Kwan-Hwa Park

Structure, specificity and function of cyclomaltodextrinase, a multispecific enzyme of the α-amylase family

Cyclomaltodextrinase (CDase, EC 3.2.1.54), maltogenic amylase (EC 3. 2.1.133), and neopullulanase (EC 3.2.1.135) are reported to be capable of hydrolyzing all or two of the following three types of substrates: cyclomaltodextrins (CDs); pullulan; and starch. These enzymes hydrolyze CDs and starch to maltose and pullulan to panose by cleavage of alpha-1,4 glycosidic bonds whereas alpha-amylases essentially lack activity on CDs and pullulan. They also catalyze transglycosylation of oligosaccharides to the C3-, C4- or C6-hydroxyl groups of various acceptor sugar molecules. The present review surveys the biochemical, enzymatic, and structural properties of three types of such enzymes as defined based on the substrate specificity toward the CDs: type I, cyclomaltodextrinase and maltogenic amylase that hydrolyze CDs much faster than pullulan and starch; type II, Thermoactinomyces vulgaris amylase II (TVA II) that hydrolyzes CDs much less efficiently than pullulan; and type III, neopullulanase that hydrolyzes pullulan efficiently, but remains to be reported to hydrolyze CDs. These three types of enzymes exhibit 40-60% amino acid sequence identity. They occur in the cytoplasm of bacteria and have molecular masses from 62 to 90 kDa which are slightly larger than those of most alpha-amylases. Multiple amino acid sequence alignment and crystal structures of maltogenic amylase and TVA II reveal the presence of an N-terminal extension of approximately 130 residues not found in alpha-amylases. This unique N-terminal domain as seen in the crystal structures apparently contributes to the active site structure leading to the distinct substrate specificity through a dimer formation. In aqueous solution, most of these enzymes show a monomer-dimer equilibrium. The present review discusses the multiple specificity in the light of the oligomerization and the molecular structures arriving at a clarified enzyme classification. Finally, a physiological role of the enzymes is proposed.

Development of reduced-fat mayonnaise using 4αGTase-modified rice starch and xanthan gum

In this study a disproportionating enzyme, 4-alpha-glucanotransferase (4alphaGTase), was used to modify the structural properties of rice starch to produce a suitable fat substitute in reduced-fat (RF) mayonnaise. The mayonnaise fat was partially substituted with the 4alphaGTase-treated starch paste at levels up to 50% in combination with xanthan gum and the physical and rheological properties of the modified RF mayonnaise samples were investigated. All mayonnaises prepared in this study exhibited shear thinning behavior and yield stress. Viscoelastic properties of mayonnaise were characterized using dynamic oscillatory shear test and it was observed that mayonnaises exhibited weak gel-like properties. The magnitude of elastic and loss moduli was also affected by 4alphaGTase-treated starch concentration and presence of xanthan gum. In relation to microstructure, RF mayonnaise prepared with 3.8 or 5.6 wt% of 4alphaGTase-treated starch and xanthan gum showed smaller droplets. The use of 5.6 wt% of 4alphaGTase-treated starch and 0.1 wt% of xanthan gum produced a RF mayonnaise with similar rheological properties and appearances as FF mayonnaise with gum. This study demonstrated a high feasibility for using 4alphaGTase-treated rice starch as a viable fat replacer in mayonnaise.

Enzymatic Analysis of an Amylolytic Enzyme from the Hyperthermophilic Archaeon Pyrococcus furiosus Reveals Its Novel Catalytic Properties as both an α-Amylase and a Cyclodextrin-Hydrolyzing Enzyme

ABSTRACT Genomic analysis of the hyperthermophilic archaeon Pyrococcus furiosus revealed the presence of an open reading frame (ORF PF1939) similar to the enzymes in glycoside hydrolase family 13. This amylolytic enzyme, designated PFTA (Pyrococcus furiosus thermostable amylase), was cloned and expressed in Escherichia coli. The recombinant PFTA was extremely thermostable, with an optimum temperature of 90°C. The substrate specificity of PFTA suggests that it possesses characteristics of both α-amylase and cyclodextrin-hydrolyzing enzyme. Like typical α-amylases, PFTA hydrolyzed maltooligosaccharides and starch to produce mainly maltotriose and maltotetraose. However, it could also attack and degrade pullulan and β-cyclodextrin, which are resistant to α-amylase, to primarily produce panose and maltoheptaose, respectively. Furthermore, acarbose, a potent α-amylase inhibitor, was drastically degraded by PFTA, as is typical of cyclodextrin-hydrolyzing enzymes. These results confirm that PFTA possesses novel catalytic properties characteristic of both α-amylase and cyclodextrin-hydrolyzing enzyme.

Properties of a Novel Thermostable Glucoamylase from the Hyperthermophilic Archaeon Sulfolobus solfataricus in Relation to Starch Processing

ABSTRACT A gene (ssg) encoding a putative glucoamylase in a hyperthermophilic archaeon, Sulfolobus solfataricus, was cloned and expressed in Escherichia coli, and the properties of the recombinant protein were examined in relation to the glucose production process. The recombinant glucoamylase was extremely thermostable, with an optimal temperature at 90°C. The enzyme was most active in the pH range from 5.5 to 6.0. The enzyme liberated β-d-glucose from the substrate maltotriose, and the substrate preference for maltotriose distinguished this enzyme from fungal glucoamylases. Gel permeation chromatography and sedimentation equilibrium analytical ultracentrifugation analysis revealed that the enzyme exists as a tetramer. The reverse reaction of the glucoamylase from S. solfataricus produced significantly less isomaltose than did that of industrial fungal glucoamylase. The glucoamylase from S. solfataricus has excellent potential for improving industrial starch processing by eliminating the need to adjust both pH and temperature.

