Crystal structure of alkalophilic asparagine 233-replaced cyclodextrin glucanotransferase complexed with an inhibitor, acarbose, at 2.0 A resolution

The product specificity of cyclodextrin glucanotransferase (CGTase) from alkalophilic Bacillus sp. #1011 is improved to near-uniformity by mutation of histidine-233 to asparagine. Asparagine 233-replaced CGTase (H233N-CGTase) no longer produces alpha-cyclodextrin, while the wild-type CGTase from the same bacterium produces a mixture of predominantly alpha-, beta-, and gamma-cyclodextrins, catalyzing the conversion of starch into cyclic or linear alpha-1,4-linked glucopyranosyl chains. In order to better understand the protein engineering of H233N-CGTase, the crystal structure of the mutant enzyme complexed with a maltotetraose analog, acarbose, was determined at 2.0 A resolution with a final crystallographic R value of 0.163 for all data. Taking a close look at the active site cleft in which the acarbose molecule is bound, the most probable reason for the improved specificity of H233N-CGTase is the removal of interactions needed to form a compact ring like a-cyclodextrin.     

Transglycosylation reactions of Bacillus stearothermophilus maltogenic amylase with acarbose and various acceptors

It was observed that Bacillus stearothermophilus maltogenic amylase cleaved the first glycosidic bond of acarbose to produce glucose and a pseudotrisaccharide (PTS) that was transferred to C-6 of the glucose to give an alpha-(1-->6) glycosidic linkage and the formation of isoacarbose. The addition of a number of different carbohydrates to the digest gave transfer products in which PTS was primarily attached alpha-(1-->6) to D-glucose, D-mannose, D-galactose, and methyl alpha-D-glucopyranoside. With D-fructopyranose and D-xylopyranose, PTS was linked alpha-(1-->5) and alpha-(1-->4), respectively. PTS was primarily transferred to C-6 of the nonreducing residue of maltose, cellobiose, lactose, and gentiobiose. Lesser amounts of alpha-(1-->3) and/or alpha-(1-->4) transfer products were also observed for these carbohydrate acceptors. The major transfer product to sucrose gave PTS linked alpha-(1-->4) to the glucose residue. alpha,alpha-Trehalose gave two major products with PTS linked alpha-(1-->6) and alpha-(1-->4). Maltitol gave two major products with PTS linked alpha-(1-->6) and alpha-(1-->4) to the glucopyranose residue. Raffinose gave two major products with PTS linked alpha-(1-->6) and alpha-(1-->4) to the D-galactopyranose residue. Maltotriose gave two major products with PTS linked alpha-(1-->6) and alpha-(1-->4) to the nonreducing end glucopyranose residue. Xylitol gave PTS linked alpha-(1-->5) as the major product and D-glucitol gave PTS linked alpha-(1-->6) as the only product. The structures of the transfer products were determined using thin-layer chromatography, high-performance ion chromatography, enzyme hydrolysis, methylation analysis and 13C NMR spectroscopy. The best acceptor was gentiobiose, followed closely by maltose and cellobiose, and the weakest acceptor was D-glucitol.     

Inhibition of beta-glycosidases by acarbose analogues containing cellobiose and lactose structures

Acarbose analogues, containing cellobiose and lactose structures, were prepared by reaction of the two disaccharides with acarbose and Bacillus stearothermophilus maltogenic amylase. The kinetics for the inhibition by the two analogues was studied for beta-glucosidase, beta-galactosidase, cyclomaltodextrin glucanosyltransferase (CGTase), and alpha-glucosidase. Both analogues were potent competitive inhibitors for beta-glucosidase, with K(I) values in the range of 0.04-2.44 microM, and the lactose analogues were good uncompetitive inhibitors for beta-galactosidase, with K(I) values in the range of 159-415 microM, while acarbose was not an inhibitor for either enzyme at 10 and 5 mM, respectively. Both analogues were also potent mixed inhibitors for CGTase, with K(I) values in the range of 0.1-9.3 microM. The lactose analogue was a 6.4-fold better competitive inhibitor for alpha-glucosidase than was acarbose.     

