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 (similar to 20 U/mg) as isolated. However, the half-life time of purified XKS1-Strep was only similar to 1.5 h at 4 degrees 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 (>= 10 kDa) 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 mu M) and Mg(2+)-ATP (1.70 +/- 0.10 mM). (C) 2011 Elsevier Inc. All rights reserved,