Construction, purification, and functional characterization of His-tagged Candida albicans glucosamine-6-phosphate synthase expressed in Escherichia coli

Olchowy, Jarosław; Kur, Krzysztof; Sachadyn, Paweł; Milewski, Sławomir

doi:10.1016/j.pep.2005.07.030

Cited by 23 publications

(17 citation statements)

References 20 publications

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“…The recombinant LipA (r-LipA) was in a soluble form, which was further purified using Ni-NTA affinity chromatography [22,23]. SDS-PAGE analysis of the purified r-LipA showed a single band corresponding to approximate 35.6 kDa (Figure 2), which is consistent with the calculated molecular mass of the recombinant protein.…”

Section: Resultsmentioning

confidence: 54%

A Novel Cold-Adapted Lipase from Sorangium cellulosum Strain So0157-2: Gene Cloning, Expression, and Enzymatic Characterization

Cheng

Qian

et al. 2011

IJMS

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Genome sequencing of cellulolytic myxobacterium Sorangium cellulosum reveals many open-reading frames (ORFs) encoding various degradation enzymes with low sequence similarity to those reported, but none of them has been characterized. In this paper, a predicted lipase gene (lipA) was cloned from S. cellulosum strain So0157-2 and characterized. lipA is 981-bp in size, encoding a polypeptide of 326 amino acids that contains the pentapeptide (GHSMG) and catalytic triad residues (Ser114, Asp250 and His284). Searching in the GenBank database shows that the LipA protein has only the 30% maximal identity to a human monoglyceride lipase. The novel lipA gene was expressed in Escherichia coli BL21 and the recombinant protein (r-LipA) was purified using Ni-NTA affinity chromatography. The enzyme hydrolyzed the p-nitrophenyl (pNP) esters of short or medium chain fatty acids (≤C10), and the maximal activity was on pNP acetate. The r- LipA is a cold-adapted lipase, with high enzymatic activity in a wide range of temperature and pH values. At 4 °C and 30 °C, the Km values of r-LipA on pNP acetate are 0.037 ± 0.001 and 0.174 ± 0.006 mM, respectively. Higher pH and temperature conditions promoted hydrolytic activity toward the pNP esters with longer chain fatty acids. Remarkably, this lipase retained much of its activity in the presence of commercial detergents and organic solvents. The results suggest that the r-LipA protein has some new characteristics potentially promising for industrial applications and S. cellulosum is an intriguing resource for lipase screening.

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Section: Resultsmentioning

confidence: 54%

A Novel Cold-Adapted Lipase from Sorangium cellulosum Strain So0157-2: Gene Cloning, Expression, and Enzymatic Characterization

Cheng

Qian

et al. 2011

IJMS

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“…Incorporation of 13 C into 2,5‐anhydromannitol, the deaminated product of GlcN, was determined from the ratio of isotopomer ions at m/z 272 (M, unlabelled) and 278 (M+6, uniformally labelled). GFAT activity was also measured in hypotonically lyzed cell extracts using the Morgan‐Elson reagent (Olchowy et al ., 2006).…”

Section: Methodsmentioning

confidence: 99%

Role of hexosamine biosynthesis in Leishmania growth and virulence

Naderer

Wee

McConville³

2008

Molecular Microbiology

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SummaryLeishmania parasites incorporate N-acetylglucosamine (GlcNAc) into surface-expressed glycosylphosphatidylinositol (GPI) glycolipids and N-linked glycans. To investigate whether these glycoconjugates are required for infectivity of promastigote and intracellular amastigote stages, we generated a Leishmania major mutant lacking the gene encoding glutamine : fructose-6-phosphate amidotransferase (GFAT). The L. major Dgfat mutant is unable to synthesize GlcN-6-phosphate de novo and is auxotrophic for GlcN or GlcNAc. GlcN starvation leads to the rapid depletion of dolichol-linked oligosaccharides and GPI precursors, hypersensitivity to elevated temperatures encountered in the mammalian host and eventual parasite death. Short-term tunicamycin treatment induces a similar hypersensitivity to temperature, indicating that N-linked glycans are required for thermotolerance and viability. L. major Dgfat promastigotes are unable to proliferate in ex vivo infected macrophages, demonstrating that GlcN(Ac) levels in the phagolysosome are low. In contrast, Dgfat amastigotes grow as well as wild-type amastigotes in macrophages and induce lesions in susceptible mice. These stages still require GlcN(Ac) for viability but can apparently scavenge all of their glucosamine requirements from the macrophage phagolysosome. These results highlight significant differences in the nutrient requirements of promastigote and amastigote stages and suggest that enzymes involved in UDP-GlcNAc biosynthesis are essential for pathogenesis in the mammalian host.

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“…The construction of the recombinant versions of the enzyme, His 6 ‐tagged at either N‐ or C‐terminus, enabled much faster and more efficient purification, but the constructs could not catalyze GlcN‐6‐P formation (Olchowy et al . ). The GlcN‐6‐P synthase activity was also dramatically reduced in the N‐terminus‐linked fusion constructs of the human enzyme (Chang et al .…”

Section: Discussionmentioning

confidence: 97%

“…; Olchowy et al . ). 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: 99%

“…Unfortunately, eukaryotic GlcN-6-P synthase is an unstable protein and purification of the wild-type (WT) enzyme is labor intensive and inefficient (Milewski et al 1999;Huynh et al 2000;Milewski 2002;Gonzalez-Ibarra et al 2010). On the other hand, the construction of fusion versions of GlcN-6-P synthase containing oligoHis tags or glutathione-S-transferase at either N-or C-terminus resulted in muteins that could be efficiently purified but were very poorly active (Chang et al 2000;Teplyakov et al 2002;Olchowy et al 2006). The only successful strategy was a construction of human GlcN-6-P synthase containing an hexaHis insert at the GAH domain (Richez et al 2007).…”

Section: Introductionmentioning

confidence: 99%

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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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Construction, purification, and functional characterization of His-tagged Candida albicans glucosamine-6-phosphate synthase expressed in Escherichia coli

Cited by 23 publications

References 20 publications

A Novel Cold-Adapted Lipase from Sorangium cellulosum Strain So0157-2: Gene Cloning, Expression, and Enzymatic Characterization

A Novel Cold-Adapted Lipase from Sorangium cellulosum Strain So0157-2: Gene Cloning, Expression, and Enzymatic Characterization

Role of hexosamine biosynthesis in Leishmania growth and virulence

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

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