Study of the inhibition of four alpha amylases by acarbose and its 4IV-alpha-maltohexaosyl and 4IV-alpha-maltododecaosyl analogues

Acarbose analogues, 4IV-maltohexaosyl acarbose (G6-Aca) and 4IV-maltododecaosyl acarbose (G12-Aca), were prepared by the reaction of cyclomaltodextrin glucanyltransferase with cyclomaltohexaose and acarbose. The inhibition kinetics of acarbose and the two acarbose analogues were studied for four different alpha-amylases: Aspergillus oryzae, Bacillus amyloliquefaciens, human salivary, and porcine pancreatic alpha-amylases. The three inhibitors showed mixed, noncompetitive inhibition, for all four alpha-amylases. The acarbose inhibition constants, Ki, for the four alpha-amylases were 270, 13, 1.27, and 0.80 microM, respectively; the Ki values for G6-Aca were 33, 37, 14, and 7 nM, respectively; and the G12-Aca Ki constants were 59, 81, 18, and 11 nM, respectively. The G6-Aca and G12-Aca analogues are the most potent alpha-amylase inhibitors observed, with Ki values one to three orders of magnitude more potent than acarbose, which itself was one to three orders of magnitude more potent than other known alpha-amylase inhibitors.     

Single amino acid mutations interchange the reaction specificities of cyclodextrin glycosyltransferase and the acarbose-modifying enzyme acarviosyl transferase

Acarviosyl transferase (ATase) from Actinoplanes sp. SE50/110 is a bacterial enzyme that transfers the acarviosyl moiety of the diabetic drug acarbose to sugar acceptors. The enzyme exhibits 42% sequence identity with cyclodextrin glycosyltransferases (CGTase), and both enzymes are members of the alpha-amylase family, a large clan of enzymes acting on starch and related compounds. ATase is virtually inactive on starch, however. In contrast, ATase is the only known enzyme to efficiently use acarbose as substrate (2 micromol min(-1) mg(-1)); acarbose is a strong inhibitor of CGTase and of most other alpha-amylase family enzymes. This distinct reaction specificity makes ATase an interesting enzyme to investigate the variation in reaction specificity of alpha-amylase family enzymes. Here we show that a G140H mutation in ATase, introducing the typical His of the conserved sequence region I of the alpha-amylase family, changed ATase into an enzyme with 4-alpha-glucanotransferase activity (3.4 micromol min(-1) mg(-1)). Moreover, this mutation introduced cyclodextrin-forming activity into ATase, converting 2% of starch into cyclodextrins. The opposite experiment, removing this typical His side chain in CGTase (H140A), introduced acarviosyl transferase activity in CGTase (0.25 micromol min(-1) mg(-1)).     

Modes of action of acarbose hydrolysis and transglycosylation catalyzed by a thermostable maltogenic amylase, the gene for which was cloned from a Thermus strain

A maltogenic amylase gene was cloned in Escherichia coli from a gram-negative thermophilic bacterium, Thermus strain IM6501. The gene encoded an enzyme (ThMA) with a molecular mass of 68 kDa which was expressed by the expression vector p6xHis119. The optimal temperature of ThMA was 60 degrees C, which was higher than those of other maltogenic amylases reported so far. Thermal inactivation kinetic analysis of ThMA indicated that it was stabilized in the presence of 10 mM EDTA. ThMA harbored both hydrolysis and transglycosylation activities. It hydrolyzed beta-cyclodextrin and starch mainly to maltose and pullulan to panose. ThMA not only hydrolyzed acarbose, an amylase inhibitor, to glucose and pseudotrisaccharide (PTS) but also transferred PTS to 17 sugar acceptors, including glucose, fructose, maltose, cellobiose, etc. Structural analysis of acarbose transfer products by using methylation, thin-layer chromatography, high-performance ion chromatography, and nuclear magnetic resonance indicated that PTS was transferred primarily to the C-6 of the acceptors and at lower degrees to the C-3 and/or C-4. The transglycosylation of sugar to methyl-alpha-D-glucopyranoside by forming an alpha-(1,3)-glycosidic linkage was demonstrated for the first time by using acarbose and ThMA. Kinetic analysis of the acarbose transfer products showed that the C-4 transfer product formed most rapidly but readily hydrolyzed, while the C-6 transfer product was stable and accumulated in the reaction mixture as the main product.     

