Faculty of Agriculture, Niigata University, Niigata 950-2181, Japan.
Carbohydr Res. 2012 Nov 1;361:49-54. doi: 10.1016/j.carres.2012.08.006. Epub 2012 Aug 21.
We identified a glycoside hydrolase family 94 homolog (ACL0729) from Acholeplasma laidlawii PG-8A as a laminaribiose (1,3-β-D-glucobiose) phosphorylase (EC 2.4.1.31). The recombinant ACL0729 produced in Escherichia coli catalyzed phosphorolysis of laminaribiose with inversion of the anomeric configuration in a typical sequential bi bi mechanism releasing α-D-glucose 1-phosphate and D-glucose. Laminaritriose (1,3-β-D-glucotriose) was not an efficient substrate for ACL0729. The phosphorolysis is reversible, enabling synthesis of 1,3-β-D-glucosyl disaccharides by reverse phosphorolysis with strict regioselectivity from α-D-glucose 1-phosphate as the donor and suitable monosaccharide acceptors (D-glucose, 2-deoxy-D-arabino-hexopyranose, D-xylose, D-glucuronic acid, 1,5-anhydro-D-glucitol, and D-mannose) with C-3 and C-4 equatorial hydroxyl groups. The D-glucose and 2-deoxy-D-arabino-hexopyranose caused significantly strong competitive substrate inhibition compared with other glucobiose phosphorylases reported, in which the acceptor competitively inhibited the binding of the donor substrate. By contrast, none of the examined disaccharides served as acceptor in the synthetic reaction.
我们从阿克氏菌 PG-8A 中鉴定出一种糖苷水解酶家族 94 同系物（ACL0729），它是一种纤维二糖（1,3-β-D-葡糖苷）磷酸化酶（EC 2.4.1.31）。在大肠杆菌中生产的重组 ACL0729 以典型的顺序双顺反子机制催化纤维二糖的磷酸解，使糖苷配基的构型反转，释放α-D-葡萄糖 1-磷酸和 D-葡萄糖。纤维三糖（1,3-β-D-葡三糖）不是 ACL0729 的有效底物。磷酸解是可逆的，使 1,3-β-D-葡萄糖基二糖能够通过从α-D-葡萄糖 1-磷酸作为供体和合适的单糖受体（D-葡萄糖、2-脱氧-D-阿拉伯己糖、D-木糖、D-葡萄糖醛酸、1,5-脱水-D-山梨醇和 D-甘露糖）以 C-3 和 C-4 平伏羟基进行严格区域选择性的反向磷酸解合成。与已报道的其他葡糖磷酸化酶相比，D-葡萄糖和 2-脱氧-D-阿拉伯己糖对该酶具有明显的强竞争性底物抑制作用，其中受体竞争性地抑制供体底物的结合。相比之下，在合成反应中，没有一种二糖可作为受体。
