Changes in the Catalytic Properties of Pyrococcus furiosus Thermostable Amylase by Mutagenesis of the Substrate Binding Sites

Site-directed mutagenesis.Variant forms of P. furiosus TA were constructed using a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). The recombinant plasmid pETPFTA-6h (34) carrying the P. furiosus TA gene (UniProt number Q8TZP8) was digested with NdeI and XhoI, and the DNA fragment containing the P. furiosus TA gene was then extracted from agarose gel and subcloned into pTKNd6xH (34). The resulting plasmid was designated pTKwtPA-6h and was used as the template for mutagenesis. The oligonucleotide sequences used for the mutagenesis reactions are summarized in Table 1. The mutations introduced into P. furiosus TA were confirmed using dideoxy chain termination sequencing with an ABI 3700 PRISM DNA sequencer (Perkin-Elmer, Wellesley, MA).

Purification of P. furiosus TA wild-type and mutant enzymes. Escherichia coli MC1061 [F⁻araD139 recA13 Δ(araABC-leu)7696 galU galK ΔlacX74 rpsL thi hsdR2 mcrB] was used as a host for the expression of wild-type P. furiosus TA and its variant forms. E. coli MC1061 transformants were grown in Luria-Bertani broth (1% [wt/vol] Bacto tryptone, 0.5% [wt/vol] yeast extract, and 0.5% [wt/vol] NaCl) supplemented with kanamycin (20 μg/ml) at 37°C for 20 h. The recombinant enzymes were purified by nickel-nitrotriacetate (Ni-NTA) affinity chromatography with a system from QIAGEN (Hilden, Germany) as previously described (34). The eluted fractions were dialyzed against 50 mM Tris-HCl buffer (pH 7.0) containing 150 mM NaCl. The purity of the enzymes was estimated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein concentrations were determined using the Bradford method (3), with bovine serum albumin as a standard.

Enzyme assay for β-CD hydrolysis.The activities of the wild-type and mutant enzymes in β-CD hydrolysis were assayed at 85°C in 50 mM sodium acetate buffer (pH 4.5) with 3,5-dinitrosalicylic acid as described by Miller (22). One hundred fifty microliters of 1% (wt/vol) β-CD and 130 μl of 50 mM sodium acetate buffer were mixed and preincubated at 85°C. Twenty microliters of the enzyme was added to the preheated solution, and then the reaction mixture was incubated for 10 min. The reaction was stopped by adding 900 μl of 3,5-dinitrosalicylic acid solution and then boiling for 5 min and cooling with iced water. The absorbance of the reaction mixture at 570 nm was measured using a spectrophotometer (UV-160A; Shimadzu Co., Tokyo, Japan). One unit of enzyme activity was defined as the amount of enzyme that produced 1 μmol of reducing sugars from the substrate per min.

Hydrolytic patterns of P. furiosus TA.To examine the hydrolytic patterns of P. furiosus TA wild-type and mutant enzymes, 0.5 ml of the purified enzyme (1.5 U of β-CD hydrolysis activity) was incubated with 0.5 ml of 1% (wt/vol) substrate in 50 mM sodium acetate buffer (pH 4.5) at 75°C for 1 h. The substrates used were CDs (α-, β-, and γ-CDs), maltooligosaccharides (maltose [G2] to G7), and soluble starch. The reaction products were analyzed on K5F silica gel plates (Whatman, Maidstone, United Kingdom) by thin-layer chromatography (TLC) with isopropyl alcohol-ethyl acetate-water (3:1:1, vol/vol/vol) as the solvent system. After two elutions, each TLC plate was dried and visualized by dipping it into 0.3% (wt/vol) N-(1-naphthyl)-ethylenediamine and 5% (vol/vol) H₂SO₄ in methanol followed by charring it for 10 min at 110°C (28).

