Purification, Characterization, and Gene Cloning of a Novel Maltosyltransferase from an Arthrobacter globiformis Strain That Produces an Alternating α-1,4- and α-1,6-Cyclic Tetrasaccharide from Starch

Abstract

A glycosyltransferase, involved in the synthesis of cyclic maltosylmaltose [CMM; cyclo-{→6)-α-d-Glcp(1→4)-α-d-Glcp(1→6)-α-d-Glcp(1→4)-α-d-Glcp(1→}] from starch, was purified to homogeneity from the culture supernatant of Arthrobacter globiformis M6. The CMM-forming enzyme had a molecular mass of 71.7 kDa and a pI of 3.6. The enzyme was most active at pH 6.0 and 50°C and was stable from pH 5.0 to 9.0 and up to 30°C. The addition of 1 mM Ca²⁺ enhanced the thermal stability of the enzyme up to 45°C. The enzyme acted on maltooligosaccharides that have degrees of polymerization of ≥3, amylose, and soluble starch to produce CMM but failed to act on cyclomaltodextrins, pullulan, and dextran. The mechanism for the synthesis of CMM from maltotetraose was determined as follows: (i) maltotetraose + maltotetraose → 6⁴-O-α-maltosyl-maltotetraose + maltose and (ii) 6⁴-O-α-maltosyl-maltotetraose → CMM + maltose. Thus, the CMM-forming enzyme was found to be a novel maltosyltransferase (6MT) catalyzing both intermolecular and intramolecular α-1,6-maltosyl transfer reactions. The gene for 6MT, designated cmmA, was isolated from a genomic library of A. globiformis M6. The cmmA gene consisted of 1,872 bp encoding a signal peptide of 40 amino acids and a mature protein of 583 amino acids with a calculated molecular mass of 64,637. The deduced amino acid sequence showed similarities to α-amylase and cyclomaltodextrin glucanotransferase. The four conserved regions common in the α-amylase family enzymes were also found in 6MT, indicating that 6MT should be assigned to this family.

Cyclic tetrasaccharide is the smallest cyclic gluco-oligosaccharide and is composed of four circularly linked d-glucose residues. A cyclic tetrasaccharide that has alternate α-1,3- and α-1,6-glucosidic linkages was first reported by Côté and Biely (6). The cyclic tetrasaccharide, cycloalternan [CA; cyclo-{→6)-α-d-Glcp(1→3)-α-d-Glcp(1→6)-α-d-Glcp(1→3)-α-d-Glcp(1→}], was produced from alternan by its degrading enzyme, called alternanase (4). Alternan is a polysaccharide mainly composed of an alternating sequence of α-1,3- and α-1,6-linked glucose residues (16) but is not commercially available. Therefore, CA has not yet been produced in large quantities.

Recently, we discovered a novel enzymatic system to produce CA from starch or maltodextrins in several bacteria and purified 6-α-glucosyltransferase (6GT; EC 2.4.1.24) and 3-α-isomaltosyltransferase (IMT; EC 2.4.1.−) (19, 3, 17). CA was synthesized from α-1,4-glucan as follows. 6GT transfers a glucose residue at the nonreducing end of α-1,4-glucan to the 6-OH of the nonreducing end glucose of another α-1,4-glucan to produce isomaltosyl-α-1,4-glucan. The isomaltosyl part of the intermediate product is transferred to another isomaltosyl-α-1,4-glucan by IMT, to synthesize isomaltosyl-α-1,3-isomaltosyl-α-1,4-glucan. IMT also cuts off and cyclizes the isomaltosyl-α-1,3-isomaltosyl part of the second intermediate to finally produce CA. We also succeeded in the mass-production of the cyclic tetrasaccharide from starch by a joint reaction of both enzymes (1). An X-ray crystal structure analysis has shown that CA is shaped like a plate with a depression on one side and has a shallow cavity in the center of its ring (5). CA can complex with ethanol on both the concave and convex sides (9). The oxidation of CA to the dicarboxylic acid yields a selective chelator for Pb²⁺, Fe²⁺, and Fe³⁺ (8). These properties of CA open the possibility of further applications, such as a carrier in drug delivery systems or a base that removes toxic metal ions.

