Glycerol 3-Phosphate Dehydrogenase: Role of the Protein Conformational Change in Activation of a Readily Reversible Enzyme-Catalyzed Hydride Transfer Reaction.

Kinetic parameters are reported for glycerol 3-phosphate dehydrogenase (GPDH)-catalyzed hydride transfer from the whole substrate glycerol 3-phosphate (G3P) or truncated substrate ethylene glycol (EtG) to NAD, and for activation of the hydride transfer reaction of EtG by phosphite dianion. These kinetic parameters were combined with parameters for enzyme-catalyzed hydride transfer in the microscopic reverse direction to give the reaction equilibrium constants Keq. Hydride transfer from G3P is favored in comparison to EtG because the carbonyl product of the former reaction is stabilized by hyperconjugative electron donation from the -CH2R keto substituent. The kinetic data show that the phosphite dianion provides the same 7.6 ± 0.1 kcal/mol stabilization of the transition states for enzyme-catalyzed reactions in the forward [reduction of NAD by EtG] and reverse [oxidation of NADH by glycolaldehyde] directions. The experimental evidence that supports a role for phosphite dianion in stabilizing the active closed form of the GPDH (EC) relative to the ca. 6 kcal/mol more unstable open form (EO) is summarized.

Immp2l Enhances the Structure and Function of Mitochondrial Gpd2 Dehydrogenase.

'Inner mitochondrial membrane peptidase 2 like' (IMMP2L) is a nuclear-encoded mitochondrial peptidase that has been conserved through evolutionary history, as has its target enzyme, 'mitochondrial glycerol phosphate dehydrogenase 2' (GPD2). IMMP2L is known to cleave the mitochondrial transit peptide from GPD2 and another nuclear-encoded mitochondrial respiratory-related protein, cytochrome C1 (CYC1). However, it is not known whether IMMP2L peptidase activates or alters the activity or respiratory-related functions of GPD2 or CYC1. Previous investigations found compelling evidence of behavioural change in the Immp2lKD-/- KO mouse, and in this study, EchoMRI analysis found that the organs of the Immp2lKD-/- KO mouse were smaller and that the KO mouse had significantly less lean mass and overall body weight compared with wildtype littermates (p < 0.05). Moreover, all organs analysed from the Immp2lKD-/- KO had lower relative levels of mitochondrial reactive oxygen species (mitoROS). The kidneys of the Immp2lKD-/- KO mouse displayed the greatest decrease in mitoROS levels that were over 50% less compared with wildtype litter mates. Mitochondrial respiration was also lowest in the kidney of the Immp2lKD-/- KO mouse compared with other tissues when using succinate as the respiratory substrate, whereas respiration was similar to the wildtype when glutamate was used as the substrate. When glycerol-3-phosphate (G3P) was used as the substrate for Gpd2, we observed ~20% and ~7% respective decreases in respiration in female and male Immp2lKD-/- KO mice over time. Together, these findings indicate that the respiratory-related functions of mGpd2 and Cyc1 have been compromised to different degrees in different tissues and genders of the Immp2lKD-/- KO mouse. Structural analyses using AlphaFold2-Multimer further predicted that the interaction between Cyc1 and mitochondrial-encoded cytochrome b (Cyb) in Complex III had been altered, as had the homodimeric structure of the mGpd2 enzyme within the inner mitochondrial membrane of the Immp2lKD-/- KO mouse. mGpd2 functions as an integral component of the glycerol phosphate shuttle (GPS), which positively regulates both mitochondrial respiration and glycolysis. Interestingly, we found that nonmitochondrial respiration (NMR) was also dramatically lowered in the Immp2lKD-/- KO mouse. Primary mouse embryonic fibroblast (MEF) cell lines derived from the Immp2lKD-/- KO mouse displayed a ~27% decrease in total respiration, comprising a ~50% decrease in NMR and a ~12% decrease in total mitochondrial respiration, where the latter was consistent with the cumulative decreases in substrate-specific mediated mitochondrial respiration reported here. This study is the first to report the role of Immp2l in enhancing Gpd2 structure and function, mitochondrial respiration, nonmitochondrial respiration, organ size and homeostasis.

