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Glucose 6-phosphate dehydrogenase (G6PDH) is one of the most important dehydrogenases responsible for generating reduced NADPH for anabolism and is also the rate-limiting enzyme in the Entner-Doudoroff pathway. For in vitro biocatalysis, G6PDH must possess both high activity and good thermostability due to requirements of efficient use and low expense of biocatalyst. Here, we used directed evolution to improve thermostability of the highly active G6PDH from Zymomonas mobilis. Four generations of random mutagenesis and Petri-dish-based double-layer screening evolved the thermolabile wild-type enzyme to the thermostable mutant Mut 4-1, which showed a more than 124-fold increase in half-life time (t1/2) at 60 °C, a 3.4 °C increase in melting temperature (Tm), and a 5 °C increase in optimal temperature (Topt), without compromising the specific activity. In addition, the thermostable mutant was conducted to generate hydrogen from maltodextrin via in vitro synthetic biosystems (ivSB), gaining a more than 8-fold improvement of productivity rate with 76% of theoretical yield at 60 °C. Thus, the engineered G6PDH has been shown to effectively regenerate NADPH at high temperatures and will be applicable for NAD(P)H regeneration in numerous in vitro biocatalysis applications.

Background: Glucose-6-phosphate dehydrogenase (G6PD) deficiency, impacting 4.9% of the population and more prevalent in Mediterranean communities, is a common enzymopathy with potential relevance to Attention Deficit/Hyperactivity Disorder (ADHD). This study investigated this association. Methods: The clinical characteristics of 7473 G6PD-deficient patients and 29,892 matched case–controls (selected at a 1:4 ratio) from a cohort of 1,031,354 within the Leumit Health Services database were analyzed using Fisher’s exact test for categorical variables and the Mann–Whitney U test for continuous variables. Results: In total, 68.7% were male. The mean duration of follow-up was 14.3 ± 6.2 years at a mean age of 29.2 ± 22.3 years. G6PD deficiency was associated with an increased risk of being diagnosed with ADHD (Odds Ratio (OR) = 1.16 [95% CI, 1.08–1.25], p < 0.001), seeking care from adult neurologists (OR = 1.30 [95% CI, 1.22–1.38], p < 0.001), and consulting adult psychiatrists (OR = 1.12 [95% CI, 1.01–1.24], p = 0.048). The use of stimulant medications among G6PD-deficient individuals was 17% higher for the methylphenidate class of drugs (OR = 1.17 [95% CI, 1.08, 1.27], p < 0.001), and there was a 16% elevated risk for amphetamine use (OR = 1.16 [95% CI, 1.03, 1.37], p = 0.047). Conclusions: G6PD deficiency signals an increased risk of ADHD diagnosis, more severe presentations of ADHD and a greater need for psychiatric medications to treat ADHD.