During the course of our screening for microorganisms that produce nonreducing oligosaccharides from starch, we have obtained a bacterial strain Arthrobacter globiformis M6 from soil (18). Structural analyses showed that the nonreducing oligosaccharide produced by the strain is a novel cyclic tetrasaccharide that differs from CA in the way of glycoside linkages. The novel cyclic tetrasaccharide, called cyclic maltosylmaltose [CMM; cyclo-{→6)-α-d-Glcp(1→4)-α-d-Glcp(1→6)-α-d-Glcp(1→4)-α-d-Glcp(1→}], has a unique structure in that four glucose residues are joined by alternate α-1,4 and α-1,6 linkages. CMM was purified as a crystal preparation from the reaction mixture containing a culture supernatant of A. globiformis M6 as an enzyme solution and soluble starch as a substrate. We report here the purification, characterization, and gene cloning of the CMM-forming enzyme from A. globiformis M6. We also describe the mechanism for the synthesis of CMM from starch and maltodextrins.

MATERIALS AND METHODS

Saccharides and enzymes.

Soluble starch was purchased from Katayama Chemical (Osaka, Japan). Maltooligosaccharides, amylose EX-I whose average degree of polymerization (DP) is 17, and CMM were prepared in our laboratory (18). 6²-O-α-Maltosyl-maltose (maltosylmaltose) was prepared from maltose by the condensation reaction of pullulanase (21). Glucoamylase (EC 3.2.1.3) from Rhizopus sp. was obtained from Seikagaku Corp. (Tokyo, Japan). Isomalto-dextranase (EC 3.2.1.94) from A. globiformis T6 was prepared according to the method of Okada et al. (20). β-Amylase (EC 3.2.1.2), pullulanase (EC 3.2.1.41), and isoamylase (EC 3.2.1.68) were of commercial grades.

Microorganism and culture conditions.

A. globiformis M6 isolated from the soil (18) was used in the present study. The bacterial strain was cultured at 27°C on a rotary shaker for 5 days in 500-ml Erlenmeyer flasks containing 100 ml of the medium composed of 1.5% Pinedex #4 (partially hydrolyzed starch; Matsutani Chemical Industry Co., Itami, Japan), 0.5% polypeptone, 0.1% yeast extract, 0.1% K₂HPO₄, 0.06% NaH₂PO₄ · 2H₂O, 0.05% MgSO₄ · 7H₂O, and 0.3% CaCO₃ (pH 6.8). After removal of the cells by centrifugation at 19,200 × g for 60 min, the supernatant of the culture broth was used for enzyme purification.

Enzyme purification.

Unless otherwise stated, all procedures of the purification were carried out at 4°C. The culture supernatant (10 liters) was brought to 60% saturation by adding solid ammonium sulfate and left overnight. The resulting precipitate was collected by centrifugation at 19,200 × g for 30 min and dissolved in 10 mM Tris-HCl buffer (pH 7.5). After dialysis overnight against the same buffer, the enzyme solution was put on a DEAE-Toyopearl 650S (Tosoh Corp., Tokyo, Japan) column (1.6 by 50 cm) equilibrated with the same buffer. Proteins adsorbed on the column were eluted with a linear gradient of 0 to 0.4 M NaCl in the same buffer at a flow rate of 2.0 ml/min. The active fractions were pooled and brought to a 1 M ammonium sulfate level by adding solid ammonium sulfate. The enzyme solution was then loaded onto a Phenyl-Toyopearl 650 M (Tosoh) column (1.6 by 5 cm) equilibrated with 20 mM acetate buffer (pH 6.0) containing 1 M ammonium sulfate. The adsorbed proteins were eluted with a linear gradient of 1 to 0 M ammonium sulfate in the same buffer at a flow rate of 1.0 ml/min. The active fractions were pooled and dialyzed against the same buffer. The resultant enzyme solution was used for further experiments as a purified preparation.

Enzyme assay.

The activity of the CMM-forming enzyme was assayed as follows. The enzyme solution (0.5 ml) was added to a substrate solution (0.5 ml) containing 2% soluble starch, 50 mM acetate buffer (pH 6.0), and 2 mM CaCl₂ and incubated at 40°C for 30 min. The reaction mixture was then boiled for 10 min to stop the reaction. Two and a half units of glucoamylase in 100 mM acetate buffer (pH 6.0) were next added to the reaction mixture and incubated at 50°C for 60 min to hydrolyze the remaining linear or branched oligosaccharides. After reboiling for 10 min, the solution was filtered by KC Prep Dura (0.45-μm pore size; Katayama Chemical) and desalted with a Micro Acilyzer G0 AC-110 (Asahi Chemical, Tokyo, Japan). Saccharides in the solution were analyzed by high-performance liquid chromatography (HPLC) as previously described (18). One unit of the CMM-forming enzyme activity was defined as the amount of enzyme that produces 1 μmol of CMM per min under the conditions described above.

