Crystal structure analysis and enzymatic characterization of γ-glutamyltranspeptidase from <i>Pseudomonas nitroreducens</i>

Hibi, Takao; Imaoka, Masashi; Shimizu, Yoichiro; Itoh, Takafumi; Wakayama, Mamoru

doi:10.1080/09168451.2018.1547104

Cited by 10 publications

(5 citation statements)

References 20 publications

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“…INTRODUCTION γ-Glutamyltransferase (GGT; EC 2.3.2.2) catalyzes the lysis of γ-glutamyl moieties in glutathione or γ-glutamylated compounds and transfers the γ-glutamyl group to acceptors, playing critical roles in the metabolic cycle of glutathione. 1 Its promising potential in the biosynthesis of various γ-glutamyl amino acid derivatives or peptides with high value and relevant physiological functions make GGT very important in the food industry and pharmaceutical field. 2 For instance, γ-glutamyl ethylamide (L-theanine), which is popularly used as a food taste enhancer, is synthesized by GGT via transpeptidation without the addition of ATP.…”

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confidence: 99%

Expression of Bacillus amyloliquefaciens γ-Glutamyltransferase in Lactococcus lactis and Immobilization on Magnetic Nanoparticles

et al. 2021

ACS Food Sci. Technol.

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γ-Glutamyltransferase (GGT) can catalyze the transfer of a γ-glutamyl group to amino acids or peptides. Bacterial GGTs have a wide substrate specificity, which means that a variety of γ-glutamyl amino acid derivatives or peptides can be synthesized under GGT catalysis. In this work, in order to enhance the expression and stability of GGT, a recombinant plasmid harboring Bacillus amyloliquefaciens ggt gene with a Usp45 signal peptide gene fused upstream was constructed and transferred into Lactococcus lactis NZ9000 to express BaGGT. After on-column hydrolysis and purification by immobilized metal affinity chromatography, 90.2 ± 0.7 mg/L of BaGGT-6His with an enzymatic activity of 57.3 ± 0.9 U/mg was purified. Subsequently, BaGGT-6His was immobilized onto magnetic mesoporous nanoparticles (Fe 3 O 4 NPs) by covalent binding. The immobilized BaGGT-6His largely retained the activity and, importantly, could be recovered and reused. After optimization of the production and immobilization conditions, the biotechnologically relevant product L-theanine was synthesized with free and immobilized BaGGT-6His.

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confidence: 99%

Expression of Bacillus amyloliquefaciens γ-Glutamyltransferase in Lactococcus lactis and Immobilization on Magnetic Nanoparticles

et al. 2021

ACS Food Sci. Technol.

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“…Enzyme assay. The hydrolysis and transpeptidation activities of GGT were measured by spectrophotometric methods and using high-performance liquid chromatography (Hibi et al, 2019). The protein concentrations were determined according to the Lowry method (Lowry et al, 1951).…”

Section: Methodsmentioning

confidence: 99%

Characterization of three γ-glutamyltranspeptidases from <i>Pseudomonas aeruginosa</i> PAO1

