PMID:21664974

Citation	Pival, SL, Birner-Gruenberger, R, Krump, C and Nidetzky, B (2011) D-Xylulose kinase from Saccharomyces cerevisiae: isolation and characterization of the highly unstable enzyme, recombinantly produced in Escherichia coli. Protein Expr. Purif. 79:223-30
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).
Links	PubMed PMC3158326 Online version:10.1016/j.pep.2011.05.018
Keywords	Amino Acid Sequence; Bacterial Proteins/pharmacology; Cloning, Molecular; Electrophoresis, Polyacrylamide Gel; Enzyme Stability/drug effects; Escherichia coli; Excipients/pharmacology; Half-Life; Kinetics; Molecular Sequence Data; Oligopeptides/genetics; Oligopeptides/metabolism; Phosphotransferases (Alcohol Group Acceptor)/genetics; Phosphotransferases (Alcohol Group Acceptor)/isolation & purification; Phosphotransferases (Alcohol Group Acceptor)/metabolism; Plasmids; Recombinant Proteins/genetics; Recombinant Proteins/isolation & purification; Recombinant Proteins/metabolism; Saccharomyces cerevisiae/enzymology; Saccharomyces cerevisiae/genetics; Transformation, Bacterial; Xylulose/metabolism
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
YEAST:XKS1		GO:0042732: D-xylose metabolic process	ECO:0000314:		P		Figure 4: Michaelis-Menton plot is used to present the Kinetic analysis data relating the ATP-dependent phosphorylation of D-xylulose by purified S. cervisiae XKS1.	complete

References

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