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Vibrio vulnificus MO6-24/O has three genes annotated as debranching enzymes or pullulanase genes. Among them, the gene encoded by VVMO6_03032 (vvde1) shares a higher similarity at the amino acid sequence level to the glycogen debranching enzymes, AmyX of Bacillus subtilis (40.5%) and GlgX of Escherichia coli (55.5%), than those encoded by the other two genes. The vvde1 gene encoded a protein with a molecular mass of 75.56 kDa and purified Vvde1 efficiently hydrolyzed glycogen and pullulan to shorter chains of maltodextrin and maltotriose (G3), respectively. However, it hydrolyzed amylopectin and soluble starch far less efficiently, and β-cyclodextrin (β-CD) only rarely. The optimal pH and temperature of Vvde1 was 6.5 and 25°C, respectively. Vvde1 was a cold-adapted debranching enzyme with more than 60% residual activity at 5°C. It could maintain stability for 2 days at 25°C and 1 day at 35°C, but it destabilized drastically at 40°C. The Vvde1 activity was inhibited considerably by Cu 2+ , Hg 2+ , and Zn 2+ , while it was slightly enhanced by Co 2+ , Ca 2+ , Ni 2+ , and Fe 2+ . The vvde1 knock-out mutant accumulated more glycogen than the wild-type in media supplemented with 1.0% maltodextrin; however, the side chain length distribution of glycogen was similar to that of the wild-type except G3, which was much more abundant in the mutant. Therefore, Vvde1 seemed to debranch glycogen with the degree of polymerization 3 (DP3) as the specific target branch length. Virulence of the pathogen against Caenorhabditis elegans was attenuated significantly by the vvde1 mutation. These results suggest that Vvde1 might be a unique glycogen debranching enzyme that is involved in both glycogen utilization and shaping of glycogen molecules, and contributes toward virulence of the pathogen., (© 2022. The Microbiological Society of Korea.)

A newly cloned 4-α-glucanotransferase (αGT) from Deinococcus geothermalis and two typical bacterial αGTs from Thermus scotoductus and Escherichia coli (MalQ) were investigated. Among 4 types of catalysis, the cyclization activity of αGTs that produces cycloamylose (CA), a valuable carbohydrate making inclusion complexes, was intensively studied. The new αGT, DgαGT, showed close protein sequence to the αGT from T. scotoductus (TsαGT). MalQ was clearly separated from the other two αGTs in the phylogenetic and the conserved regions analyses. The reaction velocities of disproportionation, cyclization, coupling, and hydrolysis of three αGTs were determined. Intriguingly, MalQ exhibited more than 100-fold lower cyclization activity than the others. To lesser extent, the disproportionation activity of MalQ was relatively low. DgαGT and TsαGT showed similar kinetics results, but TsαGT had nearly 10-fold lower hydrolysis activity than DgαGT. Due to the very low cyclizing activity of MalQ, DgαGT and TsαGT were selected for further analyses. When amylose was treated with DgαGT or TsαGT, CA with a broad DP range was generated immediately. The DP distribution of CA had a bimodal shape (DP 7 and 27 as peaks) for the both enzymes, but larger DPs of CA quickly decreased in the DgαGT. Cyclomaltopentaose, a rare cyclic sugar, was produced at early reaction stage and accumulated as the reactions went on in the both enzymes, but the increase was more profound in the TsαGT. Taken together, we clearly demonstrated the catalytic differences between αGT groups from thermophilic and pathogenic bacteria that and showed that αGTs play different roles depending on their lifestyle.

