PURIFICATION FROM FUSOBACTERIUM-MORTIFERUM ATCC-25557 OF A 6-PHOSPHORYL-O-ALPHA-D-GLUCOPYRANOSYL-6-PHOSPHOGLUCOHYDROLASE THAT HYDROLYZES MALTOSE 6-PHOSPHATE AND RELATED PHOSPHO-ALPHA-D-GLUCOSIDES

6-Phospholyl-O-alpha-D-glucopyranosyl:6-phosphoglucohydrolase (8-phospho-alpha-glucosidase) has been purified from Fusobacterium mortiferum ATCC 25557, p-Nitrophenyl-alpha-D-glucopyranoside 6-phosphate (pNP alpha Glc6P) served as the chromogenic substrate for detection and assay of enzyme activity, The O-2-sensitive, metal-dependent phospho-alpha-glucosidase was stabilized during purification by inclusion of dithiothreitol and Mn2+ ion in chromatography buffers. Various 6-phosphoryl-O-alpha-linked glucosides, including maltose 6-phosphate, pNP alpha Glc6P, trehalose 6-phosphate, and sucrose 6-phosphate, mere hydrolyzed by the enzyme to yield D-glucose 6-phosphate and aglycone moieties in a 1:1 molar ratio, 6-Phospho-alpha-glucosidase (M(r) of similar to 49,000; pI of similar to 4.9) is activated by Fe2+, Mn2+, Co2+, and Ni2+, and the maximum rate of pNP alpha Glc6P hydrolysis occurs at 40 degrees C within the pH range 7.0 to 7.5, The sequence of the first 32 amino acids of 6-phospho-alpha-glucdsidase exhibits 67% identity (90% similarity) to that deduced for the N terminus of a putative phospho-beta-glucosidase (designated ORF f212) encoded by glvG in Escherichia coli. Western blots involving highly specific polyclonal antibody against 6-phospho-alpha-glucosidase and spectrophotometric analyses with pNP alpha Glc6P revealed only low levels of the enzyme in glucose-, mannose-, or fructose-grown cells of F. mortiferum, Synthesis of 6-phospho-alpha-glucosidase increased dramatically during growth of the organism on alpha-glucosides, such as maltose, alpha-methylglucoside, trehalose, turanose, and palatinose.