Nonomura

Wang

Kikuchi

et al. 2023

J. Gen. Appl. Microbiol.

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The Pseudomonas aeruginosa strain, PAO1, has three putative -glutamyltranspeptidase (GGT) genes: ggtI, ggtII, and ggtIII. In this study, the expression of each of these genes in P. aeruginosa PAO1 was analyzed, and the properties of the corresponding GGT proteins were investigated. This is the first report on biochemical characterization of GGT paralogs from Pseudomonas species. The crude extracts prepared from P. aeruginosa PAO1 exhibited hydrolysis and transpeptidation activities of 17.3 and 65.0 mU/mg, respectively, and the transcription of each gene to mRNA was confirmed by RT-PCR. All genes were cloned, and the expression plasmids constructed were introduced into an Escherichia coli expression system. Enzyme activity of the expressed protein of ggtI (PaGGTI) was not detected in the system, while the enzyme activities of the expressed proteins derived from ggtII and ggtIII (PaGGTII and PaGGTIII, respectively) were detected. However, the enzyme activity of PaGGTII was very low and easily decreased. PaGGTII with C-terminal his-tag (PaGGTII25aa) showed increased activity and stability, and the purified enzyme consisted of a large subunit of 40 kDa and a small subunit of 28 kDa. PaGGTIII consisted of a large subunit of 37 kDa and a small subunit of 24 kDa. The maximum hydrolysis and transpeptidation activities of PaGGTII25aa were obtained at 40ºC-50ºC, and the maximum hydrolysis and transpeptidation activities of PaGGTIII were obtained at 50ºC-60ºC. These enzymes retained approximately 80% of their hydrolysis and transpeptidation activities after incubation at 50ºC for 10 min, reflecting good stability. Both PaGGTII25aa and PaGGTIII showed higher activities of hydrolysis and transpeptidation in the alkali range than in the acidic range. However, they were highly stable at a wide pH range (5-10.5).

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“…Interestingly, Pseudomonas nitroreducens GGT (PnGGT) exhibits higher hydrolytic activity than transpeptidase activity despite high sequence similarity with EcGGT (Imaoka et al, 2010). Recent crystallographic studies on PnGGT in complex with Gly-Gly acceptor demonstrated that the binding mode of Gly-Gly in the active site is probably the reason for its reduced activity as an acceptor (Hibi et al, 2019). The terminal amino group of Gly-Gly is oriented opposite to the nucleophilic active center.…”

Section: Substrate Binding Pocketmentioning

confidence: 99%

Bacterial Gamma-Glutamyl Transpeptidase, an Emerging Biocatalyst: Insights Into Structure–Function Relationship and Its Biotechnological Applications

et al. 2021

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Gamma-glutamyl transpeptidase (GGT) enzyme is ubiquitously present in all life forms and plays a variety of roles in diverse organisms. Higher eukaryotes mainly utilize GGT for glutathione degradation, and mammalian GGTs have implications in many physiological disorders also. GGTs from unicellular prokaryotes serve different physiological functions in Gram-positive and Gram-negative bacteria. In the present review, the physiological significance of bacterial GGTs has been discussed categorizing GGTs from Gram-negative bacteria like Escherichia coli as glutathione degraders and from pathogenic species like Helicobacter pylori as virulence factors. Gram-positive bacilli, however, are considered separately as poly-γ-glutamic acid (PGA) degraders. The structure–function relationship of the GGT is also discussed mainly focusing on the crystallization of bacterial GGTs along with functional characterization of conserved regions by site-directed mutagenesis that unravels molecular aspects of autoprocessing and catalysis. Only a few crystal structures have been deciphered so far. Further, different reports on heterologous expression of bacterial GGTs in E. coli and Bacillus subtilis as hosts have been presented in a table pointing toward the lack of fermentation studies for large-scale production. Physicochemical properties of bacterial GGTs have also been described, followed by a detailed discussion on various applications of bacterial GGTs in different biotechnological sectors. This review emphasizes the potential of bacterial GGTs as an industrial biocatalyst relevant to the current switch toward green chemistry.

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Crystal structure analysis and enzymatic characterization of γ-glutamyltranspeptidase from Pseudomonas nitroreducens

Cited by 10 publications

References 20 publications

Expression of Bacillus amyloliquefaciens γ-Glutamyltransferase in Lactococcus lactis and Immobilization on Magnetic Nanoparticles

Expression of Bacillus amyloliquefaciens γ-Glutamyltransferase in Lactococcus lactis and Immobilization on Magnetic Nanoparticles

Characterization of three γ-glutamyltranspeptidases from <i>Pseudomonas aeruginosa</i> PAO1

Bacterial Gamma-Glutamyl Transpeptidase, an Emerging Biocatalyst: Insights Into Structure–Function Relationship and Its Biotechnological Applications

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