We first confirmed the involvement of MalQ (4-α-glucanotransferase) in Escherichia coli glycogen breakdown by both in vitro and in vivo assays. In vivo tests of the knock-out mutant, ΔmalQ , showed that glycogen slowly decreased after the stationary phase compared to the wild-type strain, indicating the involvement of MalQ in glycogen degradation. In vitro assays incubated glycogen-mimic substrate, branched cyclodextrin (maltotetraosyl-β-CD: G4- β-CD) and glycogen phosphorylase (GlgP)-limit dextrin with a set of variable combinations of E. coli enzymes, including GlgX (debranching enzyme), MalP (maltodextrin phosphorylase), GlgP and MalQ. In the absence of GlgP, the reaction of MalP, GlgX and MalQ on substrates produced glucose-1-P (glc-1-P) 3-fold faster than without MalQ. The results revealed that MalQ led to disproportionate G4 released from GlgP-limit dextrin to another acceptor, G4, which is phosphorylated by MalP. In contrast, in the absence of MalP, the reaction of GlgX, GlgP and MalQ resulted in a 1.6-fold increased production of glc-1-P than without MalQ. The result indicated that the G4-branch chains of GlgP-limit dextrin are released by GlgX hydrolysis, and then MalQ transfers the resultant G4 either to another branch chain or another G4 that can immediately be phosphorylated into glc-1-P by GlgP. Thus, we propose a model of two possible MalQ-involved pathways in glycogen degradation. The operon structure of MalP-defecting enterobacteria strongly supports the involvement of MalQ and GlgP as alternative pathways in glycogen degradation.

We studied the activity of a debranching enzyme (TreX) from Sulfolobus solfataricus on glycogen-mimic substrates, branched maltotetraosyl-β-cyclodextrin (Glc₄-β-CD), and natural glycogen to better understand substrate transglycosylation and the effect thereof on glycogen debranching in microorganisms. The validation test of Glc₄-β-CD as a glycogen mimic substrate showed that it followed the breakdown process of the well-known yeast and rat liver extract. TreX catalyzed both hydrolysis of α-1,6-glycosidic linkages and transglycosylation at relatively high (>0.5 mM) substrate concentrations. TreX transferred maltotetraosyl moieties from the donor substrate to acceptor molecules, resulting in the formation of two positional isomers of dimaltotetraosyl-α-1,6-β-cyclodextrin [(Glc₄)₂-β-CD]; these were 6(1),6(3)- and 6(1),6(4)-dimaltotetraosyl-α-1,6-β-CD. Use of a modified Michaelis-Menten equation to study substrate transglycosylation revealed that the kcat and Km values for transglycosylation were 1.78 × 10(3) s(-1) and 3.30 mM, respectively, whereas the values for hydrolysis were 2.57 × 10(3) s(-1) and 0.206 mM, respectively. Also, enzyme catalytic efficiency (the kcat/Km ratio) increased as the degree of polymerization of branch chains rose. In the model reaction system of Escherichia coli, glucose-1-phosphate production from glycogen by the glycogen phosphorylase was elevated ∼1.45-fold in the presence of TreX compared to that produced in the absence of TreX. The results suggest that outward shifting of glycogen branch chains via transglycosylation increases the number of exposed chains susceptible to phosphorylase action. We developed a model of the glycogen breakdown process featuring both hydrolysis and transglycosylation catalyzed by the debranching enzyme.

Mutants with deletion mutations in the glg and mal gene clusters of Escherichia coli MC4100 were used to gain insight into glycogen and maltodextrin metabolism. Glycogen content, molecular mass, and branch chain distribution were analyzed in the wild type and in ΔmalP (encoding maltodextrin phosphorylase), ΔmalQ (encoding amylomaltase), ΔglgA (encoding glycogen synthase), and ΔglgA ΔmalP derivatives. The wild type showed increasing amounts of glycogen when grown on glucose, maltose, or maltodextrin. When strains were grown on maltose, the glycogen content was 20 times higher in the ΔmalP strain (0.97 mg/mg protein) than in the wild type (0.05 mg/mg protein). When strains were grown on glucose, the ΔmalP strain and the wild type had similar glycogen contents (0.04 mg/mg and 0.03 mg/mg protein, respectively). The ΔmalQ mutant did not grow on maltose but showed wild-type amounts of glycogen when grown on glucose, demonstrating the exclusive function of GlgA for glycogen synthesis in the absence of maltose metabolism. No glycogen was found in the ΔglgA and ΔglgA ΔmalP strains grown on glucose, but substantial amounts (0.18 and 1.0 mg/mg protein, respectively) were found when they were grown on maltodextrin. This demonstrates that the action of MalQ on maltose or maltodextrin can lead to the formation of glycogen and that MalP controls (inhibits) this pathway. In vitro, MalQ in the presence of GlgB (a branching enzyme) was able to form glycogen from maltose or linear maltodextrins. We propose a model of maltodextrin utilization for the formation of glycogen in the absence of glycogen synthase.