[Effect of calcium-independent phospholipase A2 on the expression of glycerol 3-phosphate dehydrogenase in human non-alcoholic fatty liver disease cells].

Objective: To explore the effect of calcium-independent phospholipase A2 (iPLA2) on the expression of mitochondrial glycerol 3-phosphate dehydrogenase (mGPDH) in human non-alcoholic fatty liver disease cells. Methods: Oleic acid was used to construct a non-alcoholic fatty liver disease cell model by inducing lipid deposition in THLE-2 cells in vitro. Simultaneously, intracellular triglyceride content, iPLA2 expression levels, and mGPDH levels were determined at various induction times (0, 24, 48, and 72 h) using a triglyceride assay kit, quantitative RT-PCR, and western blotting. The model cells were treated with bromelenol lactone, an iPLA2 inhibitor, and N-acetylcysteine, a ROS inhibitor, respectively. Following continuous culture for 24 and 48 hours, the cells were harvested, and the mRNA and protein expression levels of mGPDH were measured. Statistical analysis was performed using the t-test, one-way analysis of variance, and linear correlation. Results: The intracellular triglyceride content gradually increased (P < 0.01), the mGPDH mRNA and protein expression decreased (P < 0.01), and the iPLA2 mRNA and protein expression increased (P < 0.01) in THLE-2 cells with the prolonging time effect of oleic acid therapy. In addition, the mGPDH mRNA expression level was negatively correlated with the iPLA2 mRNA level (r = -0.878, P = 0.002). The expression levels of mGPDH mRNA and protein in the iPLA2 inhibitor group and ROS inhibitor group were increased compared with the model control group (P < 0.01). The expression of mGPDH mRNA was increased at 24 h compared with 48 h in the iPLA2 inhibitor group (P < 0.01). The expression of mGPDH mRNA was gradually increased in the ROS inhibitor group with the prolongation of inhibitor action time (P < 0.01). Compared with the two inhibitor groups, the increase in mGPDH mRNA was significantly higher in the ROS inhibitor group than that in the iPLA2 inhibitor group, and the difference was statistically significant (P < 0.01). Conclusion: iPLA2 can inhibit the expression of mGPDH in non-alcoholic fatty liver cells to a certain extent.

Purification and characterization of human glycerol 3-phosphate dehydrogenases (mitochondrial and cytosolic) by NAD+/NADH redox method.

Glycerol 3-phosphate (G3P) shuttle is composed of mGPDH and cGPDH and serves as the interface between carbohydrate- and lipid-metabolism. Recently, these metabolic enzymes have been implicated in type II diabetes mellitus but the detailed kinetic parameters and crystal structure of human mGPDH is unknown, though fewer studies on cGPDH are available. To characterize these enzymes, the human mGPDH and cGPDH genes were optimized and cloned into the pET-SUMO vector and pET-24a(+) vector, respectively, and over-expressed in Escherichia coli BL21 (DE3). However, SUMO-mGPDH was expressed as inclusion bodies. Hence, various culture parameters, solubilizing agents and expression vectors were used to solubilize the protein but they did not produce functional SUMO-mGPDH. Over-expression of SUMO-mGPDH along with molecular chaperone (pG-KJE8) produced a functional SUMO-mGPDH. The functional SUMO-mGPDH was purified and characterized using NAD+/NADH redox method. cGPDH was also over-expressed and purified for its characterization. DLS analysis and CD spectra of the purified proteins were performed. The mGPDH was a monomeric enzyme with MW of ∼74 kDa and displayed optimal activity in the Tris-HCl buffer (pH 7.4); while, cGPDH was a homodimer with a monomeric MW of ∼37 kDa and showed optimal activity in imidazole buffer (pH 8.0). The Kmapp was 0.475 mM for G3P, and 0.734 mM for DHAP. These methods may be used to characterize these enzymes to understand their role in metabolic disorders.