Synthesis of acarbose analogues by transglycosylation reactions of Leuconostoc mesenteroides B-512FMC and B-742CB dextransucrases

Two new acarbose analogues were synthesized by the reaction of acarbose with sucrose and dextransucrases from Leuconostoc mesenteroides B-512FMC and B-742CB. The major products for each reaction were subjected to yeast fermentation, and then separated and purified by Bio-Gel P2 gel permeation chromatography and descending paper chromatography. The structures of the products were determined by one- and two-dimensional 1H and 13C NMR spectroscopy and by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). B-512FMC-dextransucrase produced one major acarbose product, 2(I)-alpha-D-glucopyranosylacarbose and B-742CB-dextransucrase produced two major acarbose products, 2(I)-alpha-D-glucopyranosylacarbose and 3(IV)-alpha-D-glucopyranosylacarbose.     

Formation of covalent beta-linked carbohydrate-enzyme intermediates during the reactions catalyzed by alpha-amylases

Porcine pancreatic and Bacillus amyloliquefaciens alpha-amylases were examined for the formation of covalent carbohydrate intermediates during reaction. The enzymes were precipitated and denatured by adding 10 volumes of acetone. When these denatured enzymes were mixed with methyl alpha-6-[(3)H]-maltooligosaccharide glycosides and chromatographed on BioGel P-2, no carbohydrate was found in the protein void volume peak. When the enzymes were added to the methyl alpha-6-[(3)H]-maltooligosaccharide glycosides and allowed to react for 15s at 1 degrees C and then precipitated and denatured with 10 volumes of acetone, (3)H-labeled carbohydrates were found in the BioGel P-2 protein void volume peak, indicating the formation of enzyme-carbohydrate covalent intermediates. (1)H NMR analysis of the denatured enzyme from the reaction with methyl alpha-maltooligosaccharide glycosides confirmed that carbohydrate was attached to the denatured enzyme. (1)H NMR saturation-transfer analysis further showed that the carbohydrate was attached to the denatured enzyme by a beta-configuration. This configuration is what would be expected for an enzyme that catalyzes the hydrolysis of alpha-(1-->4) glycosidic linkages by a two-step, S(N)2 double-displacement reaction to give retention of the alpha-configuration of the substrates at the reducing-end of the products.     

Measurement of cyclomaltodextrin glucanotransferase activity by high-performance liquid chromatography using a fluorogenic substrate

A mixture of p-nitrophenyl O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D- glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D- glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D- glucopyranoside (FG5P) and p-nitrophenyl alpha-D-glucoside (GP) was incubated with cyclomaltodextrin glucanotransferase (CGTase) [EC 2.4.1.19]. Analysis of the digest by HPLC showed that the products were p-nitrophenyl O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D- glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D- glucopyranosyl-(1----4)-alpha-D-glucopyranoside (FG4P) and p-nitrophenyl alpha-D-maltoside (G2P), and no other product could be detected. Based on the reaction, a sensitive method to assay for CGTase was developed.     

