Comparison of primary structures and substrate specificities of two pullulan-hydrolyzing alpha-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 structure of Thermoactinomyces vulgaris R-47 alpha-amylase II (TVAII) hydrolyzing cyclodextrins and pullulan at 2.6 A resolution.

The crystal structure of Thermoactinomyces vulgaris R-47 alpha-Amylase II (TVAII) has been determined by multiple isomorphous replacement at 2.6 A resolution. TVAII was crystallized in an orthorhombic system with the space group P212121 and the cell dimensions a=118.5 A, b=119.5 A, c=114.5 A. There are two molecules in an asymmetric unit, related by the non-crystallographic 2-fold symmetry. Diffraction data were collected at 113 K and the cell dimensions reduced to a=114.6 A, b=117.9 A, c=114.2 A, and the model was refined against 7.0-2.6 A resolution data giving an R-factor of 0.204 (Rfree=0.272). The final model consists of 1170 amino acid residues (two molecules) and 478 water molecules with good chemical geometry. TVAII has three domains, A, B, and C, like other alpha-amylases. Domain A with a (beta/alpha)8 barrel structure and domain C with a beta-sandwich structure are very similar to those found in other alpha-amylases. Additionally, TVAII has an extra domain N composed of 121 amino acid residues at the N-terminal site, which has a beta-barrel-like structure consisting of seven antiparallel beta-strands. Domain N is one of the driving forces in the formation of the dimer structure of TVAII, but its role in the enzyme activity is still not clear. TVAII does not have the Ca2+ binding site that connects domains A and B in other alpha-amylases, rather the NZ atom of Lys299 of TVAII serves as the connector between these domains. TVAII can hydrolyze cyclodextrins and pullulan as well as starch. Based on a structural comparison with the complex between a mutant cyclodextrin glucanotransferase and a beta-cyclodextrin derivative, Phe286 located at domain B is considered the residue most likely to recognize the hydrophobic cavity of cyclodextrins. The active-site cleft of TVAII is wider and shallower than that of other alpha-amylases, and seems to be suitable for the binding of pullulan which is expected not to adopt the helical structure of amylose.

Conversion of neopullulanase-alpha-amylase from Thermoactinomyces vulgaris R-47 into an amylopullulanse-type enzyme.

TVA I, an alpha-amylase from Thermoactinomyces vulgaris R-47, is a versatile enzyme which hydrolyzes the alpha-(1-->4)-glucosidic linkages of pullulan to produce panose, known as neopullulanase activity, and the alpha-(1-->6)-glucosidic linkages of certain oligosaccharides. We modified the Ala-357, Gln-359, and Tyr-360 residues located in region II, one of the four regions conserved in alpha-amylase family enzymes, and deleted 11 consecutive amino acid residues located after the C-terminus of region II of the TVA I sequence by means of site-directed mutagenesis. The action pattern of the mutated enzyme for pullulan was greatly altered and it hydrolyzed mainly the alpha-(1-->6)-glucosidic linkages of pullulan to produce maltotriose, while the action patterns for starch and maltooligosaccharides were almost identical to those of the wild-type enzyme. This means that the mutated TVA I has lost the neopullulanase activity, and thus can be designated as an amylopullulanase-type enzyme. The kcat/Km value of the mutated enzyme for alpha-(1-->6)-glucosidic linkages was virtually unaltered, while that for alpha-(1-->4)-glucosidic linkages was about 100 times smaller than that of the wild-type enzyme.

Studies on the hydrolyzing mechanism for cyclodextrins of Thermoactinomyces vulgaris R-47 alpha-amylase 2 (TVAII). X-ray structure of the mutant E354A complexed with beta-cyclodextrin, and kinetic analyses on cyclodextrins.

Crystals of the mutant E354A of Thermoactinomyces vulgaris R-47 alpha-amylase 2 (TVAII) complexed with beta-cyclodextrin were prepared by a soaking method, and the diffraction data were collected at 100 K, using Synchrotron radiation (SPring-8). The crystals belong to an orthorhombic system with the space group P2(1)2(1)2(1) and cell dimensions a = 111.1 A, b = 117.7 A, c = 113.3 A, which is almost isomorphous with crystals of the wild-type TVAII, and the structure was refined to an R-factor = 0.208 (R(free) = 0.252) using 3.0 A resolution data. The refined structure shows that the interactions between Phe286 and two C6 atoms of beta-cyclodextrin at the hydrolyzing site are important for TVAII to recognize cyclodextrins as substrates. This observation from the X-ray structure was supported by kinetic analyses of cyclodextrins using the wild-type TVAII, the mutant F286A and F286L. These studies also suggested that the TVAII-hydrolyzing mechanism for cyclodextrins is slightly different from that for starch.

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.

Mechanism of active site exclusion in a site-specific recombinase: role of the DNA substrate in conferring half-of-the-sites activity.

