glutamate dehydrogenase
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Author(s):  
Feng Zhou ◽  
Yan Xu ◽  
Xiaoqing Mu ◽  
Yao Nie

In this study, a novel enzymatic approach to transform levulinic acid (LA), which can be obtained from biomass, into value-added (R)-4-aminopentanoic acid using an engineered glutamate dehydrogenase from Escherichia coli (EcGDH) was developed. Through crystal structure comparison, two residues (K116 and N348), especially residue 116, were identified to affect the substrate specificity of EcGDH. After targeted saturation mutagenesis, the mutant EcGDHK116C, which was active toward LA, was identified. Screening of the two-site combinatorial saturation mutagenesis library with EcGDHK116C as positive control, the kcat/Km of the obtained EcGDHK116Q/N348M for LA and NADPH were 42.0- and 7.9-fold higher, respectively, than that of EcGDHK116C. A molecular docking investigation was conducted to explain the catalytic activity of the mutants and stereoconfiguration of the product. Coupled with formate dehydrogenase, EcGDHK116Q/N348M was found to be able to convert 0.4 M LA by more than 97% in 11 h, generating (R)-4-aminopentanoic acid with >99% enantiomeric excess (ee). This dual-enzyme system used sustainable raw materials to synthesize (R)-4-aminopentanoic acid with high atom utilization as it utilizes cheap ammonia as the amino donor, and the inorganic carbonate is the sole by-product.


Biochimie ◽  
2022 ◽  
Author(s):  
Vera A. Borzova ◽  
Natalia A. Chebotareva ◽  
Nikolai N. Sluchanko ◽  
Sergey Yu Kleymenov ◽  
Kira A. Markossian ◽  
...  

Author(s):  
Maher A Shahrour ◽  
Francesco Massimo Lasorsa ◽  
Vito Porcelli ◽  
Imad Dweikat ◽  
Maria Antonietta Di Noia ◽  
...  

Abstract Context The hyperinsulinism/hyperammonemia (HI/HA) syndrome, the second most common form of congenital hyperinsulinism, has been associated to dominant mutations in GLUD1, coding for the mitochondrial enzyme glutamate dehydrogenase, that increase enzyme activity by reducing its sensitivity to allosteric inhibition by GTP. Objective To identify the underlying genetic aetiology in two siblings who presented with the biochemical features of HI/HA syndrome but did not carry pathogenic variants in GLUD1, and to determine the functional impact of the newly identified mutation. Main Outcome Measures The patients were investigated by whole exome sequencing. Yeast complementation studies and biochemical assays on the recombinant mutated protein were performed. The consequences of stable slc25a36 silencing in HeLa cells were also investigated. Results A homozygous splice site variant was identified in solute carrier family 25, member 36 (SLC25A36), encoding the pyrimidine nucleotide carrier 2 (PNC2), a mitochondrial nucleotide carrier that transports pyrimidine as well as guanine nucleotides across the inner mitochondrial membrane. The mutation leads to a 26 aa in-frame deletion in the first repeat domain of the protein which abolished transport activity. Furthermore, knockdown of slc25a36 expression in HeLa cells caused a marked reduction in the mitochondrial GTP content which likely leads to an hyperactivation of glutamate dehydrogenase in our patients. Conclusions We report for the first time a mutation in PNC2/SLC25A36 leading to HI/HA and provide functional evidence of the molecular mechanism responsible for this phenotype. Our findings underscore the importance of mitochondrial nucleotide metabolism and expand the role of mitochondrial transporters in insulin secretion.


Psychiatry ◽  
2021 ◽  
Vol 19 (4) ◽  
pp. 34-41
Author(s):  
O. K. Savushkina ◽  
E. B. Tereshkina ◽  
T. A. Prokhorova ◽  
I. S. Boksha ◽  
T. P. Safarova ◽  
...  

The aim of the study is to evaluate the activity of platelet glutamate dehydrogenase (GDH) in late-life depression compared to the healthy control group and to reveal possible correlations with clinical data. Patients and methods: 42 elderly patients (60–86 years old) with depressive episodes of different nosological categories according to ICD-10 were examined: a single depressive episode (F32.0, F32.1), a depressive episode in recurrent depressive disorder (RDD — F33.0, F33.1) and a depressive episode in bipolar affective disorder (BD — F31.3). The activity of GDH and the severity of depression (using the Hamilton depressive scale, HAMD-17, and the Hamilton scale for assessing anxiety, HARS) were evaluated twice: before the starting the course of antidepressant therapy (day 0) and on the 28th day of the treatment course. Results: patients showed a significant decrease in the activity of GDH compared to the control group (p < 0.0008). Before the treatment, GDH activity was significantly reduced compared to the control in both RDD and BD (p < 0.002 and p < 0.004), whereas after the treatment, the decreased GDH activity was observed only in patients with BD (p < 0.002). When compared with the control group, male patients showed a significant decrease in GDH activity both before and after the treatment course (p < 0.017 and p < 0.027), whereas women patients showed the decrease only before the treatment (p < 0.014). Conclusion: the decreased platelet GDH activity in elderly depressions may indicate an impairment of glutamate metabolism. Gender differences were revealed in the reversal of GDH activity level after the therapy: in men, the level of GDH activity did not recover to control values after the treatment course. An elevation in the level of GDH to control values over a 28-day course of therapy occurred only in patients with RDD, but not in patients with BD.