A new synthesis of the oligosaccharide domain of acarbose

Synthesis of the oligosaccharide domain of acarbose was reinvestigated and was optimally performed using a maltosidic acceptor, already bearing a alpha-D-Glc-(1-->4)-D-Glc bond, and a new D-fucopyranosyl donor. The crucial glycosylation step was improved by varying three different parameters and notably by focusing on the C-4 protecting group of the fucosyl residue, solvent and promoter. The resulting trisaccharide was further transformed into an electrophilic species in order to open further derivatization perspectives for designing new acarbose analogues. Substitution reactions were efficiently carried out with azide and thiocyanate anions. Two other potentially interesting trisaccharidic compounds were also synthesized, i.e. the C-4III amine and the corresponding isothiocyanate.     

Thermoanaerobacterium thermosulfurigenes cyclodextrin glycosyltransferase.

Cyclodextrin glycosyltransferase (CGTase) uses an alpha-retaining double displacement mechanism to catalyze three distinct transglycosylation reactions. To investigate these reactions as catalyzed by the CGTase from Thermoanaerobacterium thermosulfurigenes the enzyme was overproduced (8 mg.L(-1) culture) using Bacillus subtilis as a host. Detailed analysis revealed that the three reactions proceed via different kinetic mechanisms. The cyclization reaction (cyclodextrin formation from starch) is a one-substrate reaction, whereas the other two transglycosylation reactions are two-substrate reactions, which obey substituted enzyme mechanism kinetics (disproportionation reaction) or ternary complex mechanism kinetics (coupling reaction). Analysis of the effects of acarbose and cyclodextrins on the disproportionation reaction revealed that cyclodextrins are competitive inhibitors, whereas acarbose is a mixed type of inhibitor. Our results show that one molecule of acarbose binds either in the active site of the free enzyme, or at a secondary site of the enzyme-substrate complex. The mixed inhibition thus indicates the existence of a secondary sugar binding site near the active site of T. thermosulfurigenes CGTase.     

Structural features of pectic polysaccharides from the skin of Opuntia ficus-indica prickly pear fruits

After removal of the mucilage with water at room temperature, pectic polysaccharides were solubilized from Opuntia ficus-indica fruit skin, by sequential extraction with water at 60 degrees C (WSP) and EDTA solution at 60 degrees C (CSP). Polysaccharides with neutral sugar content of 0.48 and 0.36 mol/mol galacturonic acid residue were obtained, respectively, in the WSP and CSP extracts. These pectic polysaccharides were de-esterified and fractionated by anion-exchange chromatography, yielding for each extract five fractions, which were thereafter purified by size-exclusion chromatography. Two of these purified fractions were characterized by sugar analysis combined with methylation and reduction-methylation analysis. The study was then supported by (1)H and (13)C NMR spectroscopy. The results showed that the water-soluble fraction WSP3 and the EDTA soluble fraction CSP3, consisted of a disaccharide repeating unit -->2)-alpha-l-Rhap-(1-->4)-alpha-d-GalpA-(1--> backbone, with side chains attached to O-4 of the rhamnosyl residues. The side chains contained highly branched alpha-(1-->5)-linked arabinan and short linear beta-(1-->4)-linked galactan.     

游动放线菌8-22中treY基因敲除对于降低阿卡波糖C组分的作用

【目的】在阿卡波糖发酵过程中,C组分的存在严重影响阿卡波糖产品的质量,研究拟通过基因改造降低阿卡波糖C组分。【方法】通过构建treY同框敲除质粒pUAmT-YUD,以接合转移方法将其转入阿卡波糖工业菌株8-22,经同源重组将treY基因内部编码182个氨基酸的序列敲除,从而得到treY基因失活的突变株Y810。【结果】发酵结果显示突变菌株中C组分较出发菌株下降了约10倍,而阿卡波糖本身的效价末受影响。【结论】敲除treY基因可大幅降低阿卡波糖C组分的含量。研究的实施将大大简化阿卡波糖的纯化步骤,提升产品品质,降低生产成本,从而提高工业化生产的市场竞争力。研究同时还对游动放线菌的接合转移条件进行了优化,大大提高了转化效率。     

