title: Engineering Candida albicans glucosamine-6-phosphate synthase for efficient enzyme purification.
abstract: 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.
authors.affiliation: Department of Pharmaceutical Technology and Biochemistry, Gdańsk University of Technology, Poland.
authors.forename: Justyna
authors.initials: J
authors.lastname: Czarnecka
authors.forename: Karolina
authors.initials: K
authors.lastname: Kwiatkowska
authors.forename: Iwona
authors.initials: I
authors.lastname: Gabriel
authors.forename: Marek
authors.initials: M
authors.lastname: Wojciechowski
authors.forename: Sławomir
authors.initials: S
authors.lastname: Milewski
mesh_terms: D055162:Biocatalysis; D002176:Candida albicans / Q000737:chemistry / Q000201:enzymology*; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D015202:Protein Engineering; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing)