de Vos W M, Boerrigter I, van Rooyen R J, Reiche B, Hengstenberg W
Department of Biophysical Chemistry, Netherlands Institute for Dairy Research (NIZO), Ede.
J Biol Chem. 1990 Dec 25;265(36):22554-60.
The plasmid-encoded lactose genes of the Lactococcus lactis phosphotransferase system encoding Enzyme IIIlac (lacF) and Enzyme IIlac (lacE) have been identified and cloned in Escherichia coli and L. lactis. Nucleotide sequence and transcription analysis showed that these genes are organized into a lactose-inducible operon with the gene order lacF-lacE-lacG-lacX, the latter two genes encoding phospho-beta-galactosidase and a 34-kDa protein with an unknown function, respectively. The lac-operon is immediately followed by an IS element that is homologous to ISS1. Enzyme IIIlac was purified from L. lactis and determination of its NH2-terminal sequence demonstrated that the lacF gene starts with a TTG codon and encodes a 105 amino acid protein (Mr = 11416). Cross-linking studies with the purified enzyme showed that Enzyme IIIlac is active as a trimer. A mutant lacF gene was identified in strain YP2-5 and appeared to encode Enzyme IIIlac containing the missense mutation G18E. The lacF gene could be expressed under control of vector-located promoter sequences resulting in overproduction of Enzyme IIIlac in E. coli and complementation of the L. lactis lacF mutant YP2-5. The deduced amino acid sequence of Enzyme IIlac consists of 586 amino acids (Mr = 61562) and shows the characteristics of a hydrophobic, integral membrane protein. The deduced primary structures of the L. lactis Enzyme IIIlac and Enzyme IIlac are homologous to those of Staphylococcus aureus (72 and 71% identity, respectively) and Lactobacillus casei (48 and 47% identity, respectively). In contrast, the organization of the lactose genes differs significantly between those Gram-positive bacteria. Heterogramic homology in specific domains was observed between the derived amino acid sequences of the lactose-specific enzymes and that of E. coli Enzyme IIIcel and Enzyme IIcel, which suggest a common function in the transport and phosphorylation of these structurally related beta-glucosides.
乳酸乳球菌磷酸转移酶系统中编码酶Ⅲlac（lacF）和酶Ⅱlac（lacE）的质粒编码乳糖基因已在大肠杆菌和乳酸乳球菌中被鉴定并克隆。核苷酸序列和转录分析表明，这些基因被组织成一个乳糖诱导型操纵子，基因顺序为lacF-lacE-lacG-lacX，后两个基因分别编码磷酸β-半乳糖苷酶和一个功能未知的34 kDa蛋白质。lac操纵子紧接着一个与ISS1同源的IS元件。从乳酸乳球菌中纯化出酶Ⅲlac，对其NH2末端序列的测定表明，lacF基因起始于一个TTG密码子，编码一个105个氨基酸的蛋白质（Mr = 11416）。对纯化酶的交联研究表明，酶Ⅲlac以三聚体形式具有活性。在菌株YP2-5中鉴定出一个突变的lacF基因，它似乎编码含有错义突变G18E的酶Ⅲlac。lacF基因可以在载体定位的启动子序列控制下表达，从而导致大肠杆菌中酶Ⅲlac的过量产生，并对乳酸乳球菌lacF突变体YP2-5进行互补。推导的酶Ⅱlac氨基酸序列由586个氨基酸组成（Mr = 61562），显示出疏水整合膜蛋白的特征。乳酸乳球菌酶Ⅲlac和酶Ⅱlac的推导一级结构与金黄色葡萄球菌的相应结构同源（分别为72%和71%的同一性），与干酪乳杆菌的相应结构同源（分别为48%和47%的同一性）。相比之下，这些革兰氏阳性细菌中乳糖基因的组织方式有显著差异。在乳糖特异性酶的推导氨基酸序列与大肠杆菌酶Ⅲcel和酶Ⅱcel的推导氨基酸序列之间，在特定结构域观察到异源同源性，这表明在这些结构相关的β-葡萄糖苷的转运和磷酸化过程中具有共同功能。