Enzymatic synthesis of α-glucosyl-timosaponin BII catalyzed by the extremely thermophilic enzyme: Toruzyme 3.0L

Timosaponin BII (BII), a steroidal saponin showing potential anti-dementia activity, was converted into its glucosylation derivatives by Toruzyme 3.0L. Nine products with different degrees of glucosylation were purified and their structures were elucidated on the basis of ¹³C NMR, HR-ESI-MS, and FAB-MS spectra data. The active enzyme in Toruzyme 3.0L was purified to electrophoretic homogeneity by tracking BII-glycosylase activity and was identified as Cyclodextrin-glycosyltransferase (CGTase, EC 2.4.1.19) by ESI-Q-TOF MS/MS. In this work, we found that the active enzyme catalyzed the synthesis of alpha-(1→4)-linked glucosyl-BII when dextrin instead of an expensive activated sugar was used as the donor and showed a high thermal tolerance with the most favorable enzymatic activity at 100 °C. In addition, we also found that the α-amylases and CGTase, that is, GH13 family enzymes, all exhibited similar activities, which were able to catalyze glucosylation in steroidal saponins. But other kinds of amylases, such as γ-amylase (GH15 family), had no such activity under the same reaction conditions.     

Engineering cyclodextrin glycosyltransferase into a starch hydrolase with a high exo-specificity

Cyclodextrin glycosyltransferase (CGTase) enzymes from various bacteria catalyze the formation of cyclodextrins from starch. The Bacillus stearothermophilus maltogenic alpha-amylase (G2-amylase is structurally very similar to CGTases, but converts starch into maltose. Comparison of the three-dimensional structures revealed two large differences in the substrate binding clefts. (i) The loop forming acceptor subsite +3 had a different conformation, providing the G2-amylase with more space at acceptor subsite +3, and (ii) the G2-amylase contained a five-residue amino acid insertion that hampers substrate binding at the donor subsites -3/-4 (Biochemistry, 38 (1999) 8385). In an attempt to change CGTase into an enzyme with the reaction and product specificity of the G2-amylase, which is used in the bakery industry, these differences were introduced into Thermoanerobacterium thermosulfurigenes CGTase. The loop forming acceptor subsite +3 was exchanged, which strongly reduced the cyclization activity, however, the product specificity was hardly altered. The five-residue insertion at the donor subsites drastically decreased the cyclization activity of CGTase to the extent that hydrolysis had become the main activity of enzyme. Moreover, this mutant produces linear products of variable sizes with a preference for maltose and had a strongly increased exo-specificity. Thus, CGTase can be changed into a starch hydrolase with a high exo-specificity by hampering substrate binding at the remote donor substrate binding subsites.     

