Catabolism of 2-keto-3-deoxy-galactonate and the production of its enantiomers.

2-Keto-3-deoxy-galactonate (KDGal) serves as a pivotal metabolic intermediate within both the fungal D-galacturonate pathway, which is integral to pectin catabolism, and the bacterial DeLey-Doudoroff pathway for D-galactose catabolism. The presence of KDGal enantiomers, L-KDGal and D-KDGal, varies across these pathways. Fungal pathways generate L-KDGal through the reduction and dehydration of D-galacturonate, whereas bacterial pathways produce D-KDGal through the oxidation and dehydration of D-galactose. Two distinct catabolic routes further metabolize KDGal: a nonphosphorolytic pathway that employs aldolase and a phosphorolytic pathway involving kinase and aldolase. Recent findings have revealed that L-KDGal, identified in the bacterial catabolism of 3,6-anhydro-L-galactose, a major component of red seaweeds, is also catabolized by Escherichia coli, which is traditionally known to be catabolized by specific fungal species, such as Trichoderma reesei. Furthermore, the potential industrial applications of KDGal and its derivatives, such as pyruvate and D- and L-glyceraldehyde, are underscored by their significant biological functions. This review comprehensively outlines the catabolism of L-KDGal and D-KDGal across different biological systems, highlights stereospecific methods for discriminating between enantiomers, and explores industrial application prospects for producing KDGal enantiomers. KEY POINTS: • KDGal is a metabolic intermediate in fungal and bacterial pathways • Stereospecific enzymes can be used to identify the enantiomeric nature of KDGal • KDGal can be used to induce pectin catabolism or produce functional materials.

The Relationship Between GAPDH Gene Polymorphism and Risk of Acute Coronary Syndrome in South Indians with Type 2 Diabetes Mellitus.

As glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is one of the regulators of carbonyl stress, a pathogenic mechanism for diabetic complications like acute coronary syndrome (ACS), the study aimed to investigate the relationship between GAPDH gene polymorphism, GAPDH activity in red blood cell (RBC), methylglyoxal (MG) levels in plasma and ACS risk in South Indians with type 2 diabetes mellitus (T2DM). This study comprised 150 T2DM with ACS as cases and 150 T2DM without ACS as controls. The GAPDH rs1136666, rs1060620 and rs1060619 gene polymorphisms were identified by TaqMan probe assays. The RBC GAPDH activity and plasma MG levels were estimated. Cases had significantly higher plasma MG levels and lower RBC GAPDH activity than controls (P < 0.001). The distribution of rs1060620 or rs1060619 alleles and genotypes significantly differed between groups. The rs1060620 AG (OR 0.55; 95% CI 0.33-0.92; P = 0.022) or rs1060619 CT (OR 0.51; 95% CI 0.31-0.83; P = 0.007) genotype was associated with reduced ACS risk, confirmed in the over-dominant genetic model. Haplotype analyses revealed that the GAT and CGC haplotypes were associated with increased (OR 28.37; 95% CI 3.82-210.49; P = 8.51 × 10-7) and decreased (OR 0.45; 95% CI 0.24-0.86; P = 0.014) ACS risk in T2DM patients, respectively. Lower GAPDH activity was observed in the TT and CT genotypes compared to the CC genotype of rs1060619 (P < 0.001). This work established that the GAPDH rs1060620 or rs1060619 gene polymorphisms are associated with ACS risk in South Indians with T2DM.

Glyceraldehyde metabolism in mouse brain and the entry of blood-borne glyceraldehyde into the brain.

