Taguchi Yodai, Saburi Wataru, Imai Ryozo, Mori Haruhide
a Research Faculty of Agriculture , Hokkaido University , Sapporo , Japan.
b Division of Applied Genetics , National Agriculture and Food Research Organization , Tsukuba , Japan.
Biosci Biotechnol Biochem. 2017 Aug;81(8):1512-1519. doi: 10.1080/09168451.2017.1329620. Epub 2017 May 24.
Trehalose 6-phosphate phosphorylase (TrePP), a member of glycoside hydrolase family 65, catalyzes the reversible phosphorolysis of trehalose 6-phosphate (Tre6P) with inversion of the anomeric configuration to produce β-d-glucose 1-phosphate (β-Glc1P) and d-glucose 6-phosphate (Glc6P). TrePP in Lactococcus lactis ssp. lactis (LlTrePP) is, alongside the phosphotransferase system, involved in the metabolism of trehalose. In this study, recombinant LlTrePP was produced and characterized. It showed its highest reverse phosphorolytic activity at pH 4.8 and 40°C, and was stable in the pH range 5.0-8.0 and at up to 30°C. Kinetic analyses indicated that reverse phosphorolysis of Tre6P proceeded through a sequential bi bi mechanism involving the formation of a ternary complex of the enzyme, β-Glc1P, and Glc6P. Suitable acceptor substrates were Glc6P, and, at a low level, d-mannose 6-phosphate (Man6P). From β-Glc1P and Man6P, a novel sugar phosphate, α-d-Glcp-(1↔1)-α-d-Manp6P, was synthesized with 51% yield.
海藻糖6-磷酸磷酸化酶（TrePP）是糖苷水解酶家族65的成员，催化海藻糖6-磷酸（Tre6P）的可逆磷酸解反应，使异头构型发生转化，生成β-D-葡萄糖1-磷酸（β-Glc1P）和D-葡萄糖6-磷酸（Glc6P）。乳酸乳球菌亚种乳酸乳球菌（LlTrePP）中的TrePP与磷酸转移酶系统一起参与海藻糖的代谢。在本研究中，制备并表征了重组LlTrePP。它在pH 4.8和40°C时显示出最高的反向磷酸解活性，在pH范围5.0 - 8.0和高达30°C时稳定。动力学分析表明，Tre6P的反向磷酸解通过有序的双双机制进行，涉及酶、β-Glc1P和Glc6P三元复合物的形成。合适的受体底物是Glc6P，以及低水平的D-甘露糖6-磷酸（Man6P）。由β-Glc1P和Man6P合成了一种新型糖磷酸酯α-D-Glcp-(1↔1)-α-D-Manp6P，产率为51%。