Physical measurements.

The protein concentration was determined by the method of Lowry et al. using bovine serum albumin as the standard protein (14). The absorbance at 280 nm was used to monitor proteins in the column eluates. Native polyacrylamide gel electrophoresis (PAGE) was performed by the method of Davis (7). The molecular mass of the enzyme was estimated by sodium dodecyl sulfate (SDS)-PAGE performed on a 5 to 20% gradient gel according to the method of Laemmli (12). The isoelectric point was determined by gel isoelectric focusing (IEF) using a precast Ampholine PAGplate (Amersham Bioscience, Uppsala, Sweden) and IEF standards (Amersham Bioscience). Proteins were stained with Coomassie brilliant blue R-250. The N-terminal amino acid sequence of the purified enzyme was determined by using an ABI Procise 492HT protein sequencer (Applied Biosystems, California).

Isolation and identification of the intermediates from maltotetraose.

A reaction mixture (2 liter) containing 1% maltotetraose, 25 mM acetate buffer (pH 6.0), 1 mM CaCl₂, and 20 U of the purified CMM-forming enzyme was incubated at 40°C for 4 h. After the reaction was terminated by boiling for 10 min, 100 U of β-amylase was added to the reaction mixture, followed by incubation at 50°C for 16 h to convert the remaining linear maltooligosaccharides into maltose. The reaction mixture was then filtered, desalted, and concentrated to 50% (wt/wt) by evaporation. The resulting saccharide mixture was subjected to a preparative octadecylsilyl (ODS) column chromatography using a YMC-Pack ODS-A R-355-15 120A (YMC, Kyoto, Japan) column (5 by 50 cm). Elution was carried out with water at a flow rate of 30 ml/min at 25°C. The structures of the isolated saccharides were determined by mass spectrometry, methylation analysis, and enzymatic hydrolysis, as previously described (18).

Sugar analyses.

The HPLC analysis was performed by using a model LC-10A liquid chromatograph, a model RID-10A refractive index detector, and a C-R7A data processor (all from Shimadzu, Kyoto, Japan) under the following conditions: (i) column, two MCI GEL CK04SS columns connected in tandem (10 by 200 mm; Mitsubishi Chemicals, Tokyo, Japan); column temperature, 80°C; mobile phase, water; and flow rate, 0.4 ml/min; and (ii) column, YMC-Pack ODS AQ-303 (4.6 by 250 mm; YMC); column temperature, 40°C; mobile phase, water; and flow rate, 0.5 ml/min.

Gene cloning.

DNA preparation and nucleotide sequencing were performed according to the method described in our previous report (2). To obtain a partial fragment of the 6MT gene, a two-step PCR was carried out. A. globiformis M6 chromosomal DNA was used as the template for the first PCR. The sequence of the sense mix primer for the first PCR was 5′-GAYGTNATHTAYCARGT-3′ (Y = C or T; N = A, C, G, or T; H = A, C, or T; R = A or G) corresponding to the N-terminal amino acid sequence of the native 6MT, Asp-Val-Ile-Tyr-Gln-Val. The sequence of the antisense mix primer was 5′-TGRTARTTRTCRTCNGCCCA-3′ corresponding to the amino acid sequence of a lysyl endopeptidase-cleaved 6MT fragment, Gln-Tyr-Asn-Asp-Asp-Ala-Trp (MP7 in Fig. 7). After purified by using a MicroSpin S-400 HR column (Amersham Pharmacia Biotech), the DNA mixture amplified by the first step was used as the template for the second PCR. The sequence of the sense mix primer for the second PCR was 5′-TTYGARGAYGGNGAYCC-3′ corresponding to the N-terminal amino acid sequence of the native 6MT, Phe-Glu-Asp-Gly-Asp-Pro. The sequence of the antisense mix primer was 5′-GCNCKRTCRTGRTTRTC-3′ corresponding to the amino acid sequence, Ala-Arg-Asp-His-Asn-Asp (MP7). The temperature program for each cycle was 98°C for 20 s, 52°C for 2 min, and 68°C for 2 min. After 95°C for 1 min of heat treatment for DNA denaturing, 30 cycles were run. The amplified DNA fragment of ca. 0.9 kbp was purified by agarose gel electrophoresis, and then cloned into a pCR-Script Amp SK+ vector (Stratagene, CA). DNA sequencing showed that the 861-bp fragment was a portion of the 6MT gene, because the amino acid sequences of four internal peptides were found in the amino acid sequence deduced from the DNA sequence of this PCR product (data not shown). This fragment was then used as a probe for colony hybridization. The genomic DNA libraries of A. globiformis M6 were constructed with pBluescript II SK(+) (Stratagene) as a cloning vector and Escherichia coli XL2-Blue MRF′ as a host strain (22) and were screened by colony hybridization with a DIG DNA labeling and detection kit (Roche Molecular Biochemicals, Mannheim, Germany).