D-Glyceraldehyde, a reactive aldehyde metabolite of fructose and glucose, is neurotoxic in vitro by forming advanced glycation end products (AGEs) with neuronal proteins. In Alzheimer's disease brains, glyceraldehyde-containing AGEs have been detected intracellularly and in extracellular plaques. However, little information exists on how the brain handles D-glyceraldehyde metabolically or if glyceraldehyde crosses the blood-brain barrier from the circulation into the brain. We injected [U-13C]-D-glyceraldehyde intravenously into awake mice and analyzed extracts of serum and brain by 13C nuclear magnetic resonance spectroscopy. 13C-Labeling of brain lactate and glutamate indicated passage of D-glyceraldehyde from blood to brain and glycolytic and oxidative D-glyceraldehyde metabolism in brain cells. 13C-Labeling of serum glucose and lactate through hepatic metabolism of [U-13C]-D-glyceraldehyde could not explain the formation of 13C-labeled lactate and glutamate in the brain. Cerebral glyceraldehyde dehydrogenase and reductase activities, leading to the formation of D-glycerate and glycerol, respectively, were 0.27-0.28 nmol/mg/min; triokinase, which phosphorylates D-glyceraldehyde to D-glyceraldehyde-3-phosphate, has been demonstrated previously at low levels. Thus, D-glyceraldehyde metabolism toward glycolysis could proceed both through D-glycerate, glycerol, and D-glyceraldehyde-3-phosphate. The aldehyde group of D-glyceraldehyde was overwhelmingly hydrated into a diol in aqueous solution, but the diol dehydration rate greatly exceeded glyceraldehyde metabolism and did not restrict it. We conclude that (1) D-glyceraldehyde crosses the blood-brain barrier, and so blood-borne glyceraldehyde could contribute to AGE formation in the brain, (2) glyceraldehyde is taken up and metabolized by brain cells. Metabolism thus constitutes a detoxification mechanism for this reactive aldehyde, a mechanism that may be compromised in disease states.

Glyceraldehyde-3-phosphate dehydrogenase is involved in the pathogenesis of Alzheimer's disease.

One of important characteristics of Alzheimer's disease is a persistent oxidative/nitrosative stress caused by pro-oxidant properties of amyloid-beta peptide (Aß) and chronic inflammation in the brain. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is easily oxidized under oxidative stress. Numerous data indicate that oxidative modifications of GAPDH in vitro and in cell cultures stimulate GAPDH denaturation and aggregation, and the catalytic cysteine residue Cys152 is important for these processes. Both intracellular and extracellular GAPDH aggregates are toxic for the cells. Interaction of denatured GAPDH with soluble Aß results in mixed insoluble aggregates with increased toxicity. The above-described properties of GAPDH (sensitivity to oxidation and propensity to form aggregates, including mixed aggregates with Aß) determine its role in the pathogenesis of Alzheimer's disease.

GAPDH inhibition mediated by thiol oxidation in human airway epithelial cells exposed to an environmental peroxide.

Intracellular redox homeostasis in the airway epithelium is closely regulated through adaptive signaling and metabolic pathways. However, inhalational exposure to xenobiotic stressors such as secondary organic aerosols (SOA) can alter intracellular redox homeostasis. Isoprene hydroxy hydroperoxide (ISOPOOH), a ubiquitous volatile organic compound derived from the atmospheric photooxidation of biogenic isoprene, is a major contributor to SOA. We have previously demonstrated that exposure of human airway epithelial cells (HAEC) to ISOPOOH induces oxidative stress through multiple mechanisms including lipid peroxidation, glutathione oxidation, and alterations of glycolytic metabolism. Using dimedone-based reagents and copper catalyzed azo-alkynyl cycloaddition to tag intracellular protein thiol oxidation, we demonstrate that exposure of HAEC to micromolar levels of ISOPOOH induces reversible oxidation of cysteinyl thiols in multiple intracellular proteins, including GAPDH, that was accompanied by a dose-dependent loss of GAPDH enzymatic activity. These results demonstrate that ISOPOOH induces an oxidative modification of intracellular proteins that results in loss of GAPDH activity, which ultimately impacts the dynamic regulation of the intracellular redox homeostatic landscape in HAEC.

The therapeutic effect of a novel GAPDH inhibitor in mouse model of breast cancer and efficacy monitoring by molecular imaging.