Copper (Cu) is one of the toxic elements that cause environmental pollution. As a result of excessive accumulation of copper in the organism, it causes damage in various organs and tissues and hemolysis in erythrocytes. Astaxanthin (ATX) is a pigment belonging to the xanthophyll family, which is an oxygenated derivative of carotenoids. Thanks to its powerful antioxidant properties, ATX has an extraordinary potential to protect the organism against various diseases, especially cancer. The main objective of this study was to investigate the toxic effect of copper ions on the glucose 6-phosphate dehydrogenase (G6PD), 6-phospho-gluconate dehydrogenase (6PGD), glutathione reductase (GR), glutathione S-transferase (GST), and thioredoxin reductase (TrxR) enzymes and the role of astaxanthin in reducing this effect. In in vivo study, Wistar Albino male rats (n=28) were randomly divided into 4 groups: the control group, copper (Cu2+) group, astaxanthin (ATX) group, and copper + astaxanthin (Cu2++ATX) group. The results show that G6PD enzyme activity in Cu2+ group was strongly inhibited (p ˂ 0.05), while in other groups, there were no significant effects compared to the control group (p ⩾ 0.05). 6PGD enzyme activity was significantly reduced in Cu2+ group compared to that in the control group (p ˂ 0.05), and GR enzyme activity was lower in Cu2+ group compared to that in the control group (p ˂ 0.05). Similarly, when GST enzyme activity was evaluated, a strong decrease was observed in the Cu2+ group compared to that in the control group (p ˂ 0.05), while the enzyme activity in the Cu2++ATX group approached the control group (p ⩾ 0.05). When TrxR enzyme activity level was examined, a statistically significant decrease was observed in the Cu2+ and Cu2++ATX groups (p ˂ 0.05), and the enzyme activity in the ATX group was found to be close to that in the control group. When in vitro results were evaluated, it was observed that copper ions inhibited G6PD enzyme purified from rat erythrocyte tissues with IC50=1.90 μM value and Ki = 0.97 μM ± 0.082 value and the inhibition was non-competitive. From the results, it can be concluded that Cu2+ ions have an inhibitory effect on rat erythrocyte pentose phosphate pathway and antioxidant system enzymes both in vivo and in vitro, and astaxanthin reduces this effect.

Isoprenoid biosynthesis has a significant requirement for the co‐factor NADPH. Thus, increasing NADPH levels for enhancing isoprenoid yields in synthetic biology is critical. Previous efforts have focused on diverting flux into the pentose phosphate pathway or overproducing enzymes that generate NADPH. In this study, we instead focused on increasing the efficiency of enzymes that generate NADPH. We first established a robust genetic screen that allowed us to screen improved variants. The pentose phosphate pathway enzyme, glucose 6‐phosphate dehydrogenase (G6PD), was chosen for further improvement. Different gene fusions of G6PD with the downstream enzyme in the pentose phosphate pathway, 6‐phosphogluconolactonase (6PGL), were created. The linker‐less G6PD‐6PGL fusion displayed the highest activity, and although it had slightly lower activity than the WT enzyme, the affinity for G6P was higher and showed higher yields of the diterpenoid sclareol in vivo. A second gene fusion approach was to fuse G6PD to truncated HMG‐CoA reductase, the rate‐limiting step and also the major NADPH consumer in the pathway. Both domains were functional, and the fusion also yielded higher sclareol levels. We simultaneously carried out a rational mutagenesis approach with G6PD, which led to the identification of two mutants of G6PD, N403D and S238QI239F, that showed 15–25% higher activity in vitro. The diterpene sclareol yields were also increased in the strains overexpressing these mutants relative to WT G6PD, and these will be very beneficial in synthetic biology applications. This study focuses on enhancing isoprenoid yields by increasing NADPH levels. Gene fusions of S. cerevisiae glucose 6‐phosphate dehydrogenase (ScG6PD) with 6‐phosphogluconolactonase (6PGL) and truncated HMG‐CoA reductase were created, and overexpressing them yielded higher sclareol (diterpenoid). Rational mutagenesis of ScG6PD identified mutants (N403D and S238QI239F) with 15–25% higher activity and overexpression of the N403D mutant also showed increased sclareol yields.

The aim of this study was to investigate the effects of mercury chloride and boric acid on rat (Wistar albino) erythrocyte: glucose 6-phosphate dehydrogenase (G6PD), 6-phosphoglucona-te dehydrogenase (6PGD), thioredoxin reductase (TrxR), glutathione reductase (GR) and glutathione S-transferase (GST) enzymes in vivo, and the rat erythrocyte G6PD enzyme in vitro. In the in vivo study, 24 male rats were divated into three different groups: control (C), mercury chloride (M), and mercury chloride + boric acid (M + BA). At the completion of this study, a significant degree of inhibition for both G6PD and GST enzyme activity was observed in the M groups when compared to the C group ( p  < 0.05), and no significant effect was observed in the 6PGD enzyme. However, there was significantly increased TrxR and GR enzyme activity of both the M and M + BA groups ( p  < 0.05). In the in vitro study, the G6PD enzyme from rat erythrocytes was purified with 2′,5′-ADP Sepharose-4B affinity chromatography, and the effect of both mercury chloride and boric acid on the enzyme activity was investigated. The results showed that boric acid increased the G6PD enzyme activity while the mercury ions that inhibited the enzyme activity (IC 50 values of 346 μM and K i values of 387 μM) were noncompetitive.