2021 ◽  
Vol 102 (12) ◽  
Author(s):  
Jian Yang ◽  
Jia Wang ◽  
Xiaowei Peng ◽  
Chengfeng Lei ◽  
Xiulian Sun ◽  
...  

Dendrolimus punctatus causes great damage to pine forests worldwide. Dendrolimus punctatus cypovirus 1 (DpCPV-1) is an important pathogen of D. punctatus. However, the mechanism of DpCPV-1 cell entry has not been elucidated. In this study, we revealed that both GTase and MTase domains of VP3 (B-spike) and VP4 (A-spike) of DpCPV-1 interacted with the midgut proteins of Bombyx mori. Binding and competition assays revealed that GTase, MTase and VP4 played roles as viral attachment proteins. Far-Western blotting and LC-MS/MS analyses identified that heat shock protein 70 (BmHSP70), glutamate dehydrogenase (BmGDH), and angiotensin-converting enzyme (BmACE) in the midgut proteins as ligand candidates of the viral attachment proteins, and this was further verified by co-immunoprecipitation and fluorescence co-localization assays. Viral binding to the host midgut in vitro was inhibited by pre-treating B. mori midgut proteins with anti-BmHSP70, anti-BmGDH, anti-BmACE antibodies singly and in combination. Incubating DpCPV-1 virions with prokaryotically expressed BmHSP70, BmGDH, and BmACE also decreased viral attachment to the host midgut. In vivo bioassays revealed that viral infection in Helicoverpa armigera was partially neutralized by BmHSP70, BmGDH, and BmACE. Taking together, we concluded that HSP70, GDH, and ACE mediate DpCPV attachment and entry via binding to the viral attachment proteins, VP3 and VP4. The findings provide foundation for further understanding the entry mechanisms of cypoviruses.


2021 ◽  
Vol 1 ◽  
Author(s):  
Valentina Marchini ◽  
Ana I. Benítez-Mateos ◽  
David Roura Padrosa ◽  
Francesca Paradisi

A novel fusion protein has been rationally designed, combining the hexameric glutamate dehydrogenase from Clostridium symbiosum with the dimeric formate dehydrogenase from Candida boidinii. The former enzyme consumes ammonia for the reductive amination of α-ketoglutarate using NADH, while the latter biocatalyst regenerates continuously the cofactor. This enzymes fusion opens new perspectives for the detection and the removal of ammonia. The bifunctional biocatalyst has been successfully created, expressed, and then characterized. The two fused protein domains retained identical properties and catalytic activity of the individual enzymes. Additionally, the immobilization on a methacrylate resin optimized the assembly providing a reusable and stable biocatalyst. This is an example of immobilization of a fusion protein, so that efficiency and sustainability of the process are enhanced. The immobilized biocatalyst could be recycled 10 times retaining still half of the initial activity. Such preparation outperforms the co-immobilized wild-type enzymes in the conversion of 300 mM of ammonia, which could be carried out also in continuous mode.


Nutrients ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 4238
Author(s):  
Václav Tvrdý ◽  
Marcel Hrubša ◽  
Eduard Jirkovský ◽  
David Biedermann ◽  
Michal Kutý ◽  
...  

Silymarin is known for its hepatoprotective effects. Although there is solid evidence for its protective effects against Amanita phalloides intoxication, only inconclusive data are available for alcoholic liver damage. Since silymarin flavonolignans have metal-chelating activity, we hypothesized that silymarin may influence alcoholic liver damage by inhibiting zinc-containing alcohol dehydrogenase (ADH). Therefore, we tested the zinc-chelating activity of pure silymarin flavonolignans and their effect on yeast and equine ADH. The most active compounds were also tested on bovine glutamate dehydrogenase, an enzyme blocked by zinc ions. Of the six flavonolignans tested, only 2,3-dehydroderivatives (2,3-dehydrosilybin and 2,3-dehydrosilychristin) significantly chelated zinc ions. Their effect on yeast ADH was modest but stronger than that of the clinically used ADH inhibitor fomepizole. In contrast, fomepizole strongly blocked mammalian (equine) ADH. 2,3-Dehydrosilybin at low micromolar concentrations also partially inhibited this enzyme. These results were confirmed by in silico docking of active dehydroflavonolignans with equine ADH. Glutamate dehydrogenase activity was decreased by zinc ions in a concentration-dependent manner, and this inhibition was abolished by a standard zinc chelating agent. In contrast, 2,3-dehydroflavonolignans blocked the enzyme both in the absence and presence of zinc ions. Therefore, 2,3-dehydrosilybin might have a biologically relevant inhibitory effect on ADH and glutamate dehydrogenase.


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