Complexes of Thermoactinomyces vulgaris R-47 alpha-amylase 1 and pullulan model oligossacharides provide new insight into the mechanism for recognizing substrates with alpha-(1,6) glycosidic linkages

Thermoactinomyces vulgaris R-47 alpha-amylase 1 (TVAI) has unique hydrolyzing activities for pullulan with sequence repeats of alpha-(1,4), alpha-(1,4), and alpha-(1,6) glycosidic linkages, as well as for starch. TVAI mainly hydrolyzes alpha-(1,4) glycosidic linkages to produce a panose, but it also hydrolyzes alpha-(1,6) glycosidic linkages with a lesser efficiency. X-ray structures of three complexes comprising an inactive mutant TVAI (D356N or D356N/E396Q) and a pullulan model oligosaccharide (P2; [Glc-alpha-(1,6)-Glc-alpha-(1,4)-Glc-alpha-(1,4)]2 or P5; [Glc-alpha-(1,6)-Glc-alpha-(1,4)-Glc-alpha-(1,4)]5) were determined. The complex D356N/P2 is a mimic of the enzyme/product complex in the main catalytic reaction of TVAI, and a structural comparison with Aspergillus oryzaealpha-amylase showed that the (-) subsites of TVAI are responsible for recognizing both starch and pullulan. D356N/E396Q/P2 and D356N/E396Q/P5 provided models of the enzyme/substrate complex recognizing the alpha-(1,6) glycosidic linkage at the hydrolyzing site. They showed that only subsites -1 and -2 at the nonreducing end of TVAI are effective in the hydrolysis of alpha-(1,6) glycosidic linkages, leading to weak interactions between substrates and the enzyme. Domain N of TVAI is a starch-binding domain acting as an anchor in the catalytic reaction of the enzyme. In this study, additional substrates were also found to bind to domain N, suggesting that domain N also functions as a pullulan-binding domain.     

The crystal structure of Thermoactinomyces vulgaris R-47 alpha-amylase II (TVA II) complexed with transglycosylated product.

Alphan alpha-amylase (TVA II) from Thermoactinomyces vulgaris R-47 efficiently hydrolyzes alpha-1,4-glucosidic linkages of pullulan to produce panose in addition to hydrolyzing starch. TVA II also hydrolyzes alpha-1,4-glucosidic linkages of cyclodextrins and alpha-1,6-glucosidic linkages of isopanose. To clarify the basis for this wide substrate specificity of TVA II, we soaked 4(3)-alpha-panosylpanose (4(3)-P2) (a pullulan hydrolysate composed of two panosyl units) into crystals of D325N inactive mutated TVA II. We then determined the crystal structure of TVA II complexed with 4(2)-alpha-panosylpanose (4(2)-P2), which was produced by transglycosylation from 4(3)-P2, at 2.2-A resolution. The shape of the active cleft of TVA II is unique among those of alpha-amylase family enzymes due to a loop (residues 193-218) that is located at the end of the cleft around the nonreducing region and forms a 'dam'-like bank. Because this loop is short in TVA II, the active cleft is wide and shallow around the nonreducing region. It is assumed that this short loop is one of the reasons for the wide substrate specificity of TVA II. While Trp356 is involved in the binding of Glc +2 of the substrate, it appears that Tyr374 in proximity to Trp356 plays two roles: one is fixing the orientation of Trp356 in the substrate-liganded state and the other is supplying the water that is necessary for substrate hydrolysis.     

Pattern of action of Bacillus stearothermophilus neopullulanase on pullulan.

The action of neopullulanase from Bacillus stearothermophilus on many oligosaccharides was tested. The enzyme hydrolyzed not only alpha-(1----4)-glucosidic linkages but also specific alpha-(1----6)-glucosidic linkages of several branched oligosaccharides. When pullulan was used as a substrate, panose, maltose, and glucose, in that order, were produced as final products at a final molar ratio of 3:1:1. According to these results, we proposed a model for the pattern of action of neopullulanase on pullulan as follows. In the first step, the enzyme hydrolyzes only alpha-(1----4)-glucosidic linkages on the nonreducing side of alpha-(1----6) linkages of pullulan and produces panose and several intermediate products composed of some panose units. In the second step, taking 6(2)-O-alpha-(6(3)-O-alpha-glucosyl-maltotriosyl)-maltose as an example of one of the intermediate products, the enzyme hydrolyzes either alpha-(1----4) (the same position as that described above) or alpha-(1----6) linkages and produces panose or 6(3)-O-alpha-glucosyl-maltotriose plus maltose, respectively. In the third step, the alpha-(1----4) linkage of 6(3)-O-alpha-glucosyl-maltotriose is hydrolyzed by the enzyme, and glucose and another panose are produced. To confirm the model of the pattern of action, we extracted intermediate products produced from pullulan by neopullulanase and analyzed the structures by glucoamylase, pullulanase, and neopullulanase analyses. The experimental results supported the above-mentioned model of the pattern of action of neopullulanase on pullulan.     

