Thompson J, Pikis A, Ruvinov S B, Henrissat B, Yamamoto H, Sekiguchi J
Microbial Biochemistry and Genetics Unit, Oral Infection and Immunity Branch, NIDR, National Institutes of Health, Bethesda, Maryland 20892, USA.
J Biol Chem. 1998 Oct 16;273(42):27347-56. doi: 10.1074/jbc.273.42.27347.
The gene glvA (formerly glv-1) from Bacillus subtilis has been cloned and expressed in Escherichia coli. The purified protein GlvA (449 residues, Mr = 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 Mr 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 glvA. 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, Asp41, Glu111, and Glu359, are required for GlvA activity. Asp41 is located at the C terminus of a betaalphabeta fold that may constitute the dinucleotide binding domain of the protein. Glu111 and Glu359 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.
枯草芽孢杆菌的glvA基因（原glv - 1）已被克隆并在大肠杆菌中表达。纯化后的蛋白GlvA（449个残基，Mr = 50,513）是一种独特的6 - 磷酸 - 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），其活性需要NAD(H)和二价金属（Mn2 +、Fe2 +、Co2 +或Ni2 +）。枯草芽孢杆菌的6 - 磷酸 - α - 葡萄糖苷酶（EC 3.2.1.122）与针对死亡梭杆菌麦芽糖6 - 磷酸水解酶的多克隆抗体发生交叉反应，这两种蛋白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的Mr估计值与从氨基酸序列推导的50,513的分子量非常吻合。通过自动分析确定的N端前37个残基的序列与glvA翻译预测的序列完全一致。生色和荧光底物，对硝基苯基 - α - D - 吡喃葡萄糖苷6 - 磷酸和4 - 甲基伞形酮基 - α - D - 吡喃葡萄糖苷6 - 磷酸分别用于酶活性的间断测定和原位检测。定点诱变表明，Asp41、Glu111和Glu359这三个酸性残基是GlvA活性所必需的。Asp41位于一个β - α - β折叠的C端，该折叠可能构成该蛋白的二核苷酸结合结构域。在包括麦芽糖6 - 磷酸和海藻糖6 - 磷酸在内的6 - 磷酸 - α - 葡萄糖苷底物水解过程中，Glu111和Glu359可能分别作为催化酸（质子供体）和亲核试剂（碱）发挥作用。在无金属缓冲液中，GlvA以无活性的二聚体形式存在，但在Mn2 +离子存在下，这些分子缔合形成NAD(H)依赖性的催化活性四聚体。通过与其同源物的比较序列比对，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4家族。