游动放线菌Actinoplanes sp.SE50/110中阿卡波糖脱氧氨基糖单元的生物合成

【目的】解析Actinoplanes sp.SE50/110(简称SE50/110)中阿卡波糖脱氧氨基糖单元的生物合成机制。【方法】经过BLASTp分析,推测了Acb A、Acb B和Acb V负责阿卡波糖脱氧氨基糖单元的生物合成。首先,本研究在SE50/110中分别构建了acb A、acb B和acb V的同框缺失和回补突变株。然后,利用大肠杆菌BL21(DE3)/p Gro7分别对Acb A、Acb B和Acb V成功实现了可溶性表达。最后,以D-葡萄糖-1-磷酸为起始底物,通过体外催化反应,研究脱氧氨基糖单元的生物合成过程和相关蛋白的酶学性质。【结果】在SE50/110中分别缺失acb A、acb B和acb V基因后,相应突变株均丧失了阿卡波糖的合成能力,将acb A、acb B和acb V基因分别回补后,各菌株又恢复了阿卡波糖的合成能力,证明了它们均为阿卡波糖生物合成的必需基因。在体外酶促反应中,D-葡萄糖-1-磷酸-胸腺嘧啶转移酶Acb A催化D-葡萄糖-1-磷酸和d TTP合成d TDP-D-葡萄糖,对D-葡萄糖-1-磷酸的Km值为(0.185±0.053)mmol/L,Vmax为(2.366±0.217)μmol/(min·mg);对d TTP的Km值为(4.964±1.089)mmol/L,Vmax为(60.310±5.419)μmol/(min·mg)。d TDP-D-葡萄糖-4,6-脱水酶Acb B催化d TDP-D-葡萄糖转化为d TDP-4-酮基-6-脱氧-D-葡萄糖,Km值和Vmax分别为(0.353±0.089)mmol/L和(306.401±28.740)μmol/(min·mg)。氨基转移酶Acb V催化d TDP-4-酮基-6-脱氧-D-葡萄糖生成d TDP-4-氨基-4,6-双脱氧-D-葡萄糖,Km值和Vmax分别为(1.411±0.293)mmol/L和(3.447±0.279)μmol/(min·mg)。【结论】本研究阐明了阿卡波糖脱氧氨基糖单元的生物合成过程,为全面解析阿卡波糖生物合成途径奠定了基础。同时,测定了相关酶的动力学参数,为代谢工程改造SE50/110,提高阿卡波糖产量提供了重要的理论依据。     

Characterization of Bacillus stearothermophilus cyclodextrin glucanotransferase in ascorbic acid 2-O-alpha-glucoside formation

In this study, we characterized cyclodextrin glucanotransferase (CGTase) from Bacillus stearothermophilus in L-ascorbic acid-2-O-alpha-D-glucoside (AA-2G) formation and compared its enzymological properties with those of rat intestinal and rice seed alpha-glucosidases which had the ability to form AA-2G. CGTase formed AA-2G efficiently using alpha-cyclodextrin (alpha-CD) as a substrate and ascorbic acid (AA) as an acceptor. Several AA-2-oligoglucosides were also formed in this reaction mixture, and they could be converted to AA-2G by the additional treatment of glucoamylase. The optimum temperature for AA-2G formation was 70 degrees C and its optimum pH was around 5.0. CGTase also utilized beta- and gamma-CDs, maltooligosaccharides, dextrin, amylose, glycogen and starch as substrates, but not any disaccharides except maltose. CGTase showed the same acceptor specificity as two alpha-glucosidases, whereas its hydrolyzing activity towards AA-2G was very low compared with those of alpha-glucosidases. Cleavage profiles of AA-2-oligoglucosides by CGTase present a possible mechanism for AA-2G formation that CGTase transfers a glucose-hexamer to an acceptor at the first step and then a glucose is stepwisely removed from the non-reducing end of the product through glucoamylase-like action of this enzyme. These results indicate that CGTase is able to synthesize AA-2G more efficiently than rat and rice alpha-glucosidases and utilization of this enzyme makes the mass production of AA-2G possible.     

The α-amylase inhibitor acarbose does not affect the parasitoid Venturia canescens when incorporated into the diet of its host Ephestia kuehniella

Amylase inhibitors (AIs) are suitable candidates for protecting plants and their products from attacks by herbivorous and granivorous insects. However, detailed studies of the suppressive effects of AIs on target and non‐target insects are necessary before their application in post‐harvest protection. To address this issue, laboratory bioassays were used to test the effect of the non‐proteinaceous inhibitor acarbose on a stored product pest, the flour moth Ephestia kuehniella (Zeller) (Lepidoptera: Pyralidae), and its parasitoid Venturia canescens (Gravenhorst) (Hymenoptera: Ichneumonidae). Two sublethal concentrations (0.001 and 0.0001%, wt/wt) of acarbose were incorporated into the diet of parasitized and unparasitized larvae of E. kuehniella. Development time and fresh body weight of the larvae, together with the size of the wasps, were compared for insects reared on acarbose‐treated and control diets. On the diet containing 0.001% acarbose, the developmental time was longer and relative weight gains of the E. kuehniella larvae were lower, but the weight of the larvae prior to pupation was similar to that of the control. The acarbose did not have a suppressive effect on the parasitoid V. canescens; in fact the wasps that emerged from the hosts reared on a diet containing 0.0001% acarbose were on average larger and heavier than the controls. These results demonstrate that it might be possible to enhance the control of stored product pests by using both biological control and AIs.     