Structure, specificity and function of cyclomaltodextrinase, a multispecific enzyme of the alpha-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.     

Cloning of the debranching-enzyme gene from Thermoanaerobium brockii into Escherichia coli and Bacillus subtilis.

The gene for an enzyme with single or dual specificity on complex carbohydrates has been transferred from its native host (Thermoanaerobium brockii), a thermophilic anaerobe, into Escherichia coli and Bacillus subtilis. Most of the gene coding region is in a 2.2-kilobase PstI fragment that is common to the E. coli and B. subtilis chimeric vectors pCPC902 and pCPC903, respectively. Although the T. brockii debranching enzyme secreted from B. subtilis was unglycosylated and had less thermostability, more enzyme was secreted from B. subtilis (0.80 to 1.0 U/ml) than from T. brockii (0.23 U/ml). E. coli did not export any measurable enzyme. From the fermentation broth of B. subtilis containing pCPC903, three active species of the debranching enzyme were separated; two species are possibly protease digestion products of the larger protein (105,000 molecular weight). Whereas the enzyme can cleave all of the alpha-1----6 glucosidic linkages (and none of the alpha-1----4 bonds) in pullulan, it hydrolyzed mostly alpha-1----4 and very few of the alpha-1----6 linkages in starch. Upon hydrolysis of pullulan by the enzyme, only maltotriose was produced, while starch was digested to various-sized oligomers.     

Cloning, expression and biochemical characterisation of a unique thermostable pullulan-hydrolysing enzyme from the hyperthermophilic archaeon Thermococcus aggregans

The gene for a new type of pullulan hydrolase from the hyperthermophilic archaeon Thermococcus aggregans was cloned and expressed in Escherichia coli. The 2181-bp open reading frame encodes a protein of 727 amino acids. A hypothetical membrane linker region was found to be cleaved during processing in E. coli. The recombinant enzyme was purified 70-fold by heat treatment, affinity and anion exchange chromatography. Optimal activity was detected at 95 degrees C at a broad pH range from 3.5 to 8.5 with an optimum at pH 6.5. More than 35% of enzymatic activity was detected even at 120 degrees C. The enzyme was stable at 90 degrees C for several hours and exhibited a half-life of 2.5 h at 100 degrees C. Unlike all pullulan-hydrolysing enzymes described to date, the enzyme is able to attack alpha-1,6- as well as alpha-1,4-glycosidic linkages in pullulan leading to the formation of a mixture of maltotriose, panose, maltose and glucose. The enzyme is also able to degrade starch, amylose and amylopectin forming maltotriose and maltose as main products.     

Screening for and identification of starch-, amylopectin-, and pullulan-degrading activities in bifidobacterial strains

Forty-two bifidobacterial strains were screened for alpha-amylase and/or pullulanase activity by investigating their capacities to utilize starch, amylopectin, or pullulan. Of the 42 bifidobacterial strains tested, 19 were capable of degrading potato starch. Of these 19 strains, 11 were able to degrade starch and amylopectin, as well as pullulan. These 11 strains, which were shown to produce extracellular starch-degrading activities, included 5 strains of Bifidobacterium breve, 1 B. dentium strain, 1 B. infantis strain, 3 strains of B. pseudolongum, and 1 strain of B. thermophilum. Quantitative and qualitative enzyme activities were determined by measuring the concentrations of released reducing sugars and by high-performance thin-layer chromatography, respectively. These analyses confirmed both the inducible nature and the extracellular nature of the starch- and pullulan-degrading enzyme activities and showed that the five B. breve strains produced an activity that is consistent with type II pullulanase (amylopullulanase) activity, while the remaining six strains produced an activity with properties that resemble those of type III pullulan hydrolase.     

Specific detection of pullulanase type I in polyacrylamide gels

Abstract A specific detection of pullulanase type I which hydrolyzes the α-1,6-glycosidic linkages on pullulan and starch was developed using impregnation of gels with soluble starch and staining for amylose with iodine. It was a simple and highly sensitive zymogram method capable of detecting as little as 0.001 unit of pullulanase type I activity in polyacrylamide gels after electrophoresis. After fractionation of crude enzyme using DEAE ion exchange chromatography to avoid possible co-migration of amylolytic enzymes which disturb the interaction between amylose and iodine, the high and critical sensitivity of the detection was achieved. The specific detection is based on the fact that when pullulanase type I hydrolyzes the α-1,6-glycosidic bonds in soluble starch increased amounts of α-1,4-linked amylose is formed which yields more intensely blue colored conjugate with iodine. Thus, blue bands on the lighter background signal the presence of pullulanase type I. In contrast, amylolytic enzymes give 'white' bands on the lightly stained background because they remove amylose. This procedure is effective in enzyme screening to distinguish debranching enzyme (pullulanase type I) from other pullulan-degrading enzymes.     

New type of pullulanase from Bacillus stearothermophilus and molecular cloning and expression of the gene in Bacillus subtilis.

