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

Abstract

The gene glvA (formerly glv-1) from Bacillus subtilis has been cloned and expressed in Escherichia coil. The purified protein GlvA (449 residues, M(r) = 50,513) is a unique 6-phosphoryl-O-α-D- glucopyranosyl:phosphoglucohydrolase (6-phospho-α-glucosidase) that requires both NAD(H) and divalent metal (Mn2+, Fe2+, Co2+, or Ni2+) for activity. 6-Phospho-α-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-α-D-glucopyranoside 6-phosphate and 4- methylumbelliferyl-α-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 βαβ 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-α-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-α-glucosidase from B. subtilis can be assigned to the nine-member family 4 of the glycosylhydrolase superfamily.