FIG. 7.

Alignment of the amino acid sequences among 6MT from A. globiformis M6 and α-amylase family enzymes. The identical amino acid residues in each column are outlined in black boxes. The N-terminal and internal amino acid sequences of the mature 6MT are underlined. The four conserved regions (I, II, III, and IV) are lined above the amino acid sequences. Agl-6MT, 6-α-maltosyltransferase from A. globiformis M6 (the present study); Bci-CGT, cyclomaltodextrin glucanotransferase from Bacillus circulans strain 251 (P43379 in Swiss-plot); Tvu-Amy, α-amylase from Thermoactinomyces vulgaris 94-2A (Q60051).

Nucleotide sequence accession number.

The nucleotide sequence of 6MT from A. globiformis M6 has been deposited in the DDBJ/EMBL/GenBank databases under accession no. AB190187.

RESULTS

Purification of the CMM-forming enzyme.

The enzyme producing CMM from starch was purified from the culture supernatant of A. globiformis M6 by successive column chromatography on DEAE-Toyopearl 650S and Phenyl-Toyopearl 650 M, following the ammonium sulfate precipitation. The results of the purification are summarized in Table 1. Native PAGE of the purified enzyme showed a single protein band. The enzyme was purified 79-fold in a yield of 40%.

TABLE 1.

Purification of CMM-forming enzyme from A. globiformis M6

Step	Total protein (mg)	Total activity (U)	Sp act (U/mg)	Purification index (fold)	Yield (%)
Culture supernatant	1,900	240	0.13	1	100
Ammonium sulfate	300	200	0.66	5.2	83
DEAE-Toyopearl	19	140	7.3	58	58
Phenyl-Toyopearl	9.6	96	10.0	79	40

Physical and enzymatic properties.

The molecular mass of the enzyme was estimated to be 71.7 kDa by SDS-PAGE (Fig. 1). The pI of the enzyme was found to be 3.6 by gel IEF. The N-terminal sequence up to 30th residue was determined as follows: DPTTSPGPLAEGDVIYQVLVDRFEDGDPTN. The effects of pH and temperature on the activity and stability are shown in Fig. 2. The enzyme was the most active at pH 6.0 and was stable in a pH range of 5.0 to 9.0 when kept at 4°C for 24 h. The optimum temperature for the enzyme was 50°C. The enzyme was stable up to 30°C when heated at various temperatures for 60 min. The addition of 1 mM Ca²⁺ enhanced the thermal stability of the enzyme up to 45°C. At a concentration of 1 mM, Cu²⁺, Hg²⁺, Al³⁺, Fe³⁺, Pb²⁺, and EDTA inhibited the enzyme activity at a loss of 99, 98, 88, 68, 64 and 75%, respectively.

FIG. 1.

SDS-PAGE of the purified CMM-forming enzyme from A. globiformis M6. Lanes 1 and 3, molecular mass markers (from the top: myosin, 200 kDa; β-galactosidase, 116 kDa; phosphorylase b, 97.4 kDa; serum albumin, 66.2 kDa; ovalbumin, 45.0 kDa; carbonic anhydrase, 31.0 kDa; trypsin inhibitor, 21.5 kDa; lysozyme, 14.4 kDa; aprotinin, 6.5 kDa); lane 2, purified CMM-forming enzyme (2 μg).

FIG. 2.