GPD1L inhibits renal cell carcinoma progression by regulating PINK1/Parkin-mediated mitophagy.

Few approaches have been conducted in the treatment of renal cell carcinoma (RCC) after nephrectomy, resulting in a high mortality rate in urological tumours. Mitophagy is a mechanism of mitochondrial quality control that enables selective degradation of damaged and unnecessary mitochondria. Previous studies have found that glycerol-3-phosphate dehydrogenase 1-like (GPD1L) is associated with the progression of tumours such as lung cancer, colorectal cancer and oropharyngeal cancer, but the potential mechanism in RCC is still unclear. In this study, microarrays from tumour databases were analysed. The expression of GPD1L was confirmed by RT-qPCR and western blotting. The effect and mechanism of GPD1L were explored using cell counting kit 8, wound healing, invasion, flow cytometry and mitophagy-related experiments. The role of GPD1L was further confirmed in vivo. The results showed that GPD1L expression was downregulated and positively correlated with prognosis in RCC. Functional experiments revealed that GPD1L prevented proliferation, migration and invasion while promoting apoptosis and mitochondrial injury in vitro. The mechanistic results indicated that GPD1L interacted with PINK1, promoting PINK1/Parkin-mediated mitophagy. However, inhibition of PINK1 reversed GPD1L-mediated mitochondrial injury and mitophagy. Moreover, GPD1L prevented tumour growth and promoted mitophagy by activating the PINK1/Parkin pathway in vivo. Our study shows that GPD1L has a positive correlation with the prognosis of RCC. The potential mechanism involves interacting with PINK1 and regulating the PINK1/Parkin pathway. In conclusion, these results reveal that GPD1L can act as a biomarker and target for RCC diagnosis and therapy.

Mitochondrial Glycerol-3-Phosphate Dehydrogenase Restricts HBV Replication via the TRIM28-Mediated Degradation of HBx.

Hepatitis B virus (HBV) infection affects hepatic metabolism. Serum metabolomics studies have suggested that HBV possibly hijacks the glycerol-3-phosphate (G3P) shuttle. In this study, the two glycerol-3-phosphate dehydrogenases (GPD1 and GPD2) in the G3P shuttle were analyzed for determining their role in HBV replication and the findings revealed that GPD2 and not GPD1 inhibited HBV replication. The knockdown of GPD2 expression upregulated HBV replication, while GPD2 overexpression reduced HBV replication. Moreover, the overexpression of GPD2 significantly reduced HBV replication in hydrodynamic injection-based mouse models. Mechanistically, this inhibitory effect is related to the GPD2-mediated degradation of HBx protein by recruiting the E3 ubiquitin ligase TRIM28 and not to the alterations in G3P metabolism. In conclusion, this study revealed GPD2, a key enzyme in the G3P shuttle, as a host restriction factor in HBV replication. IMPORTANCE The glycerol-3-phosphate (G3P) shuttle is important for the delivery of cytosolic reducing equivalents into mitochondria for oxidative phosphorylation. The study analyzed two key components of the G3P shuttle and identified GPD2 as a restriction factor in HBV replication. The findings revealed a novel mechanism of GPD2-mediated inhibition of HBV replication via the recruitment of TRIM28 for degrading HBx, and the HBx-GPD2 interaction could be another potential therapeutic target for anti-HBV drug development.

A very rare cause of hypertrygliseridemia in infancy: a novel mutation in glycerol-3-phosphate dehydrogenase 1 (GPD1) gene.