Plastids of nongreen tissues can import carbon in the form of glucose 6-phosphate via the glucose 6-phosphate/phosphate translocator (GPT). The Arabidopsis thaliana genome contains two homologous GPT genes, AtGPT1 and AtGPT2. Both proteins show glucose 6-phosphate translocator activity after reconstitution in liposomes, and each of them can rescue the low-starch leaf phenotype of the pgi1 mutant (which lacks plastid phosphoglucoisomerase), indicating that the two proteins are also functional in planta. AtGPT1 transcripts are ubiquitously expressed during plant development, with highest expression in stamens, whereas AtGPT2 expression is restricted to a few tissues, including senescing leaves. Disruption of GPT2 has no obvious effect on growth and development under greenhouse conditions, whereas the mutations gpt1-1 and gpt1-2 are lethal. In both gpt1 lines, distorted segregation ratios, reduced efficiency of transmission in males and females, and inability to complete pollen and ovule development were observed, indicating profound defects in gametogenesis. Embryo sac development is arrested in the gpt1 mutants at a stage before the fusion of the polar nuclei. Mutant pollen development is associated with reduced formation of lipid bodies and small vesicles and the disappearance of dispersed vacuoles, which results in disintegration of the pollen structure. Taken together, our results indicate that GPT1-mediated import of glucose 6-phosphate into nongreen plastids is crucial for gametophyte development. We suggest that loss of GPT1 function results in disruption of the oxidative pentose phosphate cycle, which in turn affects fatty acid biosynthesis.

Glucose 6-phosphate dehydrogenase (G6PD) is a key enzyme catalyzing the first step of the pentose phosphate pathway which generates NADPH for anabolic pathways and protection systems in various organisms, including fish. In the present study, G6PD was purified from grass carp ( Ctenopharyngodon idella ) hepatopancreas using the methods of 2′,5′-ADP-Sepharose 4B affinity chromatography followed by DEAE Sepharose Fast Flow ion exchange chromatography. The characterization of G6PD and inhibition effects of several metal ions on G6PD activity in vitro were also determined. Grass carp hepatopancreas G6PD, with a specific activity of 18 U/mg protein, was purified 1,066-fold with a yield of 19.5 % and Mr of 71.85 kDa. The enzyme had a temperature optimum of 42 °C, pH optimum of 7.5 and 9.0. The K m values for G6-P and NADP + were determined to be 0.026, 0.0068 mM, respectively. The V max values for G6-P and NADP + were 2.20 and 2.27 μM min −1 mg protein −1 , respectively. The catalytic efficiency for G6-P and NADP as the substrates was 0.085 and 0.334 × 10 −6 min −1 mg protein −1 , respectively. Inhibition effects of metal ions on the purified G6PD activity indicated that IC 50 values of Zn +2 , Mn +2 , Al +3 , Cu +2 , and Cd +2 were 0.42, 0.54, 0.94, 1.20, and 4.17 mM, respectively. The Ki constants of Zn +2 , Al +3 , Cu +2 , and Cd +2 were 0.52, 1.12, 0.26, and 4.8 mM, respectively. Zn +2 , Al +3 , and Cd +2 showed competitive inhibition, while Cu +2 inhibited the G6PD in a noncompetitive inhibition manner. Our study provided important information about the control of the grass carp liver PPP, the biosynthesis of several important related biomolecules, and the status of detoxification systems in grass carp liver in relation to metabolism.