BACKGROUND: Breast cancer is a serious threat to women's health with high morbidity and mortality. The development of more effective therapies for the treatment of breast cancer is strongly warranted. Growing evidence suggests that targeting glucose metabolism may be a promising cancer treatment strategy. We previously identified a new glyceraldehyde-3-phosphate dehydrogenase (GAPDH) inhibitor, DC-5163, which shows great potential in inhibiting tumor growth. Here, we evaluated the anticancer potential of DC-5163 in breast cancer cells. METHODS: The effects of DC-5163 on breast cancer cells were investigated in vitro and in vivo. Seahorse, glucose uptake, lactate production, and cellular ATP content assays were performed to examine the impact of DC-5163 on cellular glycolysis. Cell viability, colony-forming ability, cell cycle, and apoptosis were assessed by CCK8 assay, colony formation assay, flow cytometry, and immunoblotting respectively. The anticancer activity of DC-5163 in vivo was evaluated in a mouse breast cancer xenograft model. RESULTS: DC-5163 suppressed aerobic glycolysis and reduced energy supply of breast cancer cells, thereby inhibiting breast cancer cell growth, inducing cell cycle arrest in the G0/G1 phase, and increasing apoptosis. The therapeutic efficacy was assessed using a breast cancer xenograft mouse model. DC-5163 treatment markedly suppressed tumor growth in vivo without inducing evident systemic toxicity. Micro-PET/CT scans revealed a notable reduction in tumor 18F-FDG and 18F-FLT uptake in the DC-5163 treatment group compared to the DMSO control group. CONCLUSIONS: Our results suggest that DC-5163 is a promising GAPDH inhibitor for suppressing breast cancer growth without obvious side effects. 18F-FDG and 18F-FLT PET/CT can noninvasively assess the levels of glycolysis and proliferation in tumors following treatment with DC-5163.

Evaluation of different glycerol fed-batch strategies in a lab-scale bioreactor for the improved production of a novel engineered ß-fructofuranosidase enzyme in Pichia pastoris.

The ß-fructofuranosidase enzyme from Aspergillus niger has been extensively used to commercially produce fructooligosaccharides from sucrose. In this study, the native and an engineered version of the ß-fructofuranosidase enzyme were expressed in Pichia pastoris under control of the glyceraldehyde-3-phosphate dehydrogenase promoter, and production was evaluated in bioreactors using either dissolved oxygen (DO-stat) or constant feed fed-batch feeding strategies. The DO-stat cultivations produced lower biomass concentrations but this resulted in higher volumetric activity for both strains. The native enzyme produced the highest volumetric enzyme activity for both feeding strategies (20.8% and 13.5% higher than that achieved by the engineered enzyme, for DO-stat and constant feed, respectively). However, the constant feed cultivations produced higher biomass concentrations and higher volumetric productivity for both the native as well as engineered enzymes due to shorter process time requirements (59 h for constant feed and 155 h for DO-stat feed). Despite the DO-stat feeding strategy achieving a higher maximum enzyme activity, the constant feed strategy would be preferred for production of the ß-fructofuranosidase enzyme using glycerol due to the many industrial advantages related to its enhanced volumetric enzyme productivity.

Increasing lipid production in Chlamydomonas reinhardtii through genetic introduction for the overexpression of glyceraldehyde-3-phosphate dehydrogenase.

Microalgae, valued for their sustainability and CO2 fixation capabilities, are emerging as promising sources of biofuels and high-value compounds. This study aimed to boost lipid production in C. reinhardtii by overexpressing chloroplast glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a key enzyme in the Calvin cycle and glycolysis, under the control of a nitrogen-inducible NIT1 promoter, to positively impact overall carbon metabolism. The standout transformant, PNG#7, exhibited significantly increased lipid production under nitrogen starvation, with biomass rising by 44% and 76% on days 4 and 16, respectively. Fatty acid methyl ester (FAME) content in PNG#7 surged by 2.4-fold and 2.1-fold, notably surpassing the wild type (WT) in lipid productivity by 3.4 and 3.7 times on days 4 and 16, respectively. Transcriptome analysis revealed a tenfold increase in transgenic GAPDH expression and significant upregulation of genes involved in fatty acid and triacylglycerol synthesis, especially the gene encoding acyl-carrier protein gene (ACP, Cre13. g577100. t1.2). In contrast, genes related to cellulose synthesis were downregulated. Single Nucleotide Polymorphism (SNP)/Indel analysis indicated substantial DNA modifications, which likely contributed to the observed extensive transcriptomic and phenotypic changes. These findings suggest that overexpressing chloroplast GAPDH, coupled with genetic modifications, effectively enhances lipid synthesis in C. reinhardtii. This study not only underscores the potential of chloroplast GAPDH overexpression in microalgal lipid synthesis but also highlights the expansive potential of metabolic engineering in microalgae for biofuel production.

Availability of individual proteins for quantitative analysis in postmortem brains preserved in two different brain banks.

