Rimmele M, Boos W
Department of Biology, University of Konstanz, Germany.
J Bacteriol. 1994 Sep;176(18):5654-64. doi: 10.1128/jb.176.18.5654-5664.1994.
The disaccharide trehalose acts as an osmoprotectant as well as a carbon source in Escherichia coli. At high osmolarity of the growth medium, the cells synthesize large amounts of trehalose internally as an osmoprotectant. However, they can also degrade trehalose as the sole source of carbon under both high- and low-osmolarity growth conditions. The modes of trehalose utilization are different under the two conditions and have to be well regulated (W. Boos, U. Ehmann, H. Forkl, W. Klein, M. Rimmele, and P. Postma, J. Bacteriol. 172:3450-3461, 1990). At low osmolarity, trehalose is transported via a trehalose-specific enzyme II of the phosphotransferase system, encoded by treB. The trehalose-6-phosphate formed internally is hydrolyzed to glucose and glucose 6-phosphate by the key enzyme of the system, trehalose-6-phosphate hydrolase, encoded by treC. We have cloned treC, contained in an operon with treB as the promoter-proximal gene. We have overproduced and purified the treC gene product and identified it as a protein consisting of a single polypeptide with an apparent molecular weight of 62,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme hydrolyzes trehalose-6-phosphate with a Km of 6 mM and a Vmax of at least 5.5 mumol of trehalose-6-phosphate hydrolyzed per min per mg of protein. The enzyme also very effectively hydrolyzes p-nitrophenyl-alpha-D-glucopyranoside, but it does not recognize trehalose, sucrose, maltose, isomaltose, or maltodextrins. treC was sequenced and found to encode a polypeptide with a calculated molecular weight of 63,781. The amino acid sequence deduced from the DNA sequence shows homology (50% identity) with those of oligo-1,6-glucosidases (sucrase-isomaltases) of Bacillus spp. but not with those of other disaccharide phosphate hydrolases. This report corrects our previous view on the function of the treC gene product as an amylotrehalase, which was based on the analysis of the metabolic products of trehalose metabolism in whole cells.
双糖海藻糖在大肠杆菌中既作为渗透保护剂又作为碳源。在生长培养基高渗透压条件下，细胞在内部合成大量海藻糖作为渗透保护剂。然而，在高渗透压和低渗透压生长条件下，它们也能将海藻糖降解作为唯一碳源。在这两种条件下，海藻糖的利用模式不同，必须得到良好调控（W. 布斯、U. 埃曼、H. 福尔克尔、W. 克莱因、M. 林梅勒和P. 波斯特马，《细菌学杂志》172:3450 - 3461, 1990）。在低渗透压下，海藻糖通过磷酸转移酶系统的一种海藻糖特异性酶II进行转运，该酶由treB编码。在细胞内部形成的海藻糖 - 6 - 磷酸被该系统的关键酶海藻糖 - 6 - 磷酸水解酶水解为葡萄糖和葡萄糖6 - 磷酸，该水解酶由treC编码。我们克隆了treC，它与treB包含在一个操纵子中，treB是启动子近端基因。我们过量表达并纯化了treC基因产物，通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳确定其为一种由单一多肽组成的蛋白质，表观分子量为62,000。该酶水解海藻糖 - 6 - 磷酸的米氏常数为6 mM，最大反应速度为每毫克蛋白质每分钟至少水解5.5 μmol海藻糖 - 6 - 磷酸。该酶也能非常有效地水解对硝基苯基 - α - D - 吡喃葡萄糖苷，但它不识别海藻糖、蔗糖、麦芽糖、异麦芽糖或麦芽糊精。对treC进行了测序，发现它编码一种计算分子量为63,781的多肽。从DNA序列推导的氨基酸序列与芽孢杆菌属的寡 - 1,6 - 葡萄糖苷酶（蔗糖 - 异麦芽糖酶）的氨基酸序列具有同源性（50% 同一性），但与其他二糖磷酸水解酶的氨基酸序列无同源性。本报告纠正了我们之前基于对全细胞中海藻糖代谢产物分析而认为treC基因产物是一种淀粉海藻糖酶的观点。
