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-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.
Thompson
J
J
Laboratory of Micobial Ecology, National Institute of Dental Research, National Institutes of Health, Bethesda, MD 20892, USA.
Gentry-Weeks
C R
CR
Nguyen
N Y
NY
Folk
J E
JE
Robrish
S A
SA
D000595:Amino Acid Sequence; D002240:Carbohydrate Sequence; D004795:Enzyme Stability; D005673:Fusobacterium / Q000201:enzymology*; D005958:Glucosephosphates / Q000378:metabolism*; D005960:Glucosides / Q000378:metabolism*; D007525:Isoelectric Focusing; D008954:Models, Biological; D008969:Molecular Sequence Data; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013403:Sugar Phosphates / Q000378:metabolism*; D000520:alpha-Glucosidases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*
D005958:Glucosephosphates; D005960:Glucosides; D013403:Sugar Phosphates; C087475:maltose 6-phosphate; C049150:maltose-6'-phosphate glucosidase; D000520:alpha-Glucosidases