Effects of pH and temperature on the activity and stability of CMM-forming enzyme. (A) Effects of pH. For the pH test, sodium acetate buffer (pH 4.0 to 6.0), sodium phosphate buffer (pH 6.0 to 7.5), and Tris-HCl buffer (pH 7.5 to 9.0) were used. The activity of the enzyme (0.08 U/ml) in 0.1 M buffer was measured under the standard assay conditions (•). To examine the pH stability, the enzyme (2.4 U/ml) was incubated at 4°C for 24 h at various pH values, and the residual activity was measured at pH 6.0 (○). (B) Effects of temperature. The activity of the enzyme (0.06 U/ml) was measured at various temperatures (•). To examine the thermal stability, the enzyme (0.08 U/ml) in the absence of Ca²⁺ (○) or in the presence of 1 mM Ca²⁺ (▵) was incubated at various temperatures for 60 min and immediately cooled. The remaining activity was then measured at 40°C.

Substrate specificity and action on maltooligosaccharides.

As shown in Table 2, CMM was produced from maltooligosaccharides with DP3 or greater, amylose, soluble starch, and glycogen. Maltotriose was a poor substrate compared to the other maltooligosaccharides. Amylose produced the highest yield (44.0%) of CMM. The enzyme did not act on cyclomaltodextrins, pullulan, and dextran. When soluble starch was used as a substrate, the addition of isoamylase as a debranching enzyme resulted in an increase of the CMM yield up to 43% (data not shown). This suggested that the reaction of the CMM-forming enzyme with starch would stop at the branching points in the amylopectin component.

TABLE 2.

Effect of substrate on CMM formation^a

Substrate	CMM yield (%)
Maltose	ND
Maltotriose	2.4
Maltotetraose	28.6
Maltopentaose	24.7
Maltohexaose	41.6
Maltoheptaose	36.6
Amylose	44.0
Soluble starch	31.4
Glycogen	29.5
Cyclodextrins	ND
Pullulan	ND
Dextran	ND

^aReaction mixtures (2 ml) containing 1% of each substrate, 25 mM acetate buffer (pH 6.0), 1 mM CaCl₂, and 0.02 U of enzyme were incubated at 40°C for 48 h. After glucoamylase treatment of the reaction mixture, the yield of CMM was measured by HPLC with a Shodex SUGAR KS-801 column. ND, not detected.

For a better understanding of the action pattern of the CMM-forming enzyme on the maltooligosaccharides, the reaction products from maltotetraose, maltopentaose, maltohexaose, and maltoheptaose were analyzed by HPLC with MCIgel CK04SS columns. As shown in Fig. 3, the elution pattern of products from maltotetraose was almost the same as that from maltohexaose. Saccharides with DP2, DP4 (contained CMM), DP6, DP8, and DP10 were significantly produced from these substrates. On the other hand, maltopentaose and maltoheptaose produced saccharides with DP3, DP4 (contained CMM), DP5, DP7, and DP9 as reaction products. It was noted that the saccharide with DP4 (CMM) was also synthesized from odd-numbered substrates, as well as from even-numbered substrates. These results suggested that the enzyme might catalyze a maltosyl transfer reaction.

FIG. 3.

HPLC profiles of the reaction of CMM-forming enzyme on maltooligosaccharides. Reaction mixtures (2 ml) containing 1% of each substrate, 25 mM acetate buffer (pH 6.0), 1 mM CaCl₂, and 0.02 U of enzyme were incubated at 40°C for 20 h. After boiling for 10 min to stop the reaction, the samples were analyzed by HPLC with MCIgel CK04SS columns. (A) Maltotetraose; (B) maltopentaose; (C) maltohexaose; (D) maltoheptaose.

To confirm this hypothesis, the reaction products from maltotetraose were analyzed by HPLC with an ODS AQ-303 column, and the changes with time of the products were monitored. The products from maltotetraose by the action of the enzyme were maltose, CMM, maltosylmaltose, and maltohexaose, together with unknown saccharides A and B (Fig. 4A). As shown in Fig. 5, the amounts of maltohexaose, saccharide A, and saccharide B increased during the initial stage of the reaction but decreased in the late stage. The amount of saccharide A after a 4-h reaction was about three- to fourfold as much as those of maltohexaose and saccharide B. Therefore, we postulated that saccharide A was the main intermediate for CMM. Treatment of the reaction mixture with pullulanase resulted in hydrolysis of maltosylmaltose, saccharide A, and saccharide B, with the concomitant production of maltose (Fig. 4B). Based on these results, it was strongly suggested that the CMM-forming enzyme mainly catalyzed the α-1,6-maltosyl transfer reaction and could be called 6-α-maltosyltransferase (6MT).