AIM: Postmortem brain research is necessary for elucidating the pathology of schizophrenia; an increasing number of studies require a combination of suitable tissue samples preserved at multiple brain banks. In this study, we examined whether a comparative study of protein expression levels can be conducted using postmortem brain samples preserved in different facilities. METHODS: We compared the demographic factors of postmortem brain samples preserved in two institutions and measured and compared the expression levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and glial fibrillary acidic protein (GFAP) in the prefrontal cortex and superior temporal gyrus. GAPDH is generally used as a loading control for western blotting, and GFAP is considered as an astrocyte marker in the brain. RESULTS: We found significant differences between the two institutions in postmortem interval, age at death, and preservation time. To reduce the effects of these differences on our measurements, the parameters were set as covariates in our analyses of covariance. Subsequently, no differences in GAPDH and GFAP expression were found between institutions. CONCLUSIONS: When studies are conducted using brain samples preserved in different brain banks, differences in demographic factors should be carefully considered and taken into account by statistical methods to minimize their impact as much as possible. Since there was no significant difference in the protein expression levels of GAPDH and GFAP in either region between the two institutions that preserved the postmortem brains, we concluded that it is possible to perform protein quantitative analysis assuming that there is no effect of difference between two institutions.

Construction of an alternative NADPH regeneration pathway improves ethanol production in Saccharomyces cerevisiae with xylose metabolic pathway.

Full conversion of glucose and xylose from lignocellulosic hydrolysates is required for obtaining a high ethanol yield. However, glucose and xylose share flux in the pentose phosphate pathway (PPP) and glycolysis pathway (EMP), with glucose having a competitive advantage in the shared metabolic pathways. In this work, we knocked down ZWF1 to preclude glucose from entering the PPP. This reduced the [NADPH] level and disturbed growth on both glucose or xylose, confirming that the oxidative PPP, which begins with Zwf1p and ultimately leads to CO2 production, is the primary source of NADPH in both glucose and xylose. Upon glucose depletion, gluconeogenesis is necessary to generate glucose-6-phosphate, the substrate of Zwf1p. We re-established the NADPH regeneration pathway by replacing the endogenous NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene TDH3 with heterogenous NADP + -GAPDH genes GDH, gapB, and GDP1. Among the resulting strains, the strain BZP1 (zwf1Δ, tdh3::GDP1) exhibited a similar xylose consumption rate before glucose depletion, but a 1.6-fold increased xylose consumption rate following glucose depletion compared to the original strain BSGX001, and the ethanol yield for total consumed sugars of BZP1 was 13.5% higher than BSGX001. This suggested that using the EMP instead of PPP to generate NADPH reduces the wasteful metabolic cycle and excess CO2 release from oxidative PPP. Furthermore, we used a copper-repressing promoter to modulate the expression of ZWF1 and optimize the timing of turning off the ZWF1, therefore, to determine the competitive equilibrium between glucose-xylose co-metabolism. This strategy allowed fast growth in the early stage of fermentation and low waste in the following stages of fermentation.

Structures of Toxic Advanced Glycation End-Products Derived from Glyceraldehyde, A Sugar Metabolite.

Advanced glycation end-products (AGEs) have recently been implicated in the onset/progression of lifestyle-related diseases (LSRDs); therefore, the suppression of AGE-induced effects may be used in both the prevention and treatment of these diseases. Various AGEs are produced by different biological pathways in the body. Glyceraldehyde (GA) is an intermediate of glucose and fructose metabolism, and GA-derived AGEs (GA-AGEs), cytotoxic compounds that accumulate and induce damage in mammalian cells, contribute to the onset/progression of LSRDs. The following GA-AGE structures have been detected to date: triosidines, GA-derived pyridinium compounds, GA-derived pyrrolopyridinium lysine dimers, methylglyoxal-derived hydroimidazolone 1, and argpyrimidine. GA-AGEs are a key contributor to the formation of toxic AGEs (TAGE) in many cells. The extracellular leakage of TAGE affects the surrounding cells via interactions with the receptor for AGEs. Elevated serum levels of TAGE, which trigger different types of cell damage, may be used as a novel biomarker for the prevention and early diagnosis of LSRDs as well as in evaluations of treatment efficacy. This review provides an overview of the structures of GA-AGEs.

Metabolic perturbation of Streptomyces albulus by introducing NADP-dependent glyceraldehyde 3-phosphate dehydrogenase.

