Characterization of bifunctional l-glutathione synthetases from Actinobacillus pleuropneumoniae and Actinobacillus succinogenes for efficient glutathione biosynthesis

Yang, Jianhua; Li, Wei; Wang, Dezheng; Wu, Hui; Li, Zhimin; Ye, Qin

doi:10.1007/s00253-016-7437-4

Cited by 13 publications

(16 citation statements)

References 31 publications

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“…GSH is produced through two sequential enzymatic reactions catalyzed by glutamate-cysteine ligase ( gsh A) and GSH synthetase ( gsh B). In some bacteria, GSH is produced by a bifunctional enzyme (GshF) catalyzing both reactions simultaneously (Yang et al, 2016). Prokaryotes characterized by the absence of GSH, produce other LMW thiols – such as mycothiol, bacillithiol and CoA – with a similar protective role to GSH (Fahey, 2013).…”

Section: Resultsmentioning

confidence: 99%

Structural Characterization of the Xi Class Glutathione Transferase From the Haloalkaliphilic Archaeon Natrialba magadii

et al. 2019

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Xi class glutathione transferases (GSTs) are a recently identified group, within this large superfamily of enzymes, specifically endowed with glutathione-dependent reductase activity on glutathionyl-hydroquinone. Enzymes belonging to this group are widely distributed in bacteria, fungi, and plants but not in higher eukaryotes. Xi class GSTs are also frequently found in archaea and here we focus on the enzyme produced by the extreme haloalkaliphilic archaeon Natrialba magadii (NmGHR). We investigated its function and stability and determined its 3D structure in the apo form by X-ray crystallography. NmGHR displays the same fold of its mesophilic counterparts, is enriched in negatively charged residues, which are evenly distributed along the surface of the protein, and is characterized by a peculiar distribution of hydrophobic residues. A distinctive feature of haloalkaliphilic archaea is their preference for γ-glutamyl-cysteine over glutathione as a reducing thiol. Indeed we found that the N. magadii genome lacks a gene coding for glutathione synthase. Analysis of NmGHR structure suggests that the thiol binding site (G-site) of the enzyme is well suited for hosting γ-glutamyl-cysteine.

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Section: Resultsmentioning

confidence: 99%

Structural Characterization of the Xi Class Glutathione Transferase From the Haloalkaliphilic Archaeon Natrialba magadii

et al. 2019

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“…It was confirmed that GshFSS exhibited the highest specific activity (841 U/mg). Interestingly, although GshFSS was not derived from a thermophilic microorganism, its activity at 45°C was higher than that of GshFST (Yang et al, 2016). This result showed its potential application for GSH synthesis.…”

Section: Characterizaton Of Gshfsmentioning

confidence: 90%

“…One unit of enzyme activity was defined as the amount of enzyme that produces 1 mM GSH per hour (Yang et al, 2016). All the measurements were performed in triplicates.…”

Section: Gshf Activity Assaymentioning

confidence: 99%

“…Most GshFs were reported to be less sensitive to high concentration of GSH compared with the γ-GCS-GS system (Yang et al, 2016). To select a suitable GshF for GSH synthesis, the catalytic characteristics of the four GshFs were investigated.…”

Section: Characterizaton Of Gshfsmentioning

confidence: 99%

“…It has been reported that most GshFs are less sensitive to feedback inhibition caused by GSH except those from L. monocytogenes and P. multocida (Gopal et al, 2005;Li et al, 2011;Vergauwen et al, 2006). Furthermore, many of them show higher specific activities than E. coli γ-GCS and GS (Yang et al, 2016). In our previous studies, a recombinant E. coli strain overexpressing GshF of S. thermophilus was constructed and production of GSH was investigated using the resting cells.…”

Section: Introductionmentioning

confidence: 99%

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One-pot synthesis of glutathione by a two-enzyme cascade using a thermophilic ATP regeneration system

Xing

Huang

et al. 2017

Journal of Biotechnology

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In vitro cascade catalysis using enzyme-based system is becoming a promising biomanufacturing platform for biofuels and biochemicals production. Glutathione is a pivotal non-protein thiol compound and widely applied in food and pharmaceutical industries. In this study, glutathione was synthesized by a bifunctional glutathione synthetase together with a thermophilic ATP regeneration system through a two-enzyme cascade in vitro. Four bifunctional glutathione synthetases from Streptococcus sanguinis, S. gordonii, S. uberis and Bacillus cereus were applied for glutathione synthesis. The bifunctional glutathione synthetase from S. sanguinis was selected and coupled with the polyphosphate kinase from Thermosynechococcus elongatus BP-1 for regenerating ATP to produce glutathione in one pot. In the optimized system, 28.5mM glutathione was produced within 5h due to efficient ATP regeneration from low-cost polyphosphate. The yield based on added l-cysteine reached 81.4% and the productivity of glutathione achieved 5.7mM/h. The one-pot system indicated a potential biotransformation platform for industrial production of glutathione.

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Efficient production of glutathione in Saccharomyces cerevisiae via a synthetic isozyme system

Jeon

Lee

Park

et al. 2022

Biotechnology Journal

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Glutathione, a tripeptide consisting of cysteine, glutamic acid, and glycine, has multiple beneficial effects on human health. Previous studies have focused on producing glutathione in Saccharomyces cerevisiae by overexpressing γ‐glutamylcysteine synthetase (GSH1) and glutathione synthetase (GSH2), which are the rate‐limiting enzymes involved in the glutathione biosynthetic pathway. However, the production yield and titer of glutathione remain low due to the feedback inhibition on GSH1. To overcome this limitation, a synthetic isozyme system consisting of a novel bifunctional enzyme (GshF) from Gram‐positive bacteria possessing both GSH1 and GSH2 activities, in addition to GSH1/GSH2, was introduced into S. cerevisiae, as GshF is insensitive to feedback inhibition. Given the HSP60 chaperonin system mismatch between bacteria and S. cerevisiae, co‐expression of Group‐I HSP60 chaperonins (GroEL and GroES) from Escherichia coli was required for functional expression of GshF. Among various strains constructed in this study, the SKSC222 strain capable of synthesizing glutathione with the synthetic isozyme system produced 240 mg L‐1 glutathione with glutathione content and yield of 4.3% and 25.6 mgglutathione/gglucose, respectively. These values were 6.6‐, 4.9‐, and 4.3‐fold higher than the corresponding values of the wild‐type strain. In a glucose‐limited fed‐batch fermentation, the SKSC222 strain produced 2.0 g L‐1 glutathione in 67 h. Therefore, this study highlights the benefits of the synthetic isozyme system in enhancing the production titer and yield of value‐added chemicals by engineered strains of S. cerevisiae.

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Characterization of bifunctional l-glutathione synthetases from Actinobacillus pleuropneumoniae and Actinobacillus succinogenes for efficient glutathione biosynthesis

Cited by 13 publications

References 31 publications

Structural Characterization of the Xi Class Glutathione Transferase From the Haloalkaliphilic Archaeon Natrialba magadii

Structural Characterization of the Xi Class Glutathione Transferase From the Haloalkaliphilic Archaeon Natrialba magadii

One-pot synthesis of glutathione by a two-enzyme cascade using a thermophilic ATP regeneration system

Efficient production of glutathione in Saccharomyces cerevisiae via a synthetic isozyme system

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