OBJECTIVES: Transient infantile hypertriglyceridemia (HTGTI) is caused by mutations in the glycerol-3-phosphate dehydrogenase 1 (GPD1) gene. HTGTI is characterized by hypertriglyceridemia, hepatomegaly, hepatic steatosis and fibrosis in infancy. Here, we reported first Turkish HTGTI patient with a novel mutation of GPD1, having hypertriglyceridemia, hepatomegaly, growth retardation and hepatic steatosis. He is the first case who needs transfusion until 6th month in GPD1. CASE PRESENTATION: A 2-month-27-day-old boy, who had growth retardation, hepatomegaly and anemia suffered to our hospital with vomiting. Triglyceride level was 1603 mg/dL (n<150). Liver transaminases were elevated and hepatic steatosis was developed. He needed transfusion with erythrocyte suspension until 6th month. Etiology could not be elucidated by clinical and biochemical parameters. A novel homozygous c.936_940del (p.His312GlnfsTer24) variant was detected in the GPD1 gene by Clinical Exome Analysis. CONCLUSIONS: GPD1 deficiency should be investigated in the presence of unexplained hypertriglyceridemia and hepatic steatosis in children especially in infants.

Uncoupled glycerol-3-phosphate shuttle in kidney cancer reveals that cytosolic GPD is essential to support lipid synthesis.

The glycerol-3-phosphate shuttle (G3PS) is a major NADH shuttle that regenerates reducing equivalents in the cytosol and produces energy in the mitochondria. Here, we demonstrate that G3PS is uncoupled in kidney cancer cells where the cytosolic reaction is ∼4.5 times faster than the mitochondrial reaction. The high flux through cytosolic glycerol-3-phosphate dehydrogenase (GPD) is required to maintain redox balance and support lipid synthesis. Interestingly, inhibition of G3PS by knocking down mitochondrial GPD (GPD2) has no effect on mitochondrial respiration. Instead, loss of GPD2 upregulates cytosolic GPD on a transcriptional level and promotes cancer cell proliferation by increasing glycerol-3-phosphate supply. The proliferative advantage of GPD2 knockdown tumor can be abolished by pharmacologic inhibition of lipid synthesis. Taken together, our results suggest that G3PS is not required to run as an intact NADH shuttle but is instead truncated to support complex lipid synthesis in kidney cancer.

Species-specific effects of the introduction of Aspergillus nidulans gfdB in osmophilic aspergilli.

Industrial fungi need a strong environmental stress tolerance to ensure acceptable efficiency and yields. Previous studies shed light on the important role that Aspergillus nidulans gfdB, putatively encoding a NAD+-dependent glycerol-3-phosphate dehydrogenase, plays in the oxidative and cell wall integrity stress tolerance of this filamentous fungus model organism. The insertion of A. nidulans gfdB into the genome of Aspergillus glaucus strengthened the environmental stress tolerance of this xerophilic/osmophilic fungus, which may facilitate the involvement of this fungus in various industrial and environmental biotechnological processes. On the other hand, the transfer of A. nidulans gfdB to Aspergillus wentii, another promising industrial xerophilic/osmophilic fungus, resulted only in minor and sporadic improvement in environmental stress tolerance and meanwhile partially reversed osmophily. Because A. glaucus and A. wentii are phylogenetically closely related species and both fungi lack a gfdB ortholog, these results warn us that any disturbance of the stress response system of the aspergilli may elicit rather complex and even unforeseeable, species-specific physiological changes. This should be taken into consideration in any future targeted industrial strain development projects aiming at the fortification of the general stress tolerance of these fungi. KEY POINTS: • A. wentii c' gfdB strains showed minor and sporadic stress tolerance phenotypes. • The osmophily of A. wentii significantly decreased in the c' gfdB strains. • Insertion of gfdB caused species-specific phenotypes in A. wentii and A. glaucus.

Production of glycerate from glucose using engineered Escherichiacoli.

