title: D-Xylulose kinase from Saccharomyces cerevisiae: isolation and characterization of the highly unstable enzyme, recombinantly produced in Escherichia coli.
abstract: The Saccharomyces cerevisiae gene encoding xylulose kinase (XKS1) was over-expressed to an abundance of ≥ 10% intracellular protein in Escherichia coli. Instability of XKS1, not pointed out in previous reports of the enzyme, prevented isolation of active enzyme in native or "tagged" form under a wide range of purification conditions. A fusion protein haboring C-terminal Strep-tag II (XKS1-Strep) displayed activity (∼20 U/mg) as isolated. However, the half-life time of purified XKS1-Strep was only ∼1.5h at 4°C and could not be enhanced substantially by an assortment of extrinsic stabilizers (osmolytes, protein, substrates). Peptide mass mapping and N-terminal sequencing showed that the recombinant protein was structurally intact, ruling out proteolytic processing and chemical modifications as possible factors to compromise the stability of the enzyme as isolated. Partial functional complementation of a largely inactive XKS1 preparation by the high-molecular mass fraction (≥ 10kDa) of cell extract prepared from an E. coli BL21 (DE3) expression host suggests a possible role for heterotropic protein-XKS1 interactions in conferring activity/stability to the enzyme. Michaelis-Menten constants of XKS1-Strep were determined: d-xylulose (210 ± 40 μM) and Mg(2+)-ATP (1.70 ± 0.10 mM).
authors.affiliation: Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12, A-8010 Graz, Austria.
authors.forename: Simone L
authors.initials: SL
authors.lastname: Pival
authors.forename: Ruth
authors.initials: R
authors.lastname: Birner-Gruenberger
authors.forename: Corinna
authors.initials: C
authors.lastname: Krump
authors.forename: Bernd
authors.initials: B
authors.lastname: Nidetzky
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000494:pharmacology*; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability / Q000187:drug effects; D004926:Escherichia coli; D005079:Excipients / Q000494:pharmacology*; D006207:Half-Life; D007700:Kinetics; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D014169:Transformation, Bacterial; D014996:Xylulose / Q000378:metabolism*
chemical_list: C099517:Ala-Trp-Arg-His-Pro-Gln-Phe-Gly-Gly; D001426:Bacterial Proteins; D005079:Excipients; D009842:Oligopeptides; D011994:Recombinant Proteins; D014996:Xylulose; D017853:Phosphotransferases (Alcohol Group Acceptor); C032700:xylulokinase