The available resources of Streptomyces represent a valuable repository of bioactive natural products that warrant exploration. Streptomyces albulus is primarily utilized in the industrial synthesis of ε-poly-L-lysine (ε-PL). In this study, the NADP-dependent glyceraldehyde 3-phosphate dehydrogenase (GapN) from Streptococcus mutans was heterologously expressed in S. albulus CICC11022, leading to elevated intracellular NADPH levels and reduced NADH and ATP concentrations. The resulting perturbation of S. albulus metabolism was comprehensively analyzed using transcriptomic and metabolomic methodologies. A decrease in production of ε-PL was observed. The expression of gapN significantly impacted on 23 gene clusters responsible for the biosynthesis of secondary metabolites. A comprehensive analysis revealed a total of 21 metabolites exhibiting elevated levels both intracellularly and extracellularly in the gapN expressing strain compared to those in the control strain. These findings underscore the potential of S. albulus to generate diverse bioactive natural products, thus offering valuable insights for the utilization of known Streptomyces resources through genetic manipulation.

Identification of the Reference Genes for Relative qRT-PCR Assay in Two Experimental Models of Rabbit and Horse Subcutaneous ASCs.

Obtaining accurate and reliable gene expression results in real-time RT-PCR (qRT-PCR) data analysis requires appropriate normalization by carefully selected reference genes, either a single or a combination of multiple housekeeping genes (HKGs). The optimal reference gene/s for normalization should demonstrate stable expression across varying conditions to diminish potential influences on the results. Despite the extensive database available, research data are lacking regarding the most appropriate HKGs for qRT-PCR data analysis in rabbit and horse adipose-derived stem cells (ASCs). Therefore, in our study, we comprehensively assessed and compared the suitability of some widely used HKGs, employing RefFinder and NormFinder, two extensively acknowledged algorithms for robust data interpretation. The rabbit and horse ASCs were obtained from subcutaneous stromal vascular fraction. ASCs were induced into tri-lineage differentiation, followed by the eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) treatment of the adipose-differentiated rabbit ASCs, while horse experimental groups were formed based on adipogenic, osteogenic, and chondrogenic differentiation. At the end of the experiment, the total mRNA was obtained and used for the gene expression evaluation of the observed factors. According to our findings, glyceraldehyde 3-phosphate dehydrogenase was identified as the most appropriate endogenous control gene for rabbit ASCs, while hypoxanthine phosphoribosyltransferase was deemed most suitable for horse ASCs. The obtained results underscore that these housekeeping genes exhibit robust stability across diverse experimental conditions, remaining unaltered by the treatments. In conclusion, the current research can serve as a valuable baseline reference for experiments evaluating gene expression in rabbit and horse ASCs. It highlights the critical consideration of housekeeping gene abundance and stability in qPCR experiments, emphasizing the need for an individualized approach tailored to the specific requirements of the study.

A comparison of the exposure system of glycidol-related chemicals on the formation of glycidol-hemoglobin adducts.

Glycidol fatty acid esters that are present in foods are degraded in vivo to the animal carcinogen glycidol, which binds to the N-terminal valine of hemoglobin (Hb) to form N-(2,3-dihydroxypropyl)valine (diHOPrVal) adducts. The existence of other chemicals that are converted to glycidol is unknown. To determine the effect of different exposure conditions on the formation of diHOPrVal adducts, several glycidol-related chemicals (3-monochloropropane-1,2-diol; 3-MCPD, epichlorohydrin, glyceraldehyde, acrylic acid, and 1,2-propanediol) were evaluated using in vitro and in vivo (single/repeated dose) methods. In vitro, the reaction of 3-MCPD or epichlorohydrin with human Hb produced 17% and 0.7% of diHOPrVal, as compared to equimolar glycidol, respectively. Following a single administration of glycidol-related compounds to ICR mice, diHOPrVal formation was observed only in the epichlorohydrin-treated group after day 5 of exposure. After 14 days of repeated dosing, the amounts of diHOPrVal produced by epichlorohydrin and 3-MCPD in vivo were <1% of diHOPrVal produced by an equal molar concentration of glycidol. Furthermore, glyceraldehyde group produced 0.2% of diHOPrVal at the same molar concentration of glycidol equivalents, in which diHOPrVal formation could not be confirmed by the in vitro assay. The results indicate the usefulness of diHOPrVal as an exposure marker for glycidol; however, the contribution of its formation in vivo by exposure to various chemicals will be necessary to validate and interpret the results.