In this study, glycerate was produced from glucose using engineered Escherichia coli BW25113. Plasmid pSR3 carrying gpd1 and gpp2 encoding two isoforms of glycerol-3-phosphate dehydrogenase from Saccharomyces cerevisiae and plasmid pLB2 carrying aldO encoding alditol oxidase from Streptomyces violaceoruber were introduced into E. coli to enable the production of glycerate from glucose via glycerol. Disruptions of garK and glxK genes in the E. coli genome were performed to minimize the consumption of glycerate produced. As a result, E. coli carrying these plasmids could produce nearly three times higher concentration of glycerate (0.50 ± 0.01 g/L) from 10 g/L glucose compared to E. coli EG_2 (0.14 ± 0.02 g/L). In M9 medium, disruption of garK and glxK resulted in an impaired growth rate with low production of glycerate, while supplementation of 0.5 g/L casamino acids and 0.5 g/L manganese sulfate to the medium replenished the growth rate and elevated the glycerate titer. Further disruption of glpF, encoding a glycerol transporter, increased the glycerate production to 0.80 ± 0.00 g/L. MR2 medium improved the glycerate production titers and specific productivities of E. coli EG_4, EG_5, and EG_6. Upscale production of glycerate was carried out in a jar fermentor with MR2 medium using E. coli EG_6, resulting in an improvement in glycerate production up to 2.37 ± 0.46 g/L with specific productivity at 0.34 ± 0.11 g-glycerate/g-cells. These results indicate that E. coli is an appropriate host for glycerate production from glucose.

Non-bioenergetic roles of mitochondrial GPD2 promote tumor progression.

Rationale: Despite growing evidence for mitochondria's involvement in cancer, the roles of specific metabolic components outside the respiratory complex have been little explored. We conducted metabolomic studies on mitochondrial DNA (mtDNA)-deficient (ρ0) cancer cells with lower proliferation rates to clarify the undefined roles of mitochondria in cancer growth. Methods and results: Despite extensive metabolic downregulation, ρ0 cells exhibited high glycerol-3-phosphate (G3P) level, due to low activity of mitochondrial glycerol-3-phosphate dehydrogenase (GPD2). Knockout (KO) of GPD2 resulted in cell growth suppression as well as inhibition of tumor progression in vivo. Surprisingly, this was unrelated to the conventional bioenergetic function of GPD2. Instead, multi-omics results suggested major changes in ether lipid metabolism, for which GPD2 provides dihydroxyacetone phosphate (DHAP) in ether lipid biosynthesis. GPD2 KO cells exhibited significantly lower ether lipid level, and their slower growth was rescued by supplementation of a DHAP precursor or ether lipids. Mechanistically, ether lipid metabolism was associated with Akt pathway, and the downregulation of Akt/mTORC1 pathway due to GPD2 KO was rescued by DHAP supplementation. Conclusion: Overall, the GPD2-ether lipid-Akt axis is newly described for the control of cancer growth. DHAP supply, a non-bioenergetic process, may constitute an important role of mitochondria in cancer.

GPD2: The relationship with cancer and neural stemness.

We previously reported that knocking down GPD2 (glycerol-3-phosphate dehydrogenase 2), responsible for the glycerol-phosphate shuttle, causes human hepatocarcinoma-derived HuH-7 cells, lowering the cancer stemness. After examining whether GPD2 expression in the other cell lines could affect their cancer stemness, this study showed that human neuroblastoma-derived SH-SY5Y cells also lower the ability of sphere formation by knocking down GPD2. This suggests that GPD2 relates to the common mechanism for maintaining cancer stem cells, as in the cases like SH-SY5Y and HuH-7 cells. In addition, knocking down GPD2 in SH-SY5Y cells showed a morphological change and increasing tendency of neuronal marker genes, including GAP43, NeuN, and TUBB3, indicating that GPD2 may contribute to not only cancer but also neural stem cell maintenance. After all, GPD2 may play a role in maintaining cancer and neural stemness, although further rigorous studies are essential to conclude this. It is expected that GPD2 will be a novel target gene for cancer therapy, stem cell research, and development.

Exploration of hub genes involved in PCOS using biological informatics methods.