HPAEC.The reaction mixture was analyzed by high-performance anion- exchange chromatography (HPAEC) using a pulsed amperometric detector (ED40; Dionex, Sunnyvale, CA). The system was equipped with a CarboPac PA-100 column (4 by 250 mm; Dionex) and run with a gradient of 0 to 0.4 M sodium acetate in 0.15 M NaOH with a flow rate of 1 ml/min.

Transglycosylation patterns of P. furiosus TA and the P. furiosus TA(H414N/G415E) mutant.To examine the transglycosylation patterns of wild-type P. furiosus TA and the mutant enzyme with amino acid changes H414N and G415E [P. furiosus TA(H414N/G415E)], 0.5 ml of each purified enzyme (3 U of β-CD hydrolysis activity) was incubated with 0.5 ml of 1% (wt/vol) substrate in 50 mM sodium acetate buffer (pH 4.5) at 75°C for 2 h. The substrates used were CDs (α-, β-, and γ-CDs), maltooligosaccharides (G2 to G7), and soluble starch. Two microliters of each reaction product was added to a Whatman K5F silica gel TLC plate and eluted twice using acetonitrile-ethyl acetate-isopropyl alcohol-water (85:20:50:70, vol/vol/vol/vol) as the solvent system. The reaction products were visualized using the same methods described above for hydrolytic pattern analyses. To verify the structures of the reaction products corresponding to P. furiosus TA(H414N/G415E), β-amylase (Sigma Co.) was added directly to the reaction mixture and the treated mixture was then incubated overnight at 37°C. The reaction mixture and the β-amylase-treated mixture were analyzed using HPAEC. The maltooligosaccharide standard was prepared from the reaction of isoamylase (Hayashibara Biochemical Laboratories, Inc., Okayama, Japan) with 0.5% (wt/vol) amylopectin (Sigma Co., St. Louis, MO) in 50 mM sodium citrate buffer (pH 4.3) for 60 h at 60°C. The standard of branched maltooligosaccharides was prepared from the reaction of pullulanase (Promozyme; Novo Nordisk, Copenhagen, Denmark) with 0.5% (wt/vol) pullulan (Sigma Co.) in 50 mM sodium citrate buffer (pH 4.3) for 10 min at 60°C.

Measurement of kinetic parameters for hydrolysis by P. furiosus TA and its derivatives.The substrate concentrations for the kinetic analyses ranged from 0.3 to 8 mM for β-CD and 0.2 to 8 mM for G7 in 50 mM sodium acetate buffer (pH 4.5). The reactions were carried out at 85°C. Aliquots (50 μl) of the mixtures were taken at 90-s intervals, and the reactions were immediately stopped by transferring each mixture into 50 μl of 0.1 M HCl and placing the mixture on ice. After neutralizing the mixture by adding 50 μl of 0.1 N NaOH, the amount of hydrolyzed substrate was analyzed quantitatively using HPAEC. GraFit version 4.0 (Erithacus Software, United Kingdom) was used to calculate kinetic parameters by the direct fitting of initial rates.

Identification of residues critical for substrate binding.GH-13 enzymes have four conserved regions. The catalytically important residues, including three catalytic residues and five key residues for substrate binding, are indicated in Fig. 1. According to the structures of GH-13 enzymes bound to their substrates, the last two residues of the second conserved region (Lys and His in α-amylases and CGTases and Asn and Glu in CD-degrading enzymes) and a hydrophobic residue in the third conserved region (Trp in α-amylases and CD-degrading enzymes and Phe in CGTases) play important roles in substrate binding to the +1 and +2 subsites (16, 25). As shown in Fig. 1, three residues are strictly conserved among the bacterial CD-degrading enzymes, Asn331, Glu332, and Trp359 (Thermus sp. strain IM6501 maltogenic amylase numbering). In addition, the last residue in the third conserved region of α-amylases and CGTases is relatively variable, while the corresponding residue in CD-degrading enzymes is a strictly conserved His (Fig. 1). However, P. furiosus TA has different amino acids at the positions listed above, and these variant residues (H414, G415, M439, and D440) are predicted to determine the substrate recognition and catalytic properties of the enzyme. For this reason, these four residues in the second and third conserved regions of P. furiosus TA were mutated to the corresponding residues (Asp, Glu, Trp, and His, respectively) typical in CD-degrading enzymes and the catalytic properties of the variant enzymes were investigated.

