Expression and purification of active human internal His6-tagged l-glutamine: d-Fructose-6P amidotransferase I

Richez, Céline; Boetzel, Joachim; Floquet, Nicolas; Kinnera, Koteshwar; Stevens, Julie M.; Badet, Bernard; Badet‐Denisot, Marie‐Ange

doi:10.1016/j.pep.2007.01.015

Cited by 33 publications

(40 citation statements)

References 39 publications

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“…Although the quantitative data are lacking, protein stabilization has always been of concern, especially during purification of the mammalian protein. 7 The identification of the hinge regions (peptide linker, C-tail) associated with the functional motions of GlmS also provides structural explanations of experimental observations, such as the high sensitivity of the enzyme to any modification of the peptide linker.…”

Section: Discussionmentioning

confidence: 99%

“…6,7 In the absence of X-ray structural data for the human enzyme, GlmS can be considered as a good model, since it has 40% global sequence identity, and almost 100% sequence identity in the functional regions (actives sites, ammonia channel). During the past decade, the crystallographic structures of GlmS have emphasized the contribution of the Q-loop (residues 73-87 in the glutaminase domain) and of the C-tail (residues 598-608 in the isomerase domain) in both ammonia channelling and ligand binding.…”

Section: Introductionmentioning

confidence: 99%

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Collective motions in Glucosamine-6-phosphate Synthase: Influence of Ligand Binding and role in Ammonia Channelling and Opening of the Fructose-6-Phosphate Binding Site

Floquet

Durand

Maigret

et al. 2009

Journal of Molecular Biology

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Collective motions in Glucosamine-6-phosphate Synthase: Influence of Ligand Binding and role in Ammonia Channelling and Opening of the Fructose-6-Phosphate Binding Site

Floquet

Durand

Maigret

et al. 2009

Journal of Molecular Biology

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“…; Richez et al . ). In fact, the enzyme does not tolerate even minimal modifications of its N‐terminus (Isupov et al .…”

Section: Discussionmentioning

confidence: 99%

“…The only successful strategy was a construction of human GlcN‐6‐P synthase containing an hexaHis insert at the GAH domain (Richez et al . ).…”

Section: Introductionmentioning

confidence: 97%

Engineering Candida albicans glucosamine‐6‐phosphate synthase for efficient enzyme purification

Czarnecka

Kwiatkowska

Gabriel

et al. 2012

J of Molecular Recognition

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Rationally designed muteins of Candida albicans glucosamine-6-phosphate synthase, an enzyme known as a promising target for antifungal chemotherapy, were constructed, overexpressed in Escherichia coli and purified to near homogeneity. To facilitate and to optimize the purification of the enzyme, three recombinant versions containing internal oligoHis fragments were constructed: (i) by substituting residues 343-348 of the interdomain undecapeptide linker with hexaHis, (ii) by replacing solvent-exposed residues 655-660 of the isomerase domain with hexaHis, and (iii) by replacing amino acids at positions 568 and 569 with His residues to generate the three-dimensional hexaHis microdomain in the enzyme quaternary structure. The resulting constructs were effectively purified to near homogeneity by rapid, one-step immobilized metal-ion affinity chromatography and demonstrated activity and catalytic properties comparable with that of the wild-type enzyme. The construct containing the 655-660 hexaHis insert was found to be a homodimeric protein, which is the first reported example of such quaternary structure of glucosamine-6-phosphate synthase of eukaryotic origin.

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“…GFATs have been isolated and characterized from numerous sources, e.g., Escherichia coli (Badet et al 1987), Aedes aegypti (Kato et al 2002), yeast (Watzele and Tanner 1989), mouse (Hu et al 2004) and Homo sapiens (McKnight et al 1992;Richez et al 2007;Li et al 2007). In mammals, two genes (GFAT1 and GFAT2) have been reported, whereas only one (GFAT1) exists in fungi (Milewski 2002).…”

Section: Introductionmentioning

confidence: 99%

Glucosamine: fructose-6-phosphate amidotransferase in the white shrimp Litopenaeus vannamei: characterization and regulation under alkaline and cadmium stress

et al. 2015

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Heavy metal residues and chemical contaminators considered as relevant sources of aquatic environmental pollutants have a generally immunosuppressive effect on aquatic organisms, depressing metabolic activities and immune response. Glutamine: fructose-6-phosphate aminotransferase (GFAT, EC2.6.1.16) is the first, and rate-limiting, enzyme in the hexosamine biosynthetic pathway, and is involved in the regulation of chitin biosynthesis and glycosylation of proteins. We have isolated and characterized GFAT from the white shrimp Litopenaeus vannamei. Amino acid sequence similarity of the Lv-GFAT (L.vannamei-GFAT) was highest to GFATs isolated from insects and mammals (83 % similarity to that of Haemaphysalis longicornis). The open-reading frame of the Lv-GFAT codes for a protein of 41.6 kDa with a calculated isoelectric point of 5.03. RT-PCR assays showed that endogenous Lv-GFAT mRNA is most strongly expressed in the intestine. Further analysis of Lv-GFAT gene expression in hepatopancreas by quantitative real-time PCR demonstrated that Lv-GFAT transcript levels increased when the shrimp were exposed to alkaline pH (9.3) and cadmium stress, but the time when its mRNA expression level peaked differed under these stresses. We also first expressed the recombinant protein of GFAT from shrimps in Escherichia coli. Western blot analyses confirmed that the Lv-GFAT protein was strongly expressed in the hepatopancreas after exposure to the LC-Cd stress. These results suggest that Lv-GFAT expression is stimulated by alkaline pH and cadmium stress and that it may play important roles in resistance of shrimp to environmental stresses.

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Expression and purification of active human internal His6-tagged l-glutamine: d-Fructose-6P amidotransferase I

Cited by 33 publications

References 39 publications

Collective motions in Glucosamine-6-phosphate Synthase: Influence of Ligand Binding and role in Ammonia Channelling and Opening of the Fructose-6-Phosphate Binding Site

Collective motions in Glucosamine-6-phosphate Synthase: Influence of Ligand Binding and role in Ammonia Channelling and Opening of the Fructose-6-Phosphate Binding Site

Engineering Candida albicans glucosamine‐6‐phosphate synthase for efficient enzyme purification

Glucosamine: fructose-6-phosphate amidotransferase in the white shrimp Litopenaeus vannamei: characterization and regulation under alkaline and cadmium stress

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