BACKGROUND: The aim of this study was to find underlying genes and their interaction mechanism crucial to the polycystic ovarian syndrome (PCOS) by analyzing differentially expressed genes (DEGs) between PCOS and non-PCOS subjects. METHODS: Gene expression data of PCOS and non-PCOS subjects were collected from gene expression omnibus (GEO) database. GEO2R were used to calculating P value and logFC. The screening threshold of DEGs was P < .05 and | FC | ≥ 1.2. GO annotation and Kyoto encyclopedia of genes and genomes (KEGG) signaling pathway enrichment analysis was performed by using DAVID (2021 Update). The protein-protein interaction (PPI) network of DEGs was constructed by using the STRING database, and the hub genes were recognized through Hubba plugin of Cytoscape software. RESULTS: PCOS and non-PCOS subjects shared a total of 174 DGEs, including 14 upregulated and 160 downregulated genes. The GO biological processes enriched by DEGs mainly involved actin cytoskeleton organization, positive regulation of NF-κB signaling pathway, and positive regulation of canonical Wnt signaling pathway. The DEGs were significantly enriched in cytoplasm, nucleus and cytosol. Their molecular functions mainly focused on protein binding, calmodulin binding and glycerol-3-phosphate dehydrogenase activity. The PI3K/Akt signaling pathway and glycosaminoglycan biosynthesis were highlighted as critical pathways enriched by DEGs. 10 hub genes were screened from the constructed PPI network, of which EGF, FN1 and TLR4 were mainly enriched in the PI3K/Akt signaling pathway. CONCLUSION: In this study, a total of 174 DEGs and 10 hub genes were identified as new candidate targets for insulin resistance (IR) in PCOS individuals, which may provide a new direction for developing novel treatment strategies for PCOS.

Atrial myocyte-derived exosomal microRNA contributes to atrial fibrosis in atrial fibrillation.

BACKGROUND: Atrial fibrosis plays a critical role in the development of atrial fibrillation (AF). Exosomes are a promising cell-free therapeutic approach for the treatment of AF. The purposes of this study were to explore the mechanisms by which exosomes derived from atrial myocytes regulate atrial remodeling and to determine whether their manipulation facilitates the therapeutic modulation of potential fibrotic abnormalities during AF. METHODS: We isolated exosomes from atrial myocytes and patient serum, and microRNA (miRNA) sequencing was used to analyze exosomal miRNAs in exosomes derived from atrial myocytes and patient serum. mRNA sequencing and bioinformatics analyses corroborated the key genes that were direct targets of miR-210-3p. RESULTS: The miRNA sequencing analysis identified that miR-210-3p expression was significantly increased in exosomes from tachypacing atrial myocytes and serum from patients with AF. In vitro, the miR-210-3p inhibitor reversed tachypacing-induced proliferation and collagen synthesis in atrial fibroblasts. Accordingly, miR-210-3p knock out (KO) reduced the incidence of AF and ameliorated atrial fibrosis induced by Ang II. The mRNA sequencing analysis and dual-luciferase reporter assay showed that glycerol-3-phosphate dehydrogenase 1-like (GPD1L) is a potential target gene of miR-210-3p. The functional analysis suggested that GPD1L regulated atrial fibrosis via the PI3K/AKT signaling pathway. In addition, silencing GPD1L in atrial fibroblasts induced cell proliferation, and these effects were reversed by a PI3K inhibitor (LY294002). CONCLUSIONS: Atrial myocyte-derived exosomal miR-210-3p promoted cell proliferation and collagen synthesis by inhibiting GPD1L in atrial fibroblasts. Preventing pathological crosstalk between atrial myocytes and fibroblasts may be a novel target to ameliorate atrial fibrosis in patients with AF.

Angiotensin II induces podocyte metabolic reprogramming from glycolysis to glycerol-3-phosphate biosynthesis.