Role of Maltogenic Amylase and Pullulanase in Maltodextrin and Glycogen Metabolism of Bacillus subtilis 168

The physiological functions of two amylolytic enzymes, a maltogenic amylase (MAase) encoded by yvdF and a debranching enzyme (pullulanase) encoded by amyX, in the carbohydrate metabolism of Bacillus subtilis 168 were investigated using yvdF, amyX, and yvdF amyX mutant strains. An immunolocalization study revealed that YvdF was distributed on both sides of the cytoplasmic membrane and in the periplasm during vegetative growth but in the cytoplasm of prespores. Small carbohydrates such as maltoheptaose and beta-cyclodextrin (beta-CD) taken up by wild-type B. subtilis cells via two distinct transporters, the Mdx and Cyc ABC transporters, respectively, were hydrolyzed immediately to form smaller or linear maltodextrins. On the other hand, the yvdF mutant exhibited limited degradation of the substrates, indicating that, in the wild type, maltodextrins and beta-CD were hydrolyzed by MAase while being taken up by the bacterium. With glycogen and branched beta-CDs as substrates, pullulanase showed high-level specificity for the hydrolysis of the outer side chains of glycogen with three to five glucosyl residues. To investigate the roles of MAase and pullulanase in glycogen utilization, the following glycogen-overproducing strains were constructed: a glg mutant with a wild-type background, yvdF glg and amyX glg mutants, and a glg mutant with a double mutant (DM) background. The amyX glg and glg DM strains accumulated significantly larger amounts of glycogen than the glg mutant, while the yvdF glg strain accumulated an intermediate amount. Glycogen samples from the amyX glg and glg DM strains exhibited average molecular masses two and three times larger, respectively, than that of glycogen from the glg mutant. The results suggested that glycogen breakdown may be a sequential process that involves pullulanase and MAase, whereby pullulanase hydrolyzes the alpha-1,6-glycosidic linkage at the branch point to release a linear maltooligosaccharide that is then hydrolyzed into maltose and maltotriose by MAase.

A novel amylolytic enzyme from Thermotoga maritima, resembling cyclodextrinase and α-glucosidase, that liberates glucose from the reducing end of the substrates☆

The gene previously designated as putative cyclodextrinase from Thermotoga maritima (TMG) was cloned and overexpressed in Escherichia coli. The recombinant TMG was partially purified and its enzymatic characteristics on various substrates were examined. The enzyme hydrolyzes various maltodextrins including maltotriose to maltoheptaose and cyclomaltodextrins (CDs) to mainly glucose and maltose. Although TMG could not degrade pullulan, it rapidly hydrolyzes acarbose, a strong amylase and glucosidase inhibitor, to acarviosine and glucose. Also, TMG initially hydrolyzes p-nitrophenyl-alpha-pentaoside to give maltopentaose and p-nitrophenol, implying that the enzyme specifically cleaves a glucose unit from the reducing end of maltooligosaccharides unlike to other glucosidases. Since its enzymatic activity is negligible if alpha-methylglucoside is present in the reducing end, the type of the residue at the reducing end of the substrate is important for the TMG activity. These results support the fact that TMG is a novel exo-acting glucosidase possessing the characteristics of both CD-/pullulan hydrolyzing enzyme and alpha-glucosidase.

Role of Maltose Enzymes in Glycogen Synthesis by Escherichia coli

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.

Structural Insight Into the Bifunctional Mechanism of the Glycogen-Debranching Enzyme Trex from the Archaeon Sulfolobus Solfataricus.

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 α-1,4-transferase and α-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 α-1,4-transferase activity and a reduced level of α-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 α-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.

Conserved expression pattern of chicken DAZL in primordial germ cells and germ-line cells

The autosomal gene deleted in azoospermia-like (DAZL), which was identified as a member of the deleted in azoospermia (DAZ) family, is homologous to the Drosophila gene BOULE. The authors investigated the sequence similarities of chicken DAZL (cDAZL) with several invertebrate and vertebrate DAZL proteins using CLUSTAL X. A comparison of the primary sequence of cDAZL with other DAZL proteins indicated significant similarities: 70-82% with reptiles, 63-68% with mammals, 51-67% with amphibians, and 42-49% with fishes. The conserved expression pattern of cDAZL was examined by reverse transcription-PCR, quantitative real-time PCR, and in situ hybridization during primordial germ cell (PGC) settlement in the gonads and germ-line development. Among several tissues examined on embryonic day E6.5, DAZL expression was detected specifically in male and female gonads. Quantitative real-time PCR and in situ hybridization revealed strong cDAZL expression in PGCs. When the PGCs differentiated into germ cells, cDAZL expression was slightly decreased; however, expression was continuously detected in germ-line cells until the adult stage. We inferred that cDAZL expression was conserved in PGCs and during germ-line differentiation until the adult stage, making them a valuable molecular marker for studies of PGC differentiation and germ-line development in chickens.

TreX from Sulfolobus solfataricus ATCC 35092 Displays Isoamylase and 4-α-Glucanotransferase Activities

A treX in the trehalose biosynthesis gene cluster of Sulfolobus solfataricus ATCC 35092 has been reported to produce TreX, which hydrolyzes the α-1,6-branch portion of amylopectin and glycogen. TreX exhibited 4-α-D-glucan transferase activity, catalyzing the transfer of α-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-α-glucanotransferase. These results suggest that TreX is a novel isoamylase possessing the properties of 4-α-glucanotransferase.