-dependent G6PD (Purwantini & Daniels, 1998). FGD belongs to an F420-dependent enzyme within the luciferase-like hydride transferase family (Nguyen et al., 2017). F420 is a hydride-transfer cofactor that is widely distributed in archaea and in actinomycetes and is found in cyanobacteria (Purwantini & Daniels, 1998; Mehta et al., 2015). The nitrogen in the F420 central ring provides a D-ribitylphospho-L-lactate elongated with several glutamate residues (from two to seven, depending on the organism; Fig. 14; Ney et al., 2017). The of F420 is lower than that of NAD(P)+; thus, it is useful to reduce NADP+ itself and other compounds. It is essential for protecting mycobacteria against a hostile environment such as anaerobic conditions, oxidative or nitrosative stress or the presence of antimicrobial compounds, and hence is critical in the development of resistance (Purwantini & Daniels, 1996; Jacobson & Walsh, 1984; Hasan et al., 2010; Jirapanjawat et al., 2016; Nguyen et al., 2017). While F420 is involved in the oxidation of energy sources such as H2 and formate in archaea, in Streptomyces spp. F420 participates in the biosynthesis of antibiotics (Ney et al., 2017; Wang et al., 2013). The cofactor also participates in the biodegradation of picrate and aflatoxins (Heiss et al., 2002; Taylor et al., 2010). Rhodococcus jostii FGD1 produced in an M. smegmatis culture has been proposed to possibly be useful for potential biotechnological applications of F420 (Fig. 15; Nguyen et al., 2017).
![[Figure 14]](va5059fig14thm.gif) | Figure 14 Proposed mechanism of the reaction catalyzed by FGD. An unknown base (:B) takes a proton from G6P, allowing hydride transfer to the F420 cofactor and oxidation of glucose 6-phosphate to 6-phosphogluconolactone; Glu109 then releases a proton to form F420H2 (for details, see Oyugi et al., 2016 ). |
![[Figure 15]](va5059fig15thm.gif) | Figure 15 Ribbon diagram of the Rhodococcus jostii FGD1 dimer [PDB entry 5lxe; reproduced from Nguyen et al. (2017 ) according to https://creativecommons.org/licenses/by/4.0/]. |
The first crystallized FGD was from M. tuberculosis: a 78 kDa homodimer with one F420 molecule per subunit (Bashiri et al., 2008). The monomer is depicted in Fig. 16(a), based on PDB entry 3b4y, and shows the complex with F420 and the substrate-competitive inhibitor citrate. The monomer is comprised of an (α/β)8 TIM barrel with the enzyme active site at the C-terminal end (Bashiri et al., 2008). The isoalloxazine ring of F420 is tightly enclosed in a bulge of strand β3 containing a nonprolyl cis-peptide bond between a serine and a valine. It has a butterfly bend of 162° that has been proposed to make the hydride-acceptor C5 atom of isoalloxazine more reactive (Bashiri et al., 2008; Oyugi et al., 2018). Mechanistic studies suggest that F420 binds before G6P, with this then stacking against the F420 ring, with the phosphate in a pocket formed by two lysines and an arginine (Fig. 16b; Bashiri et al., 2008). Glu109 is the active-site acid protonating the cofactor at position N1, while the base which abstracts hydrogen from the C1-OH of G6P, thus facilitating hydride transfer from C1 of G6P to C5 of F420, does not seem to be His40 (Figs. 14 and 16b; Oyugi et al., 2016). This histidine, together with Glu109, forms a pocket binding the F420 pyrimidine ring. Trp44 is essential to stabilize enzyme-reaction intermediates and to anchor F420, forming a hydrophobic wall of the pocket (Fig. 16b; Oyugi et al., 2016; Bashiri et al., 2008).
![[Figure 16]](va5059fig16thm.gif) | Figure 16 (a) Ribbon diagram of the M. tubercolosis FGD1 monomer with bound F420 and a citrate (red) in ball-and-stick representation (PDB entry 3b4y). IS are insertion sequences in the core barrel that are proposed to cap the active site. (b) View of the active site; water molecules are shown as red spheres (reproduced from Bashiri et al., 2008 ). |
7. Conclusions
Complementary to the X-ray crystallographic and electron cryo-microscopy structures, there have been a diversity of biochemical studies, including those on reaction mechanisms, regulation, inhibitors, natural and artificial mutants, and post-translational modifications. There is also a strong medical interest in this G6PD family. Research on the enzyme is aiming at finding a cure for parasitic diseases such as malaria and trypanosomiasis. In addition, there is research on the role of G6PD in cancer cell metabolism. On the other hand, in the wider family, FGD is considered as a target against M. tuberculosis and possesses a high biotechnological potential in biocatalysis and bioremediation. Overall, we hope that this topical review will stimulate a continuing suite of studies on the G6PD family and the wider family of enzymes. In addition, we hope that it will inform all researchers involved of the wide range of research going on and provide them with a snapshot of the 3D structures of the various types of G6PDs in the PDB.
Supporting information
Video interview with John Helliwell. DOI: https://doi.org/10.1107/S2053230X24008112/va5059sup1.mp4
Acknowledgements
We are grateful to Dr Tom Burnley of CCP-EM, Science and Technology Facilities Council, Research Complex at Harwell, United Kingdom for discussions on validation checking of electron cryo-microscopy protein structures. Open access publishing facilitated by Universita degli Studi di Ferrara, as part of the Wiley – CRUI-CARE agreement.
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