Abstract
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.
Highlights
Two electrophoretically distinct proteins with fructokinase (ATP:fructose-6-phosphotransferase)activitywere detected in Lactococcuslactis subsp. lactis K1
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
The findings of a study conducted almost 10 years ago [8]suggested that expression of scrK was regulated independently of scrA and scrB. Clarification of these issues and localization of scrK in L. lactis required the purification and determinationof the N-terminalsequence of the ATP:fructose-6-phosphotransferase.This enzyme will be designated fructokinase I, because in the course of our experiments we discovered, unexpectedly, a second enzyme which exhibited fructokinaseactivity
Summary
3) and Gram-positive bacterialspecies [4,5,6,7] including Lactococcus lactis [8, 9] depends initially upon thevectorial translocation and phosphorylationof the disaccharide mediatebdy an inducible phosphoenolpyruvate-dependent sucrose:phosphotransferasesystem(sucrose:PEP-PTS)’ [10, 11]. Raphy and gel filtration (Ultrogel AcA 54) chromatography PAGE performed after complete reduction of the sample by (Fig. 2, Miniprint Section) were efficient purification steps and the latter procedure alone resulted in a 30-fold increase in specific activity of the enzyme.Analysis of the AcA 54 preparation by SDS-PAGE (Fig. 3A, lune 5 ) revealed one major and several minor proteins. During nondenaturingequilibrium ultracentrifugation a t 25 “C,fructokinase I behavedas a larger molecule (Fig. 5, panel B) When this pattern was analyzed assuming a single component, the best fitwas obtained with a mass of 60kDa (data not shown).Because this value is incompatible with thepreviously determined subunitmass of 33 kDa, and since fittingfor a dimer of mass 66 kDa yielded significantly nonrandom residuals (data not shown), the pattern was re-analyzed with a monomer(33 kDa)-dimer model. Of potential phosphoryl donors (Table4,Miniprint Section) ATPelicited the highest rate of fructose phosphorylation, but othenrucleoside triphosphates whichserved asdonors(in decreasing
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