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J Biol Chem, Vol. 273, Issue 42, 27347-27356, October 16, 1998

The Gene glvA of Bacillus subtilis 168 Encodes a Metal-requiring, NAD(H)-dependent 6-Phospho--glucosidase
ASSIGNMENT TO FAMILY 4 OF THE GLYCOSYLHYDROLASE SUPERFAMILY

John Thompson, Andreas Pikis^¶, Sergei B. Ruvinov, Bernard Henrissat^**, Hiroki Yamamoto, and Junichi Sekiguchi

From the  Microbial Biochemistry and Genetics Unit, Oral Infection and Immunity Branch, NIDR, and the  Laboratory of Biochemistry, NHLBI, National Institutes of Health, Bethesda, Maryland 20892, the ^¶ Department of Infectious Diseases Children's National Medical Center Washington NW, DC 20010-2970, the ^** Structural Enzymology and Glycobiology Group, Architecture et Fonction des Macromolécules Biologiques, Centre National de la Recherche Scientifique, 13402 Marseille, France, and the  Department of Applied Biology, Faculty of Textile Science and Technology, Shinshu University, 3-15-1 Tokida, Ueda-shi, Nagano 386, Japan

The gene glvA (formerly glv-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--D-glucopyranosyl:phosphoglucohydrolase (6-phospho--glucosidase) that requires both NAD(H) and divalent metal (Mn²⁺, Fe²⁺, Co²⁺, or Ni²⁺⁾ for activity. 6-Phospho--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 glvA. The chromogenic and fluorogenic substrates, p-nitrophenyl--D-glucopyranoside 6-phosphate and 4-methylumbelliferyl--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 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--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--glucosidase from B. subtilis can be assigned to the nine-member family 4 of the glycosylhydrolase superfamily.

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