Compared with many different amylases that are able to hydrolyze only α-d-(1,4)- glycosidic bonds, maltogenic amylases exhibit catalytic versatility: hydrolysis of. The physiological functions of two amylolytic enzymes, a maltogenic amylase ( MAase) encoded by yvdF and a debranching enzyme (pullulanase) encoded by . Enzymatic characterization of a maltogenic amylase from. Lactobacillus gasseri ATCC expressed in Escherichia coli. Ko-Woon Oh a., Myo-Jeong Kim b.
The crystal structure of a maltogenic amylase from a Thermus strain was maltogenid at 2. However, the enzymatic hydrolysis of maltodextrins and glycogen, providing a major energy reservoir in prokaryotes, was not reflected in the model, due probably to a lack of experimental analysis. To investigate the specificity of pullulanase from B.
PTS can be recovered in pure form from an enzyme reaction mixture.
Mutagenesis of Ala, which modulates substrate subsite affinity at the catalytic interface matlogenic dimeric ThMA. To investigate the roles of MAase and pullulanase in glycogen utilization, the following glycogen-overproducing strains were constructed: After 30 minutes remove the test tube from the water bath, and add m l of the stop reagent 0. Expression of the promoter for the maltogenic amylase gene in Bacillus subtilis Associated Data Supplementary Materials [Supplemental material].
The size of the space appears to accommodate a disaccharide such as maltose rather maltoyenic, as a maltose maltogeni can be successfully docked into the space Fig. Assay conditions being as follows: We have determined the structure of maltogenic amylase from a Thermus strain ThMA.
All oxygen atoms are in red. MAases, NPases, and CDases are intracellular enzymes, which implicates their biological roles in metabolizing small oligosaccharides, including CDs imported into the cells for energy generation, and storing them in the form of branched oligosaccharides under the condition of high maltogenix of glucose or maltose. A closely linked enzyme, CGTase, is hypothesized to have evolved from an ancestral hydrolase on the basis of sequence and phylogenetic analysis When glycogen accumulates in B.
Several groups of starch-hydrolyzing enzymes are known to harbor more than single enzyme activity. Overexpression and purification of pullulanase.
The heavy atom positions were used to calculate MIR phases. The solid line indicates the calculated curve for dimeric species. Amino acids that are not conserved, compared with the ThMA sequence, are lightly shaded. Polysaccharide that may serve as a carbon and energy storage compound for sporulation in Bacillus cereus.
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Mailing address for J. The active site is a anylase and deep cleft suitable for malrogenic cyclodextrins, which are the preferred substrates to other starch materials.
Cloning and nucleotide sequence of a gene encoding a glycogen debranching enzyme in the trehalose operon from Arthrobacter sp. The two subunits, related by the molecular 2-fold axis lying on the figure, are labeled with different colors. It is surprising that the new catalytic activities are gained not by creating a new catalytic residue, but most likely by creating the extra sugar-binding space at the active site.
Glycogen in the reactions with cell extract from the amyX or yvdF amyX mutant was hardly attacked compared to that in the reaction with cell extract from the wild type.
Therefore, we postulated that MAase and pullulanase are involved in the degradation of glycogen in B. These two enzymes are ubiquitous among Bacillus spp. Four glucosyl residues remaining in the side chain of glycogen would then be preferably cleaved at the branch point by pullulanase.
Significant amounts of G7 and its degradation products were detected in the wild-type cells compared with the amounts in the mdxE cells. In many bacteria and archaea, the activities of debranching enzymes are detected throughout the whole cell cycle and are even upregulated by maltose or starch 72629 The inset shows the line fitting of the elution time versus logarithm of the molecular weight of the size markers.
This finding correlated well with the results obtained in the in vitro study and shows more specifically that pullulanase has a strong preference for maltotetraosyl side chains left at the branch point by the action of glycogen phosphorylase. Published online May Allow the mixture to stand for 15 min.
Dissolve mg maltotriose e. It was not at all straightforward to solve the structure by using the phase information derived from MR, and additional phase information was obtained from three heavy atom derivative crystals Table I by MIR multiple isomorphous replacement method. Mlatogenic reducing sugar was detected using the naphtol-H maltogejic SO 4 method Molecular cloning and biochemical characterization of the first archaeal maltogenic amylase from the hyperthermophilic archaeon Thermoplasma volcanium GSS1.
In a previous study, Spo0A, amyase master regulator determining the life cycle of B. The consequence of the dimer formation of ThMA is striking. The concentration of ThMA was 0.
Proposed model of starch and glycogen use in Bacillus spp. Heavy atom binding sites were identified by Fourier difference analysis using the phases derived from MR. However, glycogen is barely detected in B. Finally, the natural design of the transglycosylation activity of the enzyme at high concentration maltogdnic acceptor sugar molecules may now be altered for biotechnological applications to produce branched oligosaccharides with higher efficiency. Immunogold electron microscopy showed that the gold particles were present predominantly on both sides of the cytoplasmic membrane and in the periplasmic space Fig.
Based on previous results and those obtained in this study, we propose a model mltogenic the roles of MAase and pullulanase in Bacillus spp.
Crystal Structure of a Maltogenic Amylase Provides Insights into a Catalytic Versatility
S2 in the supplemental material. The cat gene was introduced into pET29b Novagen Inc.
The accumulation of glucoseP via YvdK has not been observed in cell extracts because of the immediate use of glucoseP in subsequent metabolism.
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