Recent studies have reported that Angiotensin II (Ang II) contributes to podocyte injury by interfering with metabolism. Glycolysis is essential for podocytes and glycolysis abnormality is associated with glomerular injury in chronic kidney disease (CKD). Glycerol-3-phosphate (G-3-P) biosynthesis is a shunt pathway of glycolysis, in which cytosolic glycerol-3-phosphate dehydrogenase 1 (GPD1) catalyzes dihydroxyacetone phosphate (DHAP) to generate G-3-P in the presence of the NADH. G-3-P is not only a substrate in glycerophospholipids and glyceride synthesis but also can be oxidated by mitochondrial glycerol-3-phosphate dehydrogenase (GPD2) to regenerate DHAP in mitochondria. Since G-3-P biosynthesis links to glycolysis, mitochondrial metabolism and lipid synthesis, we speculate G-3-P biosynthesis abnormality is probably involved in podocyte injury. In this study, we demonstrated that Ang II upregulated GPD1 expression and increased G-3-P and glycerophospholipid syntheses in podocytes. GPD1 knockdown protected podocytes from Ang II-induced lipid accumulation and mitochondrial dysfunction. GPD1 overexpression exacerbated Ang II-induced podocyte injury. In addition, we proved that lipid accumulation and mitochondrial dysfunction were correlated with G-3-P content in podocytes. These results suggest that Ang II upregulates GPD1 and promotes G-3-P biosynthesis in podocytes, which promote lipid accumulation and mitochondrial dysfunction in podocytes.

Enabling Role of Ligand-Driven Conformational Changes in Enzyme Evolution.

Many enzymes that show a large specificity in binding the enzymatic transition state with a higher affinity than the substrate utilize substrate binding energy to drive protein conformational changes to form caged substrate complexes. These protein cages provide strong stabilization of enzymatic transition states. Using part of the substrate binding energy to drive the protein conformational change avoids a similar strong stabilization of the Michaelis complex and irreversible ligand binding. A seminal step in the development of modern enzyme catalysts was the evolution of enzymes that couple substrate binding to a conformational change. These include enzymes that function in glycolysis (triosephosphate isomerase), the biosynthesis of lipids (glycerol phosphate dehydrogenase), the hexose monophosphate shunt (6-phosphogluconate dehydrogenase), and the mevalonate pathway (isopentenyl diphosphate isomerase), catalyze the final step in the biosynthesis of pyrimidine nucleotides (orotidine monophosphate decarboxylase), and regulate the cellular levels of adenine nucleotides (adenylate kinase). The evolution of enzymes that undergo ligand-driven conformational changes to form active protein-substrate cages is proposed to proceed by selection of variants, in which the selected side chain substitutions destabilize a second protein conformer that shows compensating enhanced binding interactions with the substrate. The advantages inherent to enzymes that incorporate a conformational change into the catalytic cycle provide a strong driving force for the evolution of flexible protein folds such as the TIM barrel. The appearance of these folds represented a watershed event in enzyme evolution that enabled the rapid propagation of enzyme activities within enzyme superfamilies.

Mitochondrial GCN5L1 regulates cytosolic redox state and hepatic gluconeogenesis via glycerol phosphate shuttle GPD2.

Hepatic gluconeogenesis is crucial for maintaining blood glucose during starvation, and a major contributor for hyperglycemia. Cellular redox state is related to mitochondrial biology and regulates conversion of specific metabolites to glucose. General control of amino acid synthesis 5 (GCN5) like-1 (GCN5L1) is a mitochondria-enriched protein which modulates glucose and amino acid metabolism. Here we show a new regulatory mode of GCN5L1 on gluconeogenesis using lactate and glycerol. We observed GCN5L1 deletion dramatically inhibited glucose production derived from glycerol and lactate, due to increased cytosolic redox state. The underlying mechanism is that GCN5L1 directly binds to the key component of mitochondrial shuttle glycerol phosphate dehydrogenase 2 (GPD2) and modulates its activity. These results have significant implications for understanding the physiological role and regulatory mechanism of mitochondrial shuttle in diabetes development and provide a novel therapeutic potential for diabetes.

A ferroptosis defense mechanism mediated by glycerol-3-phosphate dehydrogenase 2 in mitochondria.