FIG. 4.

HPLC profiles of the reaction of CMM-forming enzyme on maltotetraose. The reaction mixture (2 ml) was the same as described in Fig. 3 using maltotetraose as a substrate. After incubation at 40°C for 4 h, the reaction was stopped by boiling for 10 min. Two units of pullulanase in 25 mM acetate buffer (pH 6.0) were then added to the reaction mixture and incubated at 40°C for 20 h. Samples before and after the pullulanase treatment were analyzed by HPLC using an ODS AQ-303 column. (A) Before the pullulanase treatment; (B) after the pullulanase treatment. G2, maltose; G4, maltotetraose; G6, maltohexaose; CMM, cyclic maltosylmaltose; MM, maltosylmaltose.

FIG. 5.

Time course of the reaction products from maltotetraose. Reaction mixture (10 ml) containing 1% maltotetraose, 25 mM acetate buffer (pH 6.0), 1 mM CaCl₂, and 0.1 U of enzyme was incubated at 40°C. Samples were collected at intervals, boiled for 10 min to stop the reaction, and analyzed by HPLC with an ODS AQ-303 column. Symbols: ▵, maltose; ○, CMM; □, maltosylmaltose; ▴, maltohexaose; •, saccharide A; ▪, saccharide B.

Identification of both saccharides A and B.

Saccharide A was obtained (99.3% in purity) in a yield of 2.0 g. The mass spectrum of saccharide A showed an [M+Na]⁺ ion peak with an m/z ratio of 1,013, indicating that the saccharide has a molecular mass of 990 and consists of six glucose residues. Methylation analysis yielded 1 mol of 2,3,4,6-tetra-O-methyl glucitol, 1 mol of 2,3,4-tri-O-methyl glucitol and 4 mol of 2,3,6-tri-O-methyl glucitol. Pullulanase and isomalto-dextranase hydrolyzed the saccharide into maltose and maltotetraose, and into isopanose (6-O-α-maltosyl-glucose) and maltotriose, respectively. Based on these results, saccharide A was con- cluded to be α-d-Glcp(1→4)-α-d-Glcp(1→6)-α-d-Glcp(1→4)-α-d-Glcp(1→4)-α-d-Glcp(1→4)-α-d-Glcp (6⁴-O-α-maltosyl-maltotetraose). As for saccharide B, which was obtained 98.0% in purity in a yield of 0.2 g, the molecular weight was determined to be 1,314. The hydrolyzed products by pullulanase and isomalto-dextranase were maltose and maltohexaose, and isopanose and maltopentaose, respectively. In addition, the retention time of saccharide B on HPLC was identical to that of the main intermediate produced from maltohexaose (data not shown). These results suggest that saccharide B should be the octasaccharide, 6⁶-O-α-maltosyl-maltohexaose.

The purified 6MT from A. globiformis M6 acted on 6⁴-O-α-maltosyl-maltotetraose to produce nearly equimolar amounts of CMM and maltose. The yield of CMM from 6⁴-O-α-maltosyl-maltotetraose reached ca. 40% of the total sugar. Thus, it was demonstrated that CMM was synthesized by 6MT from maltotetraose via 6⁴-O-α-maltosyl-maltotetraose as the main intermediate. CMM was also produced from 6⁶-O-α-maltosyl-maltohexaose as a substrate.

Gene cloning.

By colony hybridization using the 861-bp fragment of the 6MT gene (cmmA) as a probe, a 4.4-kbp fragment containing the complete cmmA gene was obtained and sequenced. The cmmA gene encoded a protein with 623 amino acid residues calculated to have a molecular mass of 68,460 Da. The structural gene extended from the ATG initiation codon to the TGA stop codon, with a potential Shine-Dalgarno (SD) sequence, 5′-AGGAGA-3′. Upstream of the coding region, the putative −35 and −10 promoter sequences with a distance of 17-bp between them were observed. No possible terminator sequence was found downstream from the stop codon. The deduced amino acid sequence contained the seven internal peptide fragments (MP1-7 in Fig. 7) of 6MT protein. The N-terminal sequence of the mature 6MT started from Asp-41 of the deduced amino acid sequence, indicating that the preceding 40 residues might be a signal sequence for secretion. The molecular mass of the gene product without the putative signal sequence was calculated to be 64,637 Da in agreement with that (71.7 kDa) of the purified 6MT estimated by SDS-PAGE.

