Abstract
The gene glvA (formerly glv-1) from Bacillus subtilis has been cloned and expressed in Escherichia coli. The purified protein GlvA (449 residues, M sub(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 (Mn super(2+), Fe super(2+), Co super(2+), or Ni super(2+)) 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 sub(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- 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 super(41) Glu super(111), and Glu super(359), are required for GlvA activity. Asp super(41) is located at the C terminus of a beta alpha beta fold that may constitute the dinucleotide binding domain of the protein. Glu super(111) and Glu super(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 Mn super(2+) 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.