The gene glvA of Bacillus subtilis 168 encodes a metal-requiring, NAD(H)-dependent 6-phospho-alpha-glucosidase. Assignment to family 4 of the glycosylhydrolase superfamily.
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.
Thompson
J
J
Microbial Biochemistry and Genetics Unit, Oral Infection and Immunity Branch, NIDR, National Institutes of Health, Bethesda, Maryland 20892, USA. jthompson@yoda.nidr.nih.gov
Pikis
A
A
Ruvinov
S B
SB
Henrissat
B
B
Yamamoto
H
H
Sekiguchi
J
J
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D002413:Cations, Divalent; D002863:Chromogenic Compounds; D003001:Cloning, Molecular; D017124:Conserved Sequence; D003429:Cross Reactions; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D006026:Glycoside Hydrolases / Q000737:chemistry; D006868:Hydrolysis; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D005810:Multigene Family; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013403:Sugar Phosphates / Q000737:chemistry / Q000378:metabolism; D014461:Ultracentrifugation; D000520:alpha-Glucosidases / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000378:metabolism
D002413:Cations, Divalent; D002863:Chromogenic Compounds; D011994:Recombinant Proteins; D013403:Sugar Phosphates; C087475:maltose 6-phosphate; D009243:NAD; D006026:Glycoside Hydrolases; D000520:alpha-Glucosidases