Homology searches performed with BLASTP program revealed that 6MT showed similarities to glycoside hydrolase family 13 (GH 13) or α-amylase family enzymes (10); 38 and 31% identities to α-amylase (EC 3.2.1.1) from Thermoactinomyces vulgaris (11) and cyclomaltodextrin glucanotransferase (CGTase; EC 2.4.1.19) from Bacillus circulans (13), respectively. The four conserved regions that are common in the family enzymes were also found in 6MT (Fig. 7).

DISCUSSION

In this study, we purified and characterized a CMM-forming enzyme from the culture supernatant of A. globiformis M6 and determined that it is a novel maltosyltransferase (6MT) with a unique transfer specificity. Recently, it has been reported that two glycosyltransferases, 6GT and IMT, synergistically act on α-1,4-glucan to produce the alternating α-1,4- and α-1,6-cyclic tetrasaccharide, which is known as another cyclic tetrasaccharide, cycloalternan (CA) (19, 3, 17). CA was synthesized from α-1,4 glucan via the following three reactions: an α-1,6-glucosyl transfer reaction by 6GT and successive intermolecular and intramolecular α-1,3-isomaltosyl transfer reactions by IMT. On the other hand, the substrate specificity of the purified 6MT revealed that a single enzyme could produce the alternating α-1,4- and α-1,6-cyclic tetrasaccharide, called cyclic maltosylmaltose (CMM), from α-1,4-glucan with DP3 or more.

From the detailed analysis of the action of 6MT on maltotetraose, it was found that CMM was synthesized via the intermediate, 6⁴-O-α-maltosyl-maltotetraose. The mechanism for the synthesis of CMM from maltooligosaccharides was proposed as shown in Fig. 6. First, 6MT breaks down the α-1,4-glucosidic bond between the second and third residues from the nonreducing end of the maltooligosaccharide. The maltosyl part bound to the enzyme is then transferred to the 6-OH of the nonreducing end glucose of another molecule to produce 6-O-α-maltosyl-maltooligosaccharide. Second, 6MT cuts off the α-1,4-linkage between the fourth and fifth residues from the nonreducing end of the intermediate and cyclizes it through the intramolecular α-1,6-transglycosylation to finally produce CMM. Thus, 6MT was found to be a novel glycosyltransferase catalyzing both the intermolecular and the intramolecular α-1,6-maltosyl transfer reactions. At the same time, maltooligosaccharides, with a DP decreased by two, were formed by the 6MT reactions. If the maltooligosaccharides have a DP3 or more, they could be substrates for the enzyme again. The formation of maltohexaose from maltotetraose indicates that 6MT also has a weak α-1,4-maltosyl transferring activity (Fig. 4A).

FIG. 6.

Scheme for the formation of CMM from maltooligosaccharides by the action of 6MT. Symbols: ○, glucopyranosyl residue; •, glucose residue at reducing end; horizontal arrow, α-1,4-linkage; vertical arrow, α-1,6-linkage. n = 1, 2, 3…

A similar maltosyltransferase has been reported from the hyperthermophilic bacterium Thermotoga maritima (15). The Thermotoga enzyme is highly specialized on the α-1,4 transfer of maltosyl units from α-1,4-glucans to other α-1,4-glucans. During the course of the maltosyltransferase reaction, maltooligosaccharides with a given DP X are disproportionated into a series of products with DP X ± 2n (n = 0, 1, 2, 3, …), the chain length of the products being longer as well as shorter than the substrate by multiples of maltose units. However, the Thermotoga maltosyltransferase is different from the present maltosyltransferase from A. globiformis M6 in that it catalyzes neither the α-1,6-maltosyl transfer reaction nor the cyclization reaction.

Amino acid sequence analysis revealed that 6MT should be assigned to the α-amylase family (GH 13), unlike 6GT and IMT (2, 3, 17) that belong to GH 31. In terms of the strict transfer specificity, 6MT was similar to the Thermotoga maltosyltransferase that also belongs to GH 13. However, little sequence similarity was observed between the maltosyltransferases. From the viewpoints of sequence homology and catalyzing cyclization reaction, 6MT might resemble CGTase whose cyclization mechanism has been well studied (23, 24). Crystal and mutational analyses of 6MT will provide insight into the relationship between the structure and the function for its cyclization mechanism.