Role of H414 and G415 in the active site of P. furiosus TA.The role of His414 and Gly415 in determining the catalytic properties of P. furiosus TA was investigated by analyzing three variant enzymes modified at these residues, TA(H414N), TA(G415E), and TA(H414N/G415E). Each mutant enzyme was expressed in E. coli and purified to apparent homogeneity by Ni-NTA affinity chromatography. The hydrolysis of β-CD by TA(H414N), TA(G415E), and TA(H414N/G415E) decreased 8-fold (21 U/mg), 10-fold (17 U/mg), and 80-fold (2 U/mg), respectively, compared to that by the parent enzyme (170 U/mg). The substrate preferences of TA(H414N) and TA(G415E) were roughly the same as that of the wild-type enzyme; all three enzymes hydrolyzed β-CD into G7 as the major product plus several small maltooligosaccharides (Fig. 2A, lanes 1 and 2). However, the double mutant form [TA(H414N/G415E)] yielded a series of oligosaccharides that were longer than the maltooligosaccharides generated from the β-CD ring-opening reactions (Fig. 2A, lane 3). As CD-degrading enzymes produce predominantly α-(1,6)-transfer products, we expected the longer products to be oligosaccharides with multiple α-(1,6)-glycosidic linkages. Unexpectedly, when the reaction mixture containing P. furiosus TA(H414N/G415E) was compared to that containing maltooligosaccharide standards by HPAEC (see Materials and Methods), the transfer products had retention times identical to those of α-(1,4)-linked linear maltooligosaccharides (Fig. 2B). In contrast, the retention times of the transfer products were different from those of the products of the partial hydrolysis of pullulan by a pullulanase (Fig. 2B). To confirm their structures, the products synthesized by TA(H414N/G415E) were treated with β-amylase (Sigma, St. Louis, MO). β-Amylase completely hydrolyzed all transfer products into glucose, maltose, and maltotriose with neither unhydrolyzed long oligosaccharides nor small α-(1,6)-linked oligosaccharides (Fig. 2B). These results indicate that the transfer products had α-(1,4)-glycosidic linkages only. To further investigate the substrate specificity of TA(H414N/G415E) for the disproportion activity, the enzyme was incubated with 0.5% (wt/vol) maltooligosaccharides (G2 to G7) and CDs (α-, β-, and γ-CDs) and each reaction mixture was analyzed by TLC. Wild-type P. furiosus TA only hydrolyzed the substrates (Fig. 3A), whereas TA(H414N/G415E) successfully carried out disproportion reactions using maltotriose (G3) and longer maltooligosaccharides (Fig. 3B). Large amounts (10 to 20 U) of wild-type TA were unsuccessful at yielding any transfer products (data not shown).

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FIG. 2.

Analyses of the reaction products formed from β-CD by P. furiosus TA mutant enzymes with amino acid changes H414N, G415E, and H414N and G415E. Each enzyme (0.7 U) was reacted with 0.5% (wt/vol) β-CD in 50 mM sodium acetate buffer (pH 4.5) at 75°C for 1 h. (A) TLC analyses of the reaction mixtures containing TA(H414N), TA(G415E), and TA(H414N/G415E). Lane M, maltooligosaccharide standards; lane 1, reaction mixture with TA(H414N); lane 2, reaction mixture with TA(G415E); lane 3, reaction mixture with TA(H414N/G415E). (B) HPAEC analysis of the reaction mixture containing P. furiosus TA(H414N/G415E). MO standard, maltooligosaccharide standard; pullulan hydrolysate, products of the partial hydrolysis of pullulan by a pullulanase; after β-amylase, β-amylase-treated reaction mixture with TA(H414N/G415E).

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FIG. 3.