Glyceraldehyde 3-Phosphate Dehydrogenase on the Surface of Candida albicans and Nakaseomyces glabratus Cells-A Moonlighting Protein That Binds Human Vitronectin and Plasminogen and Can Adsorb to Pathogenic Fungal Cells via Major Adhesins Als3 and Epa6.

Candida albicans and other closely related pathogenic yeast-like fungi carry on their surface numerous loosely adsorbed "moonlighting proteins"-proteins that play evolutionarily conserved intracellular functions but also appear on the cell surface and exhibit additional functions, e.g., contributing to attachment to host tissues. In the current work, we characterized this "moonlighting" role for glyceraldehyde 3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) of C. albicans and Nakaseomyces glabratus. GAPDH was directly visualized on the cell surface of both species and shown to play a significant part in the total capacity of fungal cells to bind two selected human host proteins-vitronectin and plasminogen. Using purified proteins, both host proteins were found to tightly interact with GAPDH, with dissociation constants in an order of 10-8 M, as determined by bio-layer interferometry and surface plasmon resonance measurements. It was also shown that exogenous GAPDH tightly adheres to the surface of candidal cells, suggesting that the cell surface location of this moonlighting protein may partly result from the readsorption of its soluble form, which may be present at an infection site (e.g., due to release from dying fungal cells). The major dedicated adhesins, covalently bound to the cell wall-agglutinin-like sequence protein 3 (Als3) and epithelial adhesin 6 (Epa6)-were suggested to serve as the docking platforms for GAPDH in C. albicans and N. glabratus, respectively.

Fluorogenic Hyaluronan Nanogels Track Individual Early Protein Aggregates Originated under Oxidative Stress.

Proteins are broadly versatile biochemical materials, whose functionality is tightly related to their folding state. Native folding can be lost to yield misfolded conformations, often leading to formation of protein oligomers, aggregates, and biomolecular phase condensates. The fluorogenic hyaluronan HA-RB, a nonsulfonated glycosaminoglycan with a combination of polyanionic character and of hydrophobic spots due to rhodamine B dyes, binds to early aggregates of the model protein cytoplasmic glyceraldehyde-3-phosphate dehydrogenase 1 from Arabidopsis thaliana (AtGAPC1) since the very onset of the oligomeric phase, making them brightly fluorescent. This initial step of aggregation has, until now, remained elusive with other fluorescence- or scattering-based techniques. The information gathered from nanotracking (via light-sheet fluorescence microscopy) and from FCS in a confocal microscope converges to highlight the ability of HA-RB to bind protein aggregates from the very early steps of aggregation and with high affinity. Altogether, this fluorescence-based approach allows one to monitor and track individual early AtGAPC1 aggregates in the size range from 10 to 100 nm with high time (∼10-2 s) and space (∼250 nm) resolution.

Structure of glyceraldehyde-3-phosphate dehydrogenase from Paracoccidioides lutzii in complex with an aldonic sugar acid.

The pathogen Paracoccidioides lutzii (Pb01) is found in South America countries Colombia, Ecuador, Venezuela and Brazil, especially in the central, west, and north regions of the latter. It belongs to the Ajellomycetaceae family, Onygenales order, and is typically thermodimorphic, presenting yeast cells when it grows in animal tissues, but mycelia when in the environment, where it produces the infectious propagule. This fungus is one of the etiologic agents of Paracoccidioidomycosis (PCM), the most important endemic fungal infection in Latin America. Investigations on its genome have contributed to a better understanding about its metabolism and revealed the complexity of several metabolic glycolytic pathways. Glyceraldehyde-3-Phosphate Dehydrogenase from Paracoccidioides lutzii (PlGAPDH) is considered a moonlighting protein and participates in several biological processes of this pathogen. The enzyme was expressed and purified, as seen in SDS-PAGE gel, crystallized and had its three dimensional structure (3D) determined in complex with NAD+, a sulphate ion and d-galactonic acid, therefore, a type of 'GAA site'. It is the first GAPDH structure to show this chemical type in this site and how this protein can bind an acid derived from oxidation of a linear hexose.

Structure-based functional analysis of a novel NADPH-producing glyceraldehyde-3-phosphate dehydrogenase from Corynebacterium glutamicum.

