The gene glvA of Bacillus subtilis 168 encodes a metal-requiring, NAD (H)-dependent 6-phospho-α-glucosidase -: Assignment to family 4 of the glycosylhydrolase superfamily
The gene glvA of Bacillus subtilis 168 encodes a metal-requiring, NAD (H)-dependent 6-phospho-α-glucosidase -: Assignment to family 4 of the glycosylhydrolase superfamily
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DOI：
10.1074/jbc.273.42.27347
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发表时间：
1998-10-16
影响因子：
4.8
通讯作者：
Sekiguchi, J
中科院分区：
文献类型：
--
作者：
Thompson, J;Pikis, A;Sekiguchi, J
来自枯草芽孢杆菌的基因 gluA（以前称为 glu-1）已在大肠杆菌中克隆并表达。纯化的蛋白 GlvA（449 个残基，M-r = 50,513）是一种独特的 6-磷酰基-O-α-D-吡喃葡萄糖基：磷酸葡萄糖水解酶（6-磷酸-α-葡萄糖苷酶），需要 NAD(H) 和二价金属（Mn2+、Fe2+、Co2+ 或 Ni2+）才能发挥活性。来自枯草芽孢杆菌的 6-磷酸-α-葡萄糖苷酶 (EC 3.2.1.122) 与来自腐烂梭杆菌的麦芽糖 6-磷酸水解酶多克隆抗体发生交叉反应，两种蛋白质的氨基酸序列同一性为 73%。通过 SDS 聚丙烯酰胺凝胶电泳 (51,000) 和电喷雾质谱 (50,510) 测定的 GlvA M-r 估计值与从氨基酸序列推导的分子量 50,513 非常一致。通过自动分析确定的 N 末端前 37 个残基的序列与 gluA 翻译预测的序列完全一致。显色和荧光底物对硝基苯基-α-D-吡喃葡萄糖苷6-磷酸和4-甲基伞形基-α-D-吡喃葡萄糖苷6-磷酸分别用于酶活性的不连续测定和原位检测。定点诱变表明，GlvA 活性需要三个酸性残基：Asp(41)、Glu(111) 和 Glu(359)。 Asp(41) 位于 β α β 折叠的 C 末端，可能构成蛋白质的二核苷酸结合结构域。在 6-磷酸-α-葡萄糖苷底物（包括麦芽糖 6-磷酸和海藻糖 6-磷酸）水解过程中，Glu(111) 和 GlU(359) 可以分别充当催化酸（质子供体）和亲核试剂（碱）。在无金属缓冲液中，GlvA 作为无活性二聚体存在，但在 Mn2+ 离子存在下，这些物质 通过与其同源物的比较序列比对，来自枯草芽孢杆菌的新型 6-磷酸-α-葡萄糖苷酶可归属于糖基水解酶超家族的九成员家族 4。
The gene gluA (formerly glu-1) from Bacillus subtilis has been cloned and expressed in Escherichia coli. The purified protein GlvA (449 residues, M-r = 50,513) is a unique 6-phosphoryl-O-alpha-D-glucopyranosyl:phosphoglucohydrolase (6-phospho-alpha-glucosidase) that requires both NAD(H) and divalent metal (Mn2+, Fe2+, Co2+, or Ni2+) for activity. 6-Phospho-alpha-glucosidase (EC 3.2.1.122) from B. subtilis cross-reacts with polyclonal antibody to maltose 6-phosphate hydrolase from Fusobacterium mortiferum, and the two proteins exhibit amino acid sequence identity of 73%. Estimates for the M-r of GlvA determined by SDS-polyacrylamide gel electrophoresis (51,000) and electrospray-mass spectroscopy (50,510) were in excellent agreement with the molecular weight of 50,513 deduced from the amino acid sequence. The sequence of the first 37 residues from the N terminus determined by automated analysis agreed precisely with that predicted by translation of gluA. The chromogenic and fluorogenic substrates, p-nitrophenyl-alpha-D-glucopyranoside 6-phosphate and 4-methylumbelliferyl-alpha-D-glucopyranoside 6-phosphate were used for the discontinuous assay and in situ detection of enzyme activity, respectively. Site directed mutagenesis shows that three acidic residues, Asp(41), Glu(111), and Glu(359), are required for GlvA activity. Asp(41) is located at the C terminus of a beta alpha beta fold that may constitute the dinucleotide binding domain of the protein. Glu(111) and GlU(359) may function as the catalytic acid (proton donor) and nucleophile (base), respectively, during hydrolysis of 6-phospho-alpha-glucoside substrates including maltose 6-phosphate and trehalose 6-phosphate, In metal-free buffer, GlvA exists as an inactive dimer, but in the presence of Mn2+ ion, these species associate to form the NAD(H)-dependent catalytically active tetramer, By comparative sequence alignment with its homologs, the novel 6-phospho-alpha-glucosidase from B. subtilis can be assigned to the nine-member family 4 of the glycosylhydrolase superfamily.