Mechanisms of defense against ferroptosis (an iron-dependent form of cell death induced by lipid peroxidation) in cellular organelles remain poorly understood, hindering our ability to target ferroptosis in disease treatment. In this study, metabolomic analyses revealed that treatment of cancer cells with glutathione peroxidase 4 (GPX4) inhibitors results in intracellular glycerol-3-phosphate (G3P) depletion. We further showed that supplementation of cancer cells with G3P attenuates ferroptosis induced by GPX4 inhibitors in a G3P dehydrogenase 2 (GPD2)-dependent manner; GPD2 deletion sensitizes cancer cells to GPX4 inhibition-induced mitochondrial lipid peroxidation and ferroptosis, and combined deletion of GPX4 and GPD2 synergistically suppresses tumor growth by inducing ferroptosis in vivo. Mechanistically, inner mitochondrial membrane-localized GPD2 couples G3P oxidation with ubiquinone reduction to ubiquinol, which acts as a radical-trapping antioxidant to suppress ferroptosis in mitochondria. Taken together, these results reveal that GPD2 participates in ferroptosis defense in mitochondria by generating ubiquinol.

Glycerol-3-Phosphate Dehydrogenase: The K120 and K204 Side Chains Define an Oxyanion Hole at the Enzyme Active Site.

The cationic K120 and K204 side chains lie close to the C-2 carbonyl group of substrate dihydroxyacetone phosphate (DHAP) at the active site of glycerol-3-phosphate dehydrogenase (GPDH), and the K120 side chain is also positioned to form a hydrogen bond to the C-1 hydroxyl of DHAP. The kinetic parameters for unactivated and phosphite dianion-activated GPDH-catalyzed reduction of glycolaldehyde and acetaldehyde (AcA) show that the transition state for the former reaction is stabilized by ca 5 kcal/mole by interactions of the C-1 hydroxyl group with the protein catalyst. The K120A and K204A substitutions at wild-type GPDH result in similar decreases in kcat, but Km is only affected by the K120A substitution. These results are consistent with 3 kcal/mol stabilizing interactions between the K120 or K204 side chains and a negative charge at the C-2 oxygen at the transition state for hydride transfer from NADH to DHAP. This stabilization resembles that observed at oxyanion holes for other enzymes. There is no detectable rescue of the K204A variant by ethylammonium cation (EtNH3+), compared with the efficient rescue of the K120A variant. This is consistent with a difference in the accessibility of the variant enzyme active sites to exogenous EtNH3+. The K120A/K204A substitutions cause a (6 × 106)-fold increase in the promiscuity of wild-type hlGPDH for catalysis of the reduction of AcA compared to DHAP. This may reflect conservation of the active site for an ancestral alcohol dehydrogenase, whose relative activity for catalysis of reduction of AcA increases with substitutions that reduce the activity for reduction of the specific substrate DHAP.

The Drosophila melanogaster enzyme glycerol-3-phosphate dehydrogenase 1 is required for oogenesis, embryonic development, and amino acid homeostasis.

As the fruit fly, Drosophila melanogaster, progresses from one life stage to the next, many of the enzymes that compose intermediary metabolism undergo substantial changes in both expression and activity. These predictable shifts in metabolic flux allow the fly meet stage-specific requirements for energy production and biosynthesis. In this regard, the enzyme glycerol-3-phosphate dehydrogenase 1 (GPDH1) has been the focus of biochemical genetics studies for several decades and, as a result, is one of the most well-characterized Drosophila enzymes. Among the findings of these earlier studies is that GPDH1 acts throughout the fly lifecycle to promote mitochondrial energy production and triglyceride accumulation while also serving a key role in maintaining redox balance. Here, we expand upon the known roles of GPDH1 during fly development by examining how depletion of both the maternal and zygotic pools of this enzyme influences development, metabolism, and viability. Our findings not only confirm previous observations that Gpdh1 mutants exhibit defects in larval development, lifespan, and fat storage but also reveal that GPDH1 serves essential roles in oogenesis and embryogenesis. Moreover, metabolomics analysis reveals that a Gpdh1 mutant stock maintained in a homozygous state exhibits larval metabolic defects that significantly differ from those observed in the F1 mutant generation. Overall, our findings highlight unappreciated roles for GPDH1 in early development and uncover previously undescribed metabolic adaptations that could allow flies to survive the loss of this key enzyme.