Charbit A, Reizer J, Saier M H
Department of Biology, University of California at San Diego, La Jolla, California 92093-0116, USA.
J Biol Chem. 1996 Apr 26;271(17):9997-10003. doi: 10.1074/jbc.271.17.9997.
The fructose permease of Escherichia coli, the fructose-specific Enzyme II of the phosphoenolpyruvate-dependent phosphotransferase system (PTS), contains a duplicated IIB domain. The protein therefore consists of three distinct domains, B', B, and C (N-terminal to C-terminal), joined by flexible linkers and is thus designated FruB'BC. The N-terminal B' domain was removed using molecular genetic techniques, and the truncated Enzyme II (FruBC) was characterized relative to the wild-type enzyme both in vivo and in vitro. In vivo, FruBC exhibited depressed fermentation characteristics at low fructose concentrations. [14C]Fructose uptake measurements revealed reduced rates only when the permease was rate-limiting for transport. In vitro, FruBC exhibited a 10-fold lower affinity for its phosphoryl donating protein, the IIA-FPr diphosphoryl transfer protein (DTP), than was observed with the wild-type enzyme, and the maximal velocity of fructose phosphorylation was 7-fold depressed. Because the fructose-1-phosphate:[14C]fructose transphosphorylation reaction appeared normal, we conclude that the loss of the B' domain primarily affected phosphoryl transfer between the IIA and IIB domains of the permease. A mutant FruBC derivative with cysteine 112 replaced by serine (C112S FruBC) was inactive as a phosphoryl carrier and a sugar transport protein. Expression of the plasmid-encoded mutant protein inhibited the in vivo activity of the chromosomally encoded wild-type fructose permease, but it did not observably affect the activities of the mannitol or glucitol PTS permeases or of non-PTS sugar permeases. Further, the presence of the detergent extracted mutant protein inhibited the activity of the detergent solubilized wild-type or FruBC enzyme. In contrast, the wild-type FruB BC permease was apparently epistatic over the truncated FruBC permease in vivo. The experiments reported 1) show that the B' domain of the fructose permease functions to facilitate phosphoryl transfer between DTP and the permease, 2) establish the essentiality of cysteine 112 in the B domain of the permease, 3) provide evidence that a functional fructose permease consists of an oligomer in which both IIB domains must be active for the enzyme to catalyze normal rates of phosphoryl transfer and transport, 4) suggest that a single B' domain in the oligomeric Enzyme II is sufficient to allow high efficiency phosphoryl transfer between the IIA domain of DTP and the IIB domain of the permease, and 5) show that the B' domain is not important for oligomerization.
大肠杆菌的果糖通透酶是磷酸烯醇丙酮酸依赖性磷酸转移酶系统（PTS）中对果糖具有特异性的酶II，它包含一个重复的IIB结构域。因此，该蛋白由三个不同的结构域组成，即B'、B和C（从N端到C端），由柔性连接子相连，因此被命名为FruB'BC。利用分子遗传学技术去除了N端的B'结构域，并对截短后的酶II（FruBC）在体内和体外相对于野生型酶的特性进行了表征。在体内，FruBC在低果糖浓度下表现出较低的发酵特性。[14C]果糖摄取测量结果显示，只有当通透酶对转运起限速作用时，摄取速率才会降低。在体外，FruBC对其磷酰基供体蛋白IIA-FPr双磷酸转移蛋白（DTP）的亲和力比野生型酶低10倍，果糖磷酸化的最大速度降低了7倍。由于果糖-1-磷酸：[14C]果糖转磷酸化反应看起来正常，我们得出结论，B'结构域的缺失主要影响了通透酶IIA和IIB结构域之间的磷酰基转移。一种将半胱氨酸112替换为丝氨酸的FruBC突变衍生物（C112S FruBC）作为磷酰基载体和糖转运蛋白是无活性的。质粒编码的突变蛋白的表达抑制了染色体编码的野生型果糖通透酶的体内活性，但未明显影响甘露醇或葡糖醇PTS通透酶或非PTS糖通透酶的活性。此外，去污剂提取的突变蛋白的存在抑制了去污剂溶解的野生型或FruBC酶的活性。相反，在体内野生型FruB BC通透酶显然对截短的FruBC通透酶呈上位性。所报道的实验表明：1）果糖通透酶的B'结构域起到促进DTP和通透酶之间磷酰基转移的作用；2）确定了通透酶B结构域中半胱氨酸112的必要性；3）提供了证据表明功能性果糖通透酶由一种寡聚体组成，其中两个IIB结构域都必须具有活性，酶才能催化正常速率的磷酰基转移和转运；4）表明寡聚酶II中的单个B'结构域足以使DTP的IIA结构域和通透酶的IIB结构域之间进行高效的磷酰基转移；5）表明B'结构域对寡聚化并不重要。