The Flp site-specific recombinase assembles its active site by recruiting the catalytic tyrosine (Tyr-343) from one Flp monomer into the pro-active site containing a triad of Arg-191, His-305, and Arg-308 from a second monomer. In principle, two active sites may be assembled from a Flp dimer by simultaneous, reciprocal contribution of the shared amino acids by its constituent monomers. In practice, only one of the two active sites is assembled at a time, as would be consistent with a recombination mechanism involving two steps of single-strand exchanges. By using substrates containing strand-specific base bulges, we demonstrate that the relative disposition of their DNA arms can account for this active site exclusion. We also show that the exclusion mechanism operates only at the level of positioning Tyr-343 with respect to the pro-active site, and not at the level of orienting the labile phosphodiester bond within the DNA chain. It is not negative cooperativity of substrate binding but, rather, the substrate-induced negative cooperativity in protein orientation that accomplishes half-of-the-sites activity in the Flp system.

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

A region downstream of the gene for pullulan-hydrolyzing alpha-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 beta-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 glucoamy-lase-type enzymes with oligosaccaharide-metabolizing activity.

The deletion of amino-terminal domain in Thermoactinomyces vulgaris R-47 alpha-amylases: effects of domain N on activity, specificity, stability and dimerization.

Thermoactinomyces vulgaris R-47 alpha-amylases, TVA I and TVA II, have a domain N, which is an extra structure in the family 13 enzymes. To investigate the roles of domain N in TVAs, we constructed TVAs-deltaN mutants which are deleted in domain N, and Y14,16,68A and Y41,82,95A mutants of TVA II. TVAs-deltaN were unstable under alkaline conditions, and their thermal stabilities were 10 degrees C lower than that of wild-types. The specific activities of TVAs-deltaN for pullulan, starch, cyclodextrins, and oligosaccharides were drastically decreased, being about 1,500- to 10,000-fold smaller than those of wild-types. The kcat values of Y14,16,68A and Y41,82,95A for all tested substrates were markedly decreased, and the Km value of Y14,16,68A for alpha-CD and maltotriose were 25- and 3-fold larger, and that of Y41,82,92A for starch was 10-fold larger than that of the wild-type. TVA I and TVAs-deltaN in solution are a monomer, while TVA II is a homo-dimer, calculated by their molecular masses. These results suggest domain N in TVAs is an important structure for stabilization of enzymes, recognition and hydrolysis of substrates, and dimerization of TVA II.

Cloning and nucleotide sequence of the pullulanase gene of Thermus thermophilus HB8 and production of the enzyme in Escherichia coli.

A 3.4-kb SphI fragment carrying the pullulanase gene of Thermus thermophilus HB8 was cloned. Based on the nucleotide sequence of it and the flanking region analyzed by direct sequencing of the inverse PCR product, an expression vector was constructed. The E. coli cells harboring the plasmid produced an about 80-kDa protein having pullulanase activity, the optimum temperature of which was 70 degrees C.

Structures of Thermoactinomyces vulgaris R-47 alpha-amylase II complexed with substrate analogues.

The structures of Thermoactinomyces vulgaris R-47 alpha-amylase II mutant (d325nTVA II) complexed with substrate analogues, methyl beta-cyclodextrin (m beta-CD) and maltohexaose (G6), were solved by X-ray diffraction at 3.2 A and 3.3 A resolution, respectively. In d325nTVA II-m beta-CD complex, the orientation and binding-position of beta-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 alpha-glucan and CD-stacking causes the functional distinctions between CGTase and TVA II.

Analysis of catalytic residues of Thermoactinomyces vulgaris R-47 alpha-amylase II (TVA II) by site-directed mutagenesis.

To confirm that the catalytic residues (Asp325, Glu354, and Asp421) are necessary for the hydrolysis of starch, pullulan, and cyclodextrins, we constructed TVA II mutated by site-directed mutagenesis. The mutated enzymes (D325N, E354Q, and D421N) had markedly reduced levels of activity, less than 0.006% of the wild type, indicating that these three residues are the catalytic sites for these substrates. Even E354D had reduced levels of activity, less than 0.05% of wild type. These four mutated enzymes retained a trace of activity. From the result of hydrolysis patterns for maltohexaose, in particular, D421N, unlike D325N and E354Q, catalyzed transglycosylation rather than hydrolysis. The results suggest that Asp421 could function to capture water molecules.

A neopullulanase-type alpha-amylase gene from Thermoactinomyces vulgaris R-47.

Shimizu et al. and ourselves have reported some enzymatic properties of an alpha-amylase from Thermoactinomyces vulgaris R-47 that hydrolyzed pullulan to produce panose [M. Shimizu et al., Agric. Biol. Chem., 42, 1681-1688 (1978); Y. Sakano et al., Agric. Biol. Chem., 46, 1121-1129 (1982)]. In this study, we cloned a gene for an alpha-amylase, which was different from the one mentioned above but also hydrolyzed pullulan to produce panose, from T. vulgaris R-47, and analyzed the entire primary structure of the gene. We designated the previously reported enzyme as T. vulgaris alpha-amylase I (TVA I), and this novel enzyme as T. vulgaris alpha-amylase II (TVA II). The nucleotide sequence had an open reading frame of 1755 base pairs corresponding to a protein of 585 amino acid residues. Although this novel alpha-amylase, TVA II, hydrolyzed both pullulan and starch, the ratio of pullulan-hydrolyzing activity to starch-hydrolyzing activity of the enzyme was higher than that of TVA I, and the primary structure of the enzyme resembled neopullulanase, which scarcely hydrolyzed starch, rather than that of TVA I.