Purification and properties of fructokinase I from Lactococcus lactis. Localization of scrK on the sucrose-nisin transposon Tn5306.
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.
Thompson
J
J
Laboratory of Microbial Ecology, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892.
Sackett
D L
DL
Donkersloot
J A
JA
D050260:Carbohydrate Metabolism; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004251:DNA Transposable Elements; D004591:Electrophoresis, Polyacrylamide Gel; D005631:Fructokinases / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D005798:Genes, Bacterial; D007527:Isoenzymes / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D013294:Lactococcus lactis / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D046911:Macromolecular Substances; D008970:Molecular Weight; D011487:Protein Conformation; D015183:Restriction Mapping; D013395:Sucrose / Q000378:metabolism
D004251:DNA Transposable Elements; D007527:Isoenzymes; D046911:Macromolecular Substances; D013395:Sucrose; D005631:Fructokinases; C046173:fructokinase