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

Abstract

6-Phosphoryl-O-␣-D-glucopyranosyl:6-phosphoglucohydrolase (6-phospho-␣-glucosidase) has been purified from Fusobacterium mortiferum ATCC 25557. p-Nitrophenyl-␣-D-glucopyranoside 6-phosphate (pNP␣Glc6P) served as the chromogenic substrate for detection and assay of enzyme activity. The O 2 -sensitive, metaldependent phospho-␣-glucosidase was stabilized during purification by inclusion of dithiothreitol and Mn 2؉ ion in chromatography buffers. Various 6-phosphoryl-O-␣-linked glucosides, including maltose 6-phosphate, pNP␣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-␣-glucosidase (M r of ϳ49,000; pI of ϳ4.9) is activated by Fe 2؉, Mn 2؉, Co 2؉, and Ni 2؉, and the maximum rate of pNP␣Glc6P hydrolysis occurs at 40؇C within the pH range 7.0 to 7.5. The sequence of the first 32 amino acids of 6-phospho-␣-glucosidase exhibits 67% identity (90% similarity) to that deduced for the N terminus of a putative phospho-␤-glucosidase (designated ORF f212) encoded by glvG in Escherichia coli. Western blots involving highly specific polyclonal antibody against 6-phospho-␣-glucosidase and spectrophotometric analyses with pNP␣Glc6P revealed only low levels of the enzyme in glucose-, mannose-, or fructose-grown cells of F. mortiferum. Synthesis of 6-phospho-␣-glucosidase increased dramatically during growth of the organism on ␣-glucosides, such as maltose, ␣-methylglucoside, trehalose, turanose, and palatinose.A landmark in our understanding of carbohydrate dissimilation by microorganisms was established in 1964 by the serendipitous discovery of the phosphoenolpyruvate-dependent: sugar phosphotransferase system (PEP:PTS) by Saul Roseman and his colleagues (17, 35). Although first described for Escherichia coli, this group translocation system is now recognized as the primary mechanism for sugar accumulation by many species from both gram-negative (21, 26) and gram-positive bacterial genera (15,30,42,44). In a series of sequential transfers of the high-energy phosphoryl moiety from PEP, this multicomponent system catalyzes the simultaneous translocation and phosphorylation of sugars into the cell (21,26,36).The phosphorylated products of hexose-and polyol-specific PEP:PTSs may directly enter the energy-generating (fermentation) pathways of the organism. By contrast, further metabolism of intracellular disaccharide phosphates necessitates the initial cleavage of these compounds to yield the appropriate free and phosphorylated hexose moieties. Sugar-specific (and frequently inducible) hydrolases catalyze the cleavage of the O-glycosidic linkage of the various disaccharide phosphates. Several such enzymes have been described, including those responsible for the hydrolysis of 6-phospho-␤-galactosides (6, 14, 16), 6-phospho-␤-glucosides (38, 39, 48), sucrose 6-phosphate (sucrose 6P) (7,18,46), cellobiose 6P (22), and trehalose 6P (31).Reports of maltose-PEP:PTS activity have engendered debate (5, 12, 20, 28, 41, 49),...

Metabolism of sucrose and its five isomers by Fusobacterium mortiferum

“…In contrast to other species, F. mortiferum ferments an extraordinarily wide variety of carbohydrates (Robrish et al, 1991). Previously, in studies of maltose metabolism in F. mortiferum, we cloned and expressed a gene (malH) whose deduced sequence exhibits " 75 % residue identity with the phospho-α-glucosidase of K. pneumoniae (Thompson et al, 1995 ;Bouma et al, 1997). In the present report, we show that the gene adjacent to malH (designated malB) also encodes a putative EII(CB) protein that is " 60 % identical with AglA of K. pneumoniae.…”

“…Presently, F. mortiferum and K. pneumoniae are the only organisms known to ferment the five α--glucosyl--fructoses. Earlier we showed that MalH from F. mortiferum hydrolysed maltose 6h-phosphate (Thompson et al, 1995), and now we provide evidence for the cleavage of the phosphorylated isomers of sucrose by this enzyme. The phospho-α-glucosidase gene (malH) is adjacent to the gene malB, whose now completed sequence predicts a polypeptide that in size, domain structure, and conserved motifs (GITE and CATRLR) is characteristic of an EII(CB) transporter of the PEP : PTS.…”

“…For Western blot analysis, proteins in the cell extracts together with pre-stained markers (SeeBlue from Novex), were first separated by SDS-PAGE and then transferred to a nitrocellulose membrane. Immunodetection of phospho-α-glucosidase was performed with polyclonal antibody to MalH from F. mortiferum as described previously (Thompson et al, 1995). Molecular dynamics simulations and procedures for the determination of solvent-accessible surfaces have been described previously (Immel & Lichtenthaler, 1995 ;Thompson et al, 2001b).…”

“…Phosphorylated derivatives were biosynthesized via the PEP : PTS of permeabilized (palatinosegrown) cells of K. pneumoniae and were purified by Ba# + \ ethanol precipitation, ion-exchange and paper chromatography (Thompson et al, 2001a). The chromogenic and fluorogenic substrates p-nitrophenyl α--glucopyranoside 6-phosphate (pNP-α-G6P), p-nitrophenyl β--glucopyranoside 6-phosphate (pNP-β-G6P) and 4-methylumbelliferyl α--glucopyranoside-6-phosphate (4-MU-α-G6P) were prepared by selective phosphorylation (at C6-OH) of the parent glucosides with phosphorus oxychloride in trimethyl phosphate containing small proportions of water (Thompson et al, 1995). Glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) and hexokinase (HK, EC 2.7.1;1) were purchased from Boehringer Mannheim, and Ultrogel AcA-44 and TrisAcryl M-DEAE from Sigma.…”

Genes malh and pagl of Clostridium acetobutylicum ATCC 824 Encode NAD+- and Mn2+-dependent Phospho-α-glucosidase(s)

“…30 two genes for which the deduced open reading frames exhibited extensive homology with an NAD ϩ and metal-dependent GHF4 phospho-␣-glucosidase that we had found in several bacterial species, including Bacillus subtilis (14), Fusobacterium mortiferum (13,23,24), and Klebsiella pneumoniae (15). For purposes of distinction, we refer to the two putative phospho-␣-glucosidases in C. acetobutylicum 824 as MalH (maltose 6-phosphate hydrolase) and PagL (phospho-␣-glucosidase), respectively.…”