Yoshiyuki Sakano

Comparison of primary structures and substrate specificities of two pullulan-hydrolyzing α-amylases, TVA I and TVA II, from Thermoactinomyces vulgaris R-47

Thermoactinomyces vulgaris R-47 produces two alpha-amylases, TVA I, an extracellular enzyme, and TVA II, an intracellular enzyme. Both enzymes hydrolyze pullulan to produce panose, and also hydrolyze cyclodextrins. We cloned and sequenced the TVA I gene. The TVA I gene consisted of 1833 base pairs, and the deduced primary structure was composed of 611 amino-acid residues, including an N-terminal signal sequence consisting of 29 amino-acid residues. The similarity between the amino-acid sequence of mature TVA I with those of other pullulan/cyclodextrin-hydrolyzing enzymes, such as TVA II and Bacillus stearothermophilus neopullulanase, was only 30%, although that of TVA II with neopullulanase was 48%. TVA II prefers specific small oligosaccharides and alpha- and beta-cyclodextrins. Whereas kcat/Km values of TVA I for pullulan were larger than that of TVA II, and TVA II could not hydrolyze starch completely. TVA II was inhibited by maltose, the hydrolysate of starch, which seems to be the reason for inefficient hydrolysis of starch. These kinetic properties indicate that TVA I and TVA II have differential physiological roles in sugar metabolism extracellularly and intracellularly, respectively.

Crystal structures and structural comparison of Thermoactinomyces vulgaris R-47 alpha-amylase 1 (TVAI) at 1.6 A resolution and alpha-amylase 2 (TVAII) at 2.3 A resolution.

The X-ray crystal structures of Thermoactinomyces vulgaris R-47 alpha-amylase 1 (TVAI) and alpha-amylase 2 (TVAII) have been determined at 1.6 A and 2.3 A resolution, respectively. The structures of TVAI and TVAII have been refined, R-factor of 0.182 (R(free)=0.206) and 0.179 (0.224), respectively, with good chemical geometries. Both TVAI and TVAII have four domains, N, A, B and C, and all very similar in structure. However, there are some differences in the structures between them. Domain N of TVAI interacts strongly with domains A and B, giving a spherical shape structure to the enzyme, while domain N of TVAII is isolated from the other domains, which leads to the formation of a dimer. TVAI has three bound Ca ions, whereas TVAII has only one. TVAI has eight extra loops compared to TVAII, while TVAII has two extra loops compared to TVAI. TVAI can hydrolyze substrates more efficiently than TVAII with a high molecular mass such as starch, while TVAII is much more active against cyclodextrins than TVAI and other alpha-amylases. A structural comparison of the active sites has clearly revealed this difference in substrate specificity.

Pullulan 4-glucanohydrolase from Aspergillus niger

Abstract Pullulan 4-glucanohydrolase, a novel pullulan-hydrolyzing enzyme from Aspergillus niger , was highly purified by means of acetone precipitation, chromatography on P -cellulose and DEAE-cellulose, and gel filtration on Sephadex G-150. More than 430-fold purification was achieved through these procedures from crude extract of wheat bran culture. The enzyme can liberate a large amount of isopanose and a small amount of tetrasaccharide from pullulan. The optimum pH of the enzyme action on pullulan was 3.0–3.5 and the optimum temperature was 40 °C at pH 3.5. The enzyme activity remained intact after heating at 50 °C for 30 min at pH 3.7–4.5. The enzyme was stable at pH 2.0–8.0 on storage at 5 °C for 24 hr. The purified enzyme attacked reducing end α-1,4-glucosidic linkages adjacent to α-1,6-glucosidic linkages in pullulan, 6 3 -α-glucosylmaltotriose, 6 2 -α-maltosylmaltose and panose, to liberate isopanose, isomaltose and maltose, isopanose and glucose, and isomaltose and glucose, respectively. The molecular weight of the enzyme determined by gel filtration on Bio-Gel P-150 was about 74,000.

Immobilization of Bacillus acidopullulyticus pullulanase and properties of the immobilized pullulanases

Abstract In order to make an enzyme reactor for the production of maltosyl(α1→6)cyclodextrins (G2-CDs), Bacillus acidopullulyticus pullulanase (EC 3.2.1.41, Pase) was immobilized on various carriers. Different methods of immobilization were employed, including physical adsorption, cross-linking and ionic binding, and the enzymic properties of immobilized Pases were investigated. The immobilized enzymes prepared by adsorption on porous glass (PG-Pase), covalent binding on chitosan beads treated with glutaraldehyde (GA-CB-Pase) and ionic binding on Amberlite IRC-50 (IRC-Pase) had high Pase activities. The optimum pH of Pase shifted from 5.0, which is optimum for the native B. acidopullulyticus enzyme, to 3.5 for IRC-Pase and GA-CB-Pase, and 5.5 for PG-Pase. Km values for pullulan- and G2-α-CD-hydrolyzing activities of PG-, GA-CB- and IRC-Pases were greater than those of native Pase. The ratio of G2-α-CD-hydrolyzing activity to pullulan-hydrolyzing activity in immobilized Pase was higher than that of native Pase. The G2-α-CD-synthesizing activities of column reactors (1.6 × 10 cm) packed with PG- and GA-CB-Pases hardly decreased after continuous operation for 72 d at 60°C, but the G2-α-CD-synthesizing activity of the IRC-Pase column reactor decreased to about 50% of the initial activity after 30 d-operation (60°C).

