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J. Bacteriol., 05 1995, 2505-2512, Vol 177, No. 9
Copyright © 1995, American Society for Microbiology

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

J Thompson, CR Gentry-Weeks, NY Nguyen, JE Folk and SA Robrish
Laboratory of Micobial Ecology, National Institute of Dental Research, National Institutes of Health, Bethesda, MD 20892, USA.

6-Phosphoryl-O-alpha-D-glucopyranosyl:6-phosphoglucohydrolase (6- 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 O2-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, were 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 approximately 49,000; pI of approximately 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-glucosidase 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.

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