Thompson J, Sackett D L, Donkersloot J A
Laboratory of Microbial Ecology, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892.
J Biol Chem. 1991 Nov 25;266(33):22626-33.
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.
在乳酸乳球菌乳酸亚种K1中检测到两种具有果糖激酶（ATP：果糖-6-磷酸转移酶）活性的电泳性质不同的蛋白质。果糖激酶I是在该生物体以蔗糖为碳源生长时特异性诱导产生的，而果糖激酶II则在以核糖、半乳糖、麦芽糖和乳果糖为碳源生长时去阻遏表达。果糖激酶I被纯化了约1000倍，达到电泳纯（比活性为112单位/毫克）。报道了该酶的氨基酸组成、N端序列、核苷三磷酸以及金属需求。超速离心分析表明，该酶主要是由33.5 kDa（±5%）的亚基组成的二聚体。当完全还原时，果糖激酶I在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳中迁移为单一蛋白（Mr = 32,000），但在没有还原剂的情况下，检测到两条多肽（表观Mr = 29,000和31,000）。等电聚焦也显示出两条多肽（pI 5.6和5.8），并且这两种蛋白都催化果糖和甘露糖的磷酸化。杂交研究表明：（i）一个缺乏果糖激酶I基因（scrK）的蔗糖阴性突变体保留了果糖激酶II活性，（ii）scrK与分别编码蔗糖酶IIscr和蔗糖-6-磷酸水解酶的scrA和scrB紧密连锁。在乳酸乳球菌K1中，这些基因和N5-(1-羧乙基)-L-鸟氨酸合酶基因（ceo）按ceo-scrKAB的顺序编码在蔗糖-乳链菌肽转座子Tn5306上。