Red cell diseases encompass a group of inherited or acquired erythrocyte disorders that affect the structure, function, or production of red blood cells (RBCs). These disorders can lead to various clinical manifestations, including anemia, hemolysis, inflammation, and impaired oxygen-carrying capacity. Oxidative stress, characterized by an imbalance between the production of reactive oxygen species (ROS) and the antioxidant defense mechanisms, plays a significant role in the pathophysiology of red cell diseases. In this review, we discuss the most relevant oxidant species involved in RBC damage, the enzymatic and low molecular weight antioxidant systems that protect RBCs against oxidative injury, and finally, the role of oxidative stress in different red cell diseases, including sickle cell disease, glucose 6-phosphate dehydrogenase deficiency, and pyruvate kinase deficiency, highlighting the underlying mechanisms leading to pathological RBC phenotypes.

Excessive poly(ADP-ribose) (PAR) polymerase-1 (PARP-1) activation kills cells via a cell-death process designated “parthanatos” in which PAR induces the mitochondrial release and nuclear translocation of apoptosis-inducing factor to initiate chromatinolysis and cell death. Accompanying the formation of PAR are the reduction of cellular NAD ⁺ and energetic collapse, which have been thought to be caused by the consumption of cellular NAD ⁺ by PARP-1. Here we show that the bioenergetic collapse following PARP-1 activation is not dependent on NAD ⁺ depletion. Instead PARP-1 activation initiates glycolytic defects via PAR-dependent inhibition of hexokinase, which precedes the NAD ⁺ depletion in N -methyl- N -nitroso- N -nitroguanidine (MNNG)-treated cortical neurons. Mitochondrial defects are observed shortly after PARP-1 activation and are mediated largely through defective glycolysis, because supplementation of the mitochondrial substrates pyruvate and glutamine reverse the PARP-1–mediated mitochondrial dysfunction. Depleting neurons of NAD ⁺ with FK866, a highly specific noncompetitive inhibitor of nicotinamide phosphoribosyltransferase, does not alter glycolysis or mitochondrial function. Hexokinase, the first regulatory enzyme to initiate glycolysis by converting glucose to glucose-6-phosphate, contains a strong PAR-binding motif. PAR binds to hexokinase and inhibits hexokinase activity in MNNG-treated cortical neurons. Preventing PAR formation with PAR glycohydrolase prevents the PAR-dependent inhibition of hexokinase. These results indicate that bioenergetic collapse induced by overactivation of PARP-1 is caused by PAR-dependent inhibition of glycolysis through inhibition of hexokinase.

• As the central carbon uptake pathway in photosynthetic cells, the Calvin–Benson cycle is amongthe most important biochemical cycles for life on Earth. A carbon flux of anaplerotic origin (i.e. through the chloroplast-localized oxidative branch of the pentose phosphate pathway) into the Calvin–Benson cycle was proposed recently. • Here, we measured intramolecular deuterium abundances in leaf starch of Helianthus annuus grown at varying ambient CO₂ concentrations, Cₐ. Additionally, we modelled deuterium fractionations expected for the anaplerotic pathway and compared modelled with measured fractionations. • We report deuterium fractionation signals at H¹ and H² of starch glucose. Below a Cₐ change point, these signals increase with decreasing Cₐ consistent with modelled fractionations by anaplerotic flux. Under standard conditions (Cₐ = 450 ppm corresponding to intercellular CO₂ concentrations, C i, of 328 ppm), we estimate negligible anaplerotic flux. At Cₐ = 180 ppm (C i = 140 ppm), more than 10% of the glucose-6-phosphate entering the starch biosynthesis pathway is diverted into the anaplerotic pathway. • In conclusion, we report evidence consistent with anaplerotic carbon flux into the Calvin–Benson cycle in vivo. We propose the flux may help to: maintain high levels of ribulose 1,5-bisphosphate under source-limited growth conditions to facilitate photorespiratory nitrogen assimilation required to build-up source strength; and counteract oxidative stress.