A new type of pullulanase which mainly produced panose from pullulan was found in Bacillus stearothermophilus and purified. The enzyme can hydrolyze pullulan efficiently and only hydrolyzes a small amount of starch. When pullulan was used as a substrate, the main product was panose and small amounts of glucose and maltose were simultaneously produced. By using pTB522 as a vector plasmid, the enzyme gene was cloned and expressed in Bacillus subtilis. Since the enzyme from the recombinant plasmid carrier could convert pullulan into not only panose but also glucose and maltose, we concluded that these reactions were due to the single enzyme. The new pullulanase, with a molecular weight of 62,000, was fairly thermostable. The optimum temperature was 60 to 65 degrees C, and about 90% of the enzyme activity was retained even after treatment at 60 degrees C for 60 min. The optimum pH for the enzyme was 6.0.     

Pullulanase type I from Fervidobacterium pennavorans Ven5: cloning, sequencing, and expression of the gene and biochemical characterization of the recombinant enzyme

The gene encoding the type I pullulanase from the extremely thermophilic anaerobic bacterium Fervidobacterium pennavorans Ven5 was cloned and sequenced in Escherichia coli. The pulA gene from F. pennavorans Ven5 had 50.1% pairwise amino acid identity with pulA from the anaerobic hyperthermophile Thermotoga maritima and contained the four regions conserved among all amylolytic enzymes. The pullulanase gene (pulA) encodes a protein of 849 amino acids with a 28-residue signal peptide. The pulA gene was subcloned without its signal sequence and overexpressed in E. coli under the control of the trc promoter. This clone, E. coli FD748, produced two proteins (93 and 83 kDa) with pullulanase activity. A second start site, identified 118 amino acids downstream from the ATG start site, with a Shine-Dalgarno-like sequence (GGAGG) and TTG translation initiation codon was mutated to produce only the 93-kDa protein. The recombinant purified pullulanases (rPulAs) were optimally active at pH 6 and 80 degrees C and had a half-life of 2 h at 80 degrees C. The rPulAs hydrolyzed alpha-1,6 glycosidic linkages of pullulan, starch, amylopectin, glycogen, alpha-beta-limited dextrin. Interestingly, amylose, which contains only alpha-1,4 glycosidic linkages, was not hydrolyzed by rPulAs. According to these results, the enzyme is classified as a debranching enzyme, pullulanase type I. The extraordinary high substrate specificity of rPulA together with its thermal stability makes this enzyme a good candidate for biotechnological applications in the starch-processing industry.     

Enzymatic properties and regulation of ZPU1, the maize pullulanase-type starch debranching enzyme

Starch debranching enzymes (DBE) are required for mobilization of carbohydrate reserves and for the normal structural organization of storage glucan polymers. Two isoforms, the pullulanase-type DBEs and the isoamylase-type DBEs, are both highly conserved in plants. To address DBE functions in starch assembly and breakdown, this study characterized the biochemical activity of ZPU1, a pullulanase-type DBE that is the product of the maize Zpu1 gene. Assays showed directly that recombinant ZPU1 (ZPU1r) expressed in Escherichia coli functions as a pullulanase-type enzyme, and 1H-NMR spectroscopy demonstrated that ZPU1r specifically hydrolyzes alpha(1-->6) branch linkages. Preferred substrates for ZPU1r hydrolytic activity were determined, as were pH, temperature, and thermal stability optima. Kinetic properties of ZPU1r with respect to two substrates, beta-limit dextrin and pullulan, were determined. ZPU1 activity was increased by incubation with thioredoxin h, and native activity was decreased in mutants that accumulate soluble sugars, suggesting potential regulatory mechanisms.     

Analysis of the active center of Bacillus stearothermophilus neopullulanase.

The active center of the neopullulanase from Bacillus stearothermophilus was analyzed by means of site-directed mutagenesis. The amino acid residues located in the active center of the neopullulanase were tentatively identified according to a molecular model of Taka-amylase A and homology analysis of the amino acid sequences of neopullulanse, Taka-amylase A, and other amylolytic enzymes. When amino acid residues Glu and Asp, corresponding to the putative catalytic sites, were replaced by the oppositely charged (His) or noncharged (Gln or Asn) amino acid residue, neopullulanase activities toward alpha-(1----4)- and alpha-(1----6)-glucosidic linkages disappeared. When the amino acids corresponding to the putative substrate-binding sites were replaced, the specificities of the mutated neopullulanases toward alpha-(1----4)- and alpha-(1----6)-glucosidic linkages were obviously different from that of the wild-type enzyme. This finding proves that one active center of neopullulanase participated in the dual activity toward alpha-(1----4)- and alpha-(1----6)-glucosidic linkages. Pullulan is a linear glucan of maltotriosyl units linked through alpha-(1----6)-glucosidic linkages. The production ratio of panose from pullulan was significantly increased by using the mutated neopullulanase which exhibited higher specificity toward the alpha-(1----4)-glucosidic linkage. In contrast, the production ratio of panose was obviously decreased by using the mutated neopullulanse which exhibited higher specificity toward the alpha-(1----6)-glucosidic linkage.