Thompson J, Robrish S A, Bouma C L, Freedberg D I, Folk J E
Laboratory of Microbial Ecology, NIDR, National Institutes of Health, Bethesda, Maryland 20892, USA.
J Bacteriol. 1997 Mar;179(5):1636-45. doi: 10.1128/jb.179.5.1636-1645.1997.
6-Phosphoryl-beta-D-glucopyranosyl:6-phosphoglucohydrolase (P-beta-glucosidase, EC 3.2.1.86) has been purified from Fusobacterium mortiferum. Assays for enzyme activity and results from Western immunoblots showed that P-beta-glucosidase (Mr, 53,000; pI, 4.5) was induced by growth of F. mortiferum on beta-glucosides. The novel chromogenic and fluorogenic substrates, p-nitrophenyl-beta-D-glucopyranoside-6-phosphate (pNPbetaGlc6P) and 4-methylumbelliferyl-beta-D-glucopyranoside-6-phosphate (4MUbetaGlc6P), respectively, were used for the assay of P-beta-glucosidase activity. The enzyme hydrolyzed several P-beta-glucosides, including the isomeric disaccharide phosphates cellobiose-6-phosphate, gentiobiose-6-phosphate, sophorose-6-phosphate, and laminaribiose-6-phosphate, to yield glucose-6-phosphate and appropriate aglycons. The kinetic parameters for each substrate are reported. P-beta-glucosidase from F. mortiferum was inactivated by 6-phosphoglucono-delta-lactone (P-glucono-delta-lactone) derived via oxidation of glucose 6-phosphate. The pbgA gene that encodes P-beta-glucosidase from F. mortiferum has been cloned and sequenced. The first 42 residues deduced from the nucleotide sequence matched those determined for the N terminus by automated Edman degradation of the purified enzyme. From the predicted sequence of 466 amino acids, two catalytically important glutamyl residues have been identified. Comparative alignment of the amino acid sequences of P-beta-glucosidase from Escherichia coli and F. mortiferum indicates potential binding sites for the inhibitory P-glucono-delta-lactone to the enzyme from F. mortiferum.
6-磷酸-β-D-吡喃葡萄糖基：6-磷酸葡萄糖水解酶（P-β-葡萄糖苷酶，EC 3.2.1.86）已从死亡梭杆菌中纯化出来。酶活性测定和蛋白质免疫印迹结果表明，P-β-葡萄糖苷酶（分子量53,000；等电点4.5）是在死亡梭杆菌以β-葡萄糖苷为底物生长时被诱导产生的。分别使用新型生色和荧光底物对硝基苯基-β-D-吡喃葡萄糖苷-6-磷酸（pNPβGlc6P）和4-甲基伞形酮基-β-D-吡喃葡萄糖苷-6-磷酸（4MUβGlc6P）来测定P-β-葡萄糖苷酶的活性。该酶可水解多种P-β-葡萄糖苷，包括异构二糖磷酸纤维二糖-6-磷酸、龙胆二糖-6-磷酸、槐糖-6-磷酸和昆布二糖-6-磷酸，生成6-磷酸葡萄糖和相应的糖苷配基。报告了每种底物的动力学参数。死亡梭杆菌的P-β-葡萄糖苷酶可被由6-磷酸葡萄糖氧化产生的6-磷酸葡糖酸-δ-内酯（P-葡糖酸-δ-内酯）灭活。已克隆并测序了编码死亡梭杆菌P-β-葡萄糖苷酶的pbgA基因。从核苷酸序列推导的前42个残基与通过对纯化酶进行自动埃德曼降解确定的N端序列一致。从预测的466个氨基酸序列中，已鉴定出两个具有催化重要性的谷氨酰残基。大肠杆菌和死亡梭杆菌的P-β-葡萄糖苷酶氨基酸序列的比对表明，抑制性P-葡糖酸-δ-内酯与死亡梭杆菌的该酶存在潜在的结合位点。
