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

Abstract

The genome of Clostridium acetobutylicum 824 contains two genes encoding NAD ؉ , Mn 2؉ , and dithiothreitol-dependent phospho-␣-glucosidases that can be assigned to family 4 of the glycosylhydrolase superfamily. The two genes, designated malh (maltose 6-phosphate hydrolase) and pagl (phospho-␣-glucosidase), respectively, reside in separate operons that also encode proteins of the phosphoenolpyruvate-dependent:sugar phosphotransferase system. C. acetobutylicum grows on a variety of ␣-linked glucosides, including maltose, methyl-␣-D-glucoside, and the five isomers of sucrose. In the presence of the requisite cofactors, extracts of these cells readily hydrolyzed the chromogenic substrate p-nitrophenyl-␣-D-glucopyranoside 6-phosphate, but whether hydrolysis reflected expression of enzymes encoded by the malh or pagl genes was not discernible by spectrophotometric analysis or polyacrylamide gel electrophoresis. Resolution of this question required the cloning of the malh and pagl genes, and subsequent high expression, purification, and characterization of maltose-6 -phosphate hydrolase (MalH) and phospho-␣-glucosidase (PagL), respectively. MalH and PagL exhibit 50% residue identity, and in solution are tetramers comprising similar sized (ϳ50 kDa) subunits. The two proteins cross-react with polyclonal rabbit antibody against phospho-␣-glucosidase from Fusobacterium mortiferum. Purified MalH and PagL cleaved p-nitrophenyl-␣-D-glucopyranoside 6-phosphate with comparable efficiency, but only MalH catalyzed the hydrolysis of disaccharide 6 -phosphates formed via the phosphoenolpyruvate-dependent:sugar phosphotransferase system. Importantly, analysis of the proteome of C. acetobutylicum 824 by electrospray ionization-mass spectrometry confirmed expression of MalH during growth on many ␣-glucosides tested. Site-directed changes C169S and D170N yielded full-length, but catalytically inactive MalH. Of the two putative operons, our findings suggest that only proteins encoded by the mal operon participate in the dissimilation of maltose and related O-␣-linked glucosides by C. acetobutylicum 824.

Genetic Requirements for Growth of Escherichia coli K12 on Methyl-α-D-glucopyranoside and the Five α-D-Glucosyl-D-fructose Isomers of Sucrose

“…Detection of AglB ActivityNative (nondenaturing) electrophoresis and SDS-PAGE experiments were carried out in the Novex X-Cell mini system as described previously (26,27). When polypeptide-containing slices were to be excised for tryptic digestion and LC-MS/MS, the SDS-polyacrylamide gels were fixed, stained with Colloidal Blue staining kit (Invitrogen), and destained in distilled water.…”

“…A continuous spectrophotometric method (27) was used to follow hydrolysis of ␣-glucoside-6-P substrates. This glucose-6-phosphate dehydrogenase/NADP ϩ -coupled assay monitors the release of Glc6P, and the 1-ml assay contained the following: 0.…”

ThesimOperon Facilitates the Transport and Metabolism of Sucrose Isomers inLactobacillus caseiATCC 334

“…In contrast to the commonly observed dissimilation of sucrose, it was assumed for a long time that microorganisms are unable to metabolize the five linkage-isomeric ␣-D-glucosyl-D-fructose isomers of sucrose, namely, trehalulose, turanose, maltulose, leucrose, and palatinose. However, studies in our laboratory have proved that this assumption is invalid by showing that several species belonging to both gram-positive and gram-negative genera readily utilize these isomeric disaccharides as energy sources for growth (23,30,34,36,37). Remarkably, besides the five sucrose isomers, these organisms also transport a variety of related O-␣-linked glycosides, including maltose, isomaltose, maltitol, and ␣-methyl-D-glucoside, by the ␣-glucoside-specific PEP-dependent PTS.…”

“…Remarkably, besides the five sucrose isomers, these organisms also transport a variety of related O-␣-linked glycosides, including maltose, isomaltose, maltitol, and ␣-methyl-D-glucoside, by the ␣-glucoside-specific PEP-dependent PTS. Furthermore, the accumulated ␣-glu-coside-6-phosphates are hydrolyzed by a novel NAD ϩ -and Mn 2ϩ -dependent phospho-␣-glucosidase (Pagl) (EC 3.2.1.122) that is assigned to the unique GH family 4 (GH4) (3,14,27,34,36,39).…”

Characterization ofBacillus haloduransα-Galactosidase Mel4A Encoded by themel4AGene (BH2228)

“…[4][5][6] To determine the nucleotide, metal ion, and reducing agent requirements of rMel4A, the enzyme activity was measured with p-nitrophenyl--D-galactopyranoside (pNP-Gal) as the substrate in 100 mM Tris-HCl buffer (pH 7.4) containing combinations of these reagents. The reaction mixture was incubated at 37 C for 5 min, and the reaction was stopped by the addition of borate buffer (pH 9.8).…”