Acceptor specificity of 4-alpha-glucanotransferases of mammalian glycogen debranching enzymes

Glycogen debranching enzyme (GDE) has two distinct active sites for its 4-alpha-glucanotransferase and amylo-alpha-1,6-glucosidase activities. The GDE 4-alpha-glucanotransferases of mammals show stringent donor specificity; only alpha-glucans with an alpha-1,6-linked maltotetraosyl or maltotriosyl branch function as donors of a maltotriosyl or maltosyl residue. In this study, we investigated the acceptor specificity of the 4-alpha-glucanotransferases using methyl alpha-maltooligosides, p-nitrophenyl alpha-maltooligosides, and pyridylaminated maltooligosaccharides of various sizes as the acceptor substrates, and phosphorylase limit dextrin as the donor substrate. High-performance liquid chromatography analysis of the transfer products indicated that maltotriosyl and maltosyl residues were specifically transferred from phosphorylase limit dextrin to acceptors with a maltopentaosyl residue comprising a nonreducing-end. These results suggest that the acceptor binding sites in the active sites of mammalian GDE 4-alpha-glucanotransferases are composed of tandem subsites that are geometrically complementary to five glucose residues.     

Crystal structure of Thermotoga maritima 4-alpha-glucanotransferase and its acarbose complex: implications for substrate specificity and catalysis

4-alpha-Glucanotransferase (GTase) is an essential enzyme in alpha-1,4-glucan metabolism in bacteria and plants. It catalyses the transfer of maltooligosaccharides from an 1,4-alpha-D-glucan molecule to the 4-hydroxyl group of an acceptor sugar molecule. The crystal structures of Thermotoga maritima GTase and its complex with the inhibitor acarbose have been determined at 2.6A and 2.5A resolution, respectively. The GTase structure consists of three domains, an N-terminal domain with the (beta/alpha)(8) barrel topology (domain A), a 65 residue domain, domain B, inserted between strand beta3 and helix alpha6 of the barrel, and a C-terminal domain, domain C, which forms an antiparallel beta-structure. Analysis of the complex of GTase with acarbose has revealed the locations of five sugar-binding subsites (-2 to +3) in the active-site cleft lying between domain B and the C-terminal end of the (beta/alpha)(8) barrel. The structure of GTase closely resembles the family 13 glycoside hydrolases and conservation of key catalytic residues previously identified for this family is consistent with a double-displacement catalytic mechanism for this enzyme. A distinguishing feature of GTase is a pair of tryptophan residues, W131 and W218, which, upon the carbohydrate inhibitor binding, form a remarkable aromatic "clamp" that captures the sugar rings at the acceptor-binding sites +1 and +2. Analysis of the structure of the complex shows that sugar residues occupying subsites from -2 to +2 engage in extensive interactions with the protein, whereas the +3 glucosyl residue makes relatively few contacts with the enzyme. Thus, the structure suggests that four subsites, from -2 to +2, play the dominant role in enzyme-substrate recognition, consistent with the observation that the smallest donor for T.maritima GTase is maltotetraose, the smallest chain transferred is a maltosyl unit and that the smallest residual fragment after transfer is maltose. A close similarity between the structures of GTase and oligo-1,6-glucosidase has allowed the structural features that determine differences in substrate specificity of these two enzymes to be analysed.