Corynebacterium glutamicum is an industrial workhorse applied in the production of valuable biochemicals. In the process of bio-based chemical production, improving cofactor recycling and mitigating cofactor imbalance are considered major solutions for enhancing the production yield and efficiency. Although, glyceraldehyde-3-phosphate dehydrogenase (GapDH), a glycolytic enzyme, can be a promising candidate for a sufficient NADPH cofactor supply, however, most microorganisms have only NAD-dependent GapDHs. In this study, we performed functional characterization and structure determination of novel NADPH-producing GapDH from C. glutamicum (CgGapX). Based on the crystal structure of CgGapX in complex with NADP cofactor, the unique structural features of CgGapX for NADP stabilization were elucidated. Also, N-terminal additional region (Auxiliary domain, AD) appears to have an effect on enzyme stabilization. In addition, through structure-guided enzyme engineering, we developed a CgGapX variant that exhibited 4.3-fold higher kcat, and 1.2-fold higher kcat/KM values when compared with wild-type. Furthermore, a bioinformatic analysis of 100 GapX-like enzymes from 97 microorganisms in the KEGG database revealed that the GapX-like enzymes possess a variety of AD, which seem to determine enzyme stability. Our findings are expected to provide valuable information for supplying NADPH cofactor pools in bio-based value-added chemical production.

Development of a Highly Selective NAD+ -Dependent Glyceraldehyde Dehydrogenase and its Application in Minimal Cell-Free Enzyme Cascades.

Anthropogenic climate change has been caused by over-exploitation of fossil fuels and CO2 emissions. To counteract this, the chemical industry has shifted its focus to sustainable chemical production and the valorization of renewable resources. However, the biggest challenges in biomanufacturing are technical efficiency and profitability. In our minimal cell-free enzyme cascade generating pyruvate as the central intermediate, the NAD+ -dependent, selective oxidation of D-glyceraldehyde was identified as a key reaction step to improve the overall cascade flux. Successive genome mining identified one candidate enzyme with 24-fold enhanced activity and another whose stability is unaffected in 10 % (v/v) ethanol, the final product of our model cascade. Semi-rational engineering improved the substrate selectivity of the enzyme up to 21-fold, thus minimizing side reactions in the one-pot enzyme cascade. The final biotransformation of D-glucose showed a continuous linear production of ethanol (via pyruvate) to a final titer of 4.9 % (v/v) with a molar product yield of 98.7 %. Due to the central role of pyruvate in diverse biotransformations, the optimized production module has great potential for broad biomanufacturing applications.

Role of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in autophagy activation following subarachnoid hemorrhage.

BACKGROUND: Early brain injury (EBI) refers to a severe brain injury that occurs within hours to days after subarachnoid hemorrhage (SAH). Neuronal damage in EBI is considered a key factor leading to poor prognosis. Currently, our understanding of the mechanisms of neuronal damage, such as neuronal autophagy, is still incomplete. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key enzyme in metabolism and plays an important role in autophagy. Based on this, this study will further explore the regulation of autophagy by GAPDH after SAH, which may provide a new treatment strategy for improving the prognosis of SAH patients. METHODS: The rat SAH model was established by endovascular puncturing, and the trend of autophagy in hippocampal neurons at different time points was discussed. Additionally, an in vitro SAH model was created using the oxygenated hemoglobin and hippocampal neuronal HT22 cell line. Through siRNA and overexpression adenovirus techniques, we further investigated the relationship between the key enzyme GAPDH and autophagy in the in vitro SAH model. RESULTS: We observed significant neuronal damage in the hippocampus 24 h after SAH, and the proteomics showed significant enrichment of autophagy-related pathways at this time point. Further studies showed that the expression of LC3 and Beclin1 peaked at 24 h, and the nuclear translocation of GAPDH occurred simultaneously with SAH-induced neuronal autophagy. Our in vitro SAH model confirmed the role of GAPDH in regulating the level of autophagy in HT22 cells. Knockdown of GAPDH significantly reduced the level of autophagy, while overexpression of GAPDH increased the level of autophagy. CONCLUSION: This study shows the trend of autophagy in hippocampal neurons after SAH, and reveals the regulatory role of GAPDH in SAH-induced autophagy. However, further studies are needed to reveal the exact mechanism of GAPDH in the nuclear translocation regulation of autophagy and validate in animal models.