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 α‐amylase 1 (TVAI) has unique hydrolyzing activities for pullulan with sequence repeats of α‐(1,4), α‐(1,4), and α‐(1,6) glycosidic linkages, as well as for starch. TVAI mainly hydrolyzes α‐(1,4) glycosidic linkages to produce a panose, but it also hydrolyzes α‐(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‐α‐(1,6)‐Glc‐α‐(1,4)‐Glc‐α‐(1,4)]2 or P5; [Glc‐α‐(1,6)‐Glc‐α‐(1,4)‐Glc‐α‐(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 oryzaeα‐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 α‐(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 α‐(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.

Novel glucoamylase-type enzymes from Thermoactinomyces vulgaris and Methanococcus jannaschii whose genes are found in the flanking region of the α-amylase genes

Abstract. A region downstream of the gene for pullulan-hydrolyzing α-amylase, TVA II, of Thermoactinomyces vulgaris R-47 was sequenced, and an open reading frame encoding an enzyme homologous to glucoamylase was found. The nucleotide sequence of this enzyme, designated TGA, consists of 1,953 base pairs corresponding to a protein of 651 amino acid residues. The TGA gene was subcloned and expressed in Escherichia coli. Enzymatic analyses showed that, like other glucoamylases, TGA produced β-D-glucose from its substrate. However, TGA hydrolyzed maltooligosaccharides such as maltotetraose and maltose more efficiently than starch, while fungal glucoamylases preferred starch to maltooligosaccharides. The primary structure of TGA resembled a putative glucoamylase from the hyperthermophilic archaeon Methanococcus jannaschii (MGA), while homologies between TGA and the fungal glucoamylases were low. The enzymatic properties of recombinant MGA produced in E. coli cells were similar to those of TGA. These findings indicate that TGA and MGA are novel glucoamylase-type enzymes with oligosaccaharide-metabolizing activity.

Structures of Thermoactinomyces vulgaris R-47 α-Amylase II Complexed with Substrate Analogues

The structures of Thermoactinomyces vulgaris R-47 α-amylase II mutant (d325nTVA II) complexed with substrate analogues, methyl β-cyclodextrin (mβ-CD) and maltohexaose (G6), were solved by X-ray diffraction at 3.2Å and 3.3Å resolution, respectively. In d325nTVA II-mβ-CD complex, the orientation and binding-position of β-CD in TVA II were identical to those in cyclodextin glucanotransferase (CGTase). The active site residues were essentialy conserved, while there are no residues corresponding to Tyr89, Phe183, and His233 of CGTase in TVA II. In d325nTVA II-G6 complex, the electron density maps of two glucosyl units at the non-reducing end were disordered and invisible. The four glucosyl units of G6 were bound to TVA II as in CGTase, while the others were not stacked and were probably flexible. The residues of TVA II corresponding to Tyr89, Lys232, and His233 of CGTase were completely lacking. These results suggest that the lack of the residues related to α-glucan and CD-stacking causes the functional distinctions between CGTase and TVA II.

Enzymic preparation of panose and isopanose from pullulan

Abstract The trisaccharides panose and isopanose were prepared in good yield from enzymic hydrolyzates of pullulan. Pullulan was hydrolyzed by the purified alpha amylase preparation of Thermoactinomyces vulgaris R-47. The digest was applied to a carbon-Celite column and eluted with a linear gradient of 1-propanol from 0 to 5%. From the trisaccharide fractions eluted, panose was prepared in about 70% yield. Pullulan was also hydrolysed by purified isopullulanase (EC 3.2.1.57 pullulan 4-glucanohydrolase) of Aspergillus niger ATCC-9642, and isopanose was prepared in about 90% yield by using the same technique as that for the preparation of panose.

Purification, characterization, and subsite affinities of Thermoactinomyces vulgaris R-47 maltooligosaccharide-metabolizing enzyme homologous to glucoamylases.

A maltooligosaccharide-metabolizing enzyme from Thermoactinomyces vulgaris R-47 (TGA) homologous to glucoamylases does not degrade starch efficiently unlike most glucoamylases such as fungal glucoamylases (Uotsu-Tomita et al., Appl. Microbiol. Biotechnol., 56, 465–473 (2001)). In this study, we purified and characterized TGA, and determined the subsite affinities of the enzyme. The optimal pH and temperature of the enzyme are 6.8 and 60°C, respectively. Activity assays with 0.4% substrate showed that TGA was most active against maltotriose, but did not prefer soluble starch. Kinetic analysis using maltooligosaccharides ranging from maltose to maltoheptaose revealed that TGA has high catalytic efficiency for maltotriose and maltose. Based on the kinetics, subsite affinities were determined. The A 1+A 2 value of this enzyme was highly positive whereas A 4–A 6 values were negative and little affinity was detected at subsites 3 and 7. Thus, the subsite structure of TGA is different from that of any other GA. The results indicate that TGA is a metabolizing enzyme specific for small maltooligosaccharides.

Role of Phe286 in the recognition mechanism of cyclomaltooligosaccharides (cyclodextrins) by Thermoactinomyces vulgaris R-47 alpha-amylase 2 (TVAII). X-ray structures of the mutant TVAIIs, F286A and F286Y, and kinetic analyses of the Phe286-replaced mutant TVAIIs.

Phe286 located in the center of the active site of alpha-amylase 2 from Thermoactinomyces vulgaris R-47 (TVAII) plays an important role in the substrate recognition for cyclomaltooligosaccharides (cyclodextrins). The X-ray structures of mutant TVAIIs with the replacement of Phe286 by Ala (F286A) and Tyr (F286Y) were determined at 3.2 A resolution. Their structures have no significant differences from that of the wild-type enzyme. The kinetic analyses of Phe286-replaced variants showed that the variants with non-aromatic residues, Ala (F286A) and Leu (F286L), have lower enzymatic activities than those with aromatic residues, Tyr (F286Y) and Trp (F286W), and the replacement of Phe286 affects enzymatic activities for CDs more than those for starch.