Purification and properties of fructokinase I from Lactococcus lactis. Localization of scrK on the sucrose-nisin transposon Tn5306

• Published in: The Journal of biological chemistry 1991-11, Vol.266 (33), p.22626-22633
• Main Authors: THOMPSON, J, SACKETT, D. L, DONKERSLOOT, J. A
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Biological and medical sciences; Carbohydrate Metabolism; Chromatography, Affinity; Chromatography, Gel; Chromatography, Ion Exchange; DNA Transposable Elements; Electrophoresis, Polyacrylamide Gel; Enzymes and enzyme inhibitors; Fructokinases - genetics; Fructokinases - isolation & purification; Fructokinases - metabolism; Fundamental and applied biological sciences. Psychology; genes; Genes, Bacterial; Isoenzymes - genetics; Isoenzymes - isolation & purification; Isoenzymes - metabolism; Kinetics; Lactococcus lactis; Lactococcus lactis - enzymology; Lactococcus lactis - genetics; Lactococcus lactis - growth & development; Macromolecular Substances; Molecular Weight; Protein Conformation; Restriction Mapping; Sucrose - metabolism; Transferases; transposons
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)54617-2

Two electrophoretically distinct proteins with fructokinase (ATP:fructose-6-phosphotransferase) activity were detected in Lactococcus lactis subsp. lactis K1. Whereas fructokinase I was induced specifically by growth of the organism on sucrose, fructokinase II was derepressed during growth on ribose, galactose, maltose, and lactulose. Fructokinase I was purified about 1000-fold to electrophoretic homogeneity (specific activity 112 units/mg). The amino acid composition, N-terminal sequence, nucleoside triphosphate, and metal requirement(s) of the enzyme are reported. Ultracentrifugal analysis showed that the enzyme was primarily dimeric with subunits of 33.5 kDa (+/- 5%). When completely reduced, fructokinase I migrated as a single protein (Mr = 32,000) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but in the absence of reducing agent two polypeptides (apparent Mr = 29,000 and 31,000) were detected. Isoelectric focusing also revealed two polypeptides (pI 5.6 and 5.8), and both species catalyzed the phosphorylation of fructose and mannose. Hybridization studies showed that: (i) a sucrose-negative mutant lacking the fructokinase I gene (scrK) retained fructokinase II activity and (ii) scrK is closely linked to scrA and scrB which encode Enzyme IIScr and sucrose-6-phosphate hydrolase, respectively. In L. lactis K1, these genes and the N5-(1-carboxyethyl)-L-ornithine synthase gene (ceo) are encoded on the sucrose-nisin transposon Tn5306 in the order ceo-scrKAB.