Alpert C A, Chassy B M
Laboratory for Microbial Ecology, National Institute for Dental Research, National Institutes of Health, Bethesda, Maryland 20892.
J Biol Chem. 1990 Dec 25;265(36):22561-8.
The gene coding for the lactose-specific Enzyme II of the Lactobacillus casei phosphoenolpyruvate-dependent phosphotransferase system, lacE, has been isolated by molecular cloning and expressed in Escherichia coli. The DNA sequence of the lacE gene and the deduced amino acid sequence are presented. The putative translation product comprises a hydrophobic protein of 577 amino acids with a calculated molecular mass of 62,350 Da. The deduced polypeptide has a high degree of sequence similarity with the corresponding lactose-specific enzymes II of Staphylococcus aureus and Lactococcus lactis. The sequence surrounding cysteine 483 was strongly conserved in the three proteins. The identity of the lacE product as the Enzyme IIlacL.casei was demonstrated by in vitro lactose phosphorylation assays using the protein expressed in E. coli. Single replacement of each of the histidine and cysteine residues by site-directed mutagenesis pointed to cysteine 483 as an amino acid residue essential for the phosphoryl group transfer reaction.
通过分子克隆分离出了干酪乳杆菌磷酸烯醇丙酮酸依赖性磷酸转移酶系统中乳糖特异性酶II的编码基因lacE，并在大肠杆菌中进行了表达。给出了lacE基因的DNA序列和推导的氨基酸序列。推测的翻译产物是一种由577个氨基酸组成的疏水蛋白，计算分子量为62350 Da。推导的多肽与金黄色葡萄球菌和乳酸乳球菌相应的乳糖特异性酶II具有高度的序列相似性。三种蛋白质中围绕半胱氨酸483的序列高度保守。使用在大肠杆菌中表达的蛋白质进行的体外乳糖磷酸化试验证明了lacE产物作为干酪乳杆菌酶IIlacL的身份。通过定点诱变对组氨酸和半胱氨酸残基进行单取代表明，半胱氨酸483是磷酰基转移反应所必需的氨基酸残基。