TLC analyses of the reaction products formed from various substrates by wild-type P. furiosus TA (A) and P. furiosus TA(H414N/G415E) (B). Lanes M, maltooligosaccharide standards (glucose to maltoheptaose); lanes 1, maltose; lanes 2, maltotriose; lanes 3, maltotetraose; lanes 4, maltopentaose; lanes 5, maltohexaose; lanes 6, maltoheptaose, lanes 7, α-CD; lanes 8, β-CD; lanes 9, γ-CD. Each enzyme (1.5 U) was reacted with each substrate at a concentration of 0.5% (wt/vol) in 50 mM sodium acetate buffer (pH 4.5) at 75°C for 2 h.

The role of Met439 and Asp440 in the active site of P. furiosus TA.To clarify the role of Met439 and Asp440 in the catalytic activity of P. furiosus TA, these residues were mutated to Trp and His, respectively. Recombinant TA(M439W) could not be produced in a soluble form in E. coli (data not shown); however, the variant enzymes TA(D440H) and TA(M439W/D440H) were successfully produced in a soluble form and purified to apparent homogeneity by Ni-NTA affinity chromatography. By using 0.5% (wt/vol) β-CD as the substrate, P. furiosus TA(D440H) and TA(M439W/D440H) were shown to have 50% [86 U/mg of TA(D440H)] and 13% [22 U/mg of TA(M439W/D440H)] of the hydrolyzing activity of wild-type TA (170 U/mg). To explore the catalytic properties of the variant forms, we compared the hydrolysis patterns of the wild-type and variant enzymes by using 0.5% (wt/vol) β-CD as the substrate with equal numbers of enzyme units (0.1 U of enzyme per 1 mg of substrate). Time course analysis of the reaction mixture showed that wild-type P. furiosus TA simply opened the rings of the β-CD without significantly degrading the resulting G7 products (Fig. 4). The same result was obtained in our previous experiments (33). In contrast, TA(D440H) and TA(M439W/D440H) liberated various small maltooligosaccharides from the CDs (Fig. 4). In addition, the double mutant TA(M439W/D440H) hydrolyzed G7 faster than TA(D440H). These results imply that M439 and D440 each contribute to the preference of wild-type P. furiosus TA for cyclic over linear substrates.

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FIG. 4.

TLC analyses of the reaction products of wild-type P. furiosus TA (PFTA WT), P. furiosus TA(D440H) (PFTA-D440H), and P. furiosus TA(M439W/D440H) (PFTA-M439W/D440H). Lane M contains maltooligosaccharide standards (glucose to maltoheptaose). Each aliquot was taken at different reaction times of 0, 2, 10, 20, and 60 min. Each enzyme (0.5 U) was reacted with β-CD at a concentration of 0.5% (wt/vol) in 50 mM sodium acetate buffer (pH 4.5) at 75°C.

To verify the effects of the mutations on substrate specificity, kinetic analyses of the hydrolysis of β-CD and G7 were conducted using the wild-type and mutant enzymes. The kinetic parameters of P. furiosus TA(D440H) for G7 were not greatly altered compared to those of wild-type TA (Table 2); however, the catalytic efficiency of the enzyme (k_cat/K_m ratio) for β-CD was reduced from 100 to 16 s⁻¹ mM⁻¹ due to a dramatic decrease in the k_cat. Interestingly, the introduction of an additional mutation (M439W) into TA(D440H) led to enhanced hydrolysis of G7 without significantly changing the catalytic efficiency of the enzyme for β-CD (Table 2). Overall, replacing M439 and D440 in P. furiosus TA with Trp and His increased the ratio of the catalytic efficiency for G7 to that for β-CD [(k_cat/K_m ratio for G7)/(k_cat/K_m ratio for β-CD)] by 26-fold relative to the ratio of the catalytic efficiencies of wild-type TA for these substrates. These results suggest that M439 and D440 are critical for the recognition and hydrolysis of linear and cyclic maltodextrins, respectively, by P. furiosus TA.