Glycogen serves as major energy storage in most living organisms. GlgX, with its gene in the glycogen degradation operon, functions in glycogen catabolism by selectively catalyzing the debranching of polysaccharide outer chains in bacterial glycosynthesis. GlgX hydrolyzes alpha-1,6-glycosidic linkages of phosphorylase-limit dextrin containing only three or four glucose subunits produced by glycogen phosphorylase. To understand its mechanism and unique substrate specificity toward short branched alpha-polyglucans, we determined the structure of GlgX from Escherichia Coli K12 at 2.25 A resolution. The structure reveals a monomer consisting of three major domains with high structural similarity to the subunit of TreX, the oligomeric bifunctional glycogen debranching enzyme (GDE) from Sulfolobus. In the overlapping substrate binding groove, conserved residues Leu270, Asp271, and Pro208 block the cleft, yielding a shorter narrow GlgX cleft compared to that of TreX. Residues 207-213 form a unique helical conformation that is observed in both GlgX and TreX, possibly distinguishing GDEs from isoamylases and pullulanases. The structural feature observed at the substrate binding groove provides a molecular explanation for the unique substrate specificity of GlgX for G4 phosphorylase-limit dextrin and the discriminative activity of TreX and GlgX toward substrates of varying lengths.

A novel debranching enzyme from Nostoc punctiforme PCC73102 (NPDE) exhibits hydrolysis activity toward both alpha-(1,6)- and alpha-(1,4)-glucosidic linkages. The action patterns of NPDE revealed that branched chains are released first, and the resulting maltooligosaccharides are then hydrolyzed. Analysis of the reaction with maltooligosaccharide substrates labeled with (14)C-glucose at the reducing end shows that NPDE specifically liberates glucose from the reducing end. Kinetic analyses showed that the hydrolytic activity of NPDE is greatly affected by the length of the substrate. The catalytic efficiency of NPDE increased considerably upon using substrates that can occupy at least eight glycone subsites such as maltononaose and maltooctaosyl-alpha-(1,6)-beta-cyclodextrin. These results imply that NPDE has a unique subsite structure consisting of -8 to +1 subsites. Given its unique subsite structure, side chains shorter than maltooctaose in amylopectin were resistant to hydrolysis by NPDE, and the population of longer side chains was reduced.

TreX is an archaeal glycogen-debranching enzyme that exists in two oligomeric states in solution, as a dimer and tetramer. Unlike its homologs, TreX from Sulfolobus solfataricus shows dual activities for alpha-1,4-transferase and alpha-1,6-glucosidase. To understand this bifunctional mechanism, we determined the crystal structure of TreX in complex with an acarbose ligand. The acarbose intermediate was covalently bound to Asp363, occupying subsites -1 to -3. Although generally similar to the monomeric structure of isoamylase, TreX exhibits two different active-site configurations depending on its oligomeric state. The N terminus of one subunit is located at the active site of the other molecule, resulting in a reshaping of the active site in the tetramer. This is accompanied by a large shift in the "flexible loop" (amino acids 399-416), creating connected holes inside the tetramer. Mutations in the N-terminal region result in a sharp increase in alpha-1,4-transferase activity and a reduced level of alpha-1,6-glucosidase activity. On the basis of geometrical analysis of the active site and mutational study, we suggest that the structural lid (acids 99-97) at the active site generated by the tetramerization is closely associated with the bifunctionality and in particular with the alpha-1,4-transferase activity. These results provide a structural basis for the modulation of activities upon TreX oligomerization that may represent a common mode of action for other glycogen-debranching enzymes in higher organisms.

A treX in the trehalose biosynthesis gene cluster of Sulfolobus solfataricus ATCC 35092 has been reported to produce TreX, which hydrolyzes the alpha-1,6-branch portion of amylopectin and glycogen. TreX exhibited 4-alpha-D-glucan transferase activity, catalyzing the transfer of alpha-1,4-glucan oligosaccharides from one molecule to another in the case of linear maltooligosaccharides (G3-G7), and it produced cyclic glucans from amylopectin and amylose like 4-alpha-glucanotransferase. These results suggest that TreX is a novel isoamylase possessing the properties of 4-alpha-glucanotransferase.