Search

The catalytic mechanism of cyclodextrin glycosyltransferase, a member of the a-amylase family, is reviewed. The focus is put on the bond cleavage mechanism, the nature of the transition state and of the covalent intermediate, and on the stereo-electronic and lateral protonation contributions to catalysis. The functions in catalysis of the absolutely conserved residues in this family are discussed. Finally, the fascinating capability of cyclodextrin glycosyltransferase to produce cyclodextrins from linear starch oligosaccharide chains is reviewed, together with protein engineering studies to modify the enzyme's product specificity. (C) 2002 Elsevier Science Inc. All rights reserved.

The catalytic mechanism of cyclodextrin glycosyltransferase, a member of the a-amylase family, is reviewed. The focus is put on the bond cleavage mechanism, the nature of the transition state and of the covalent intermediate, and on the stereo-electronic and lateral protonation contributions to catalysis. The functions in catalysis of the absolutely conserved residues in this family are discussed. Finally, the fascinating capability of cyclodextrin glycosyltransferase to produce cyclodextrins from linear starch oligosaccharide chains is reviewed, together with protein engineering studies to modify the enzyme's product specificity. (C) 2002 Elsevier Science Inc. All rights reserved.

Cyclodextrin-glycosyltransferase (CGTase) catalyzes the formation of alpha-, beta-, and gamma-cyclodextrins (cyclic alpha-(1,4)-linked oligosaccharides of 6, 7, or 8 glucose residues, respectively) from starch. Nine substrate binding subsites were observed in an x-ray structure of the CGTase from Bacillus circulans strain 251 complexed with a maltononaose substrate. Subsite -6 is conserved in CGTases, suggesting its importance for the reactions catalyzed by the enzyme. To investigate this in detail, we made six mutant CGTases (Y167F, G179L, G180L, N193G, N193L, and G179L/G180L). All subsite -6 mutants had decreased k(cat) values for beta-cyclodextrin formation, as well as for the disproportionation and coupling reactions, but not for hydrolysis. Especially G179L, G180L, and G179L/G180L affected the transglycosylation activities, most prominently for the coupling reactions. The results demonstrate that (i) subsite -6 is important for all three CGTase-catalyzed transglycosylation reactions, (ii) Gly-180 is conserved because of its importance for the circularization of the linear substrates, (iii) it is possible to independently change cyclization and coupling activities, and (iv) substrate interactions at subsite -6 activate the enzyme in catalysis via an induced-fit mechanism. This article provides for the first time definite biochemical evidence for such an induced-fit mechanism in the alpha-amylase family.

The enzyme cyclodextrin glycosyltransferase is closely related to alpha-amylases but has the unique ability to produce cyclodextrins (circular alpha(1-->4)-linked glucoses) from starch. To characterize this specificity we determined a 1.8-Angstrom structure of an E257Q/D229N mutant cyclodextrin glycosyltransferase in complex with its product gamma-cyclodextrin, which reveals for the first time how cyclodextrin is competently bound. Across subsites -2, -1, and fl, the cyclodextrin ring binds in a twisted mode similar to linear sugars, giving rise to deformation of its circular symmetry. At subsites -3 and +2, the cyclodextrin binds in a manner different from linear sugars. Sequence comparisons and site-directed mutagenesis experiments support the conclusion that subsites -3 and +2 confer the cyclization activity in addition to subsite -6 and Tyr-195, On this basis, a role of the individual residues during the cyclization reaction cycle is proposed.

The enzyme cyclodextrin glycosyltransferase is closely related to alpha-amylases but has the unique ability to produce cyclodextrins (circular alpha(1-->4)-linked glucoses) from starch. To characterize this specificity we determined a 1.8-Angstrom structure of an E257Q/D229N mutant cyclodextrin glycosyltransferase in complex with its product gamma-cyclodextrin, which reveals for the first time how cyclodextrin is competently bound. Across subsites -2, -1, and fl, the cyclodextrin ring binds in a twisted mode similar to linear sugars, giving rise to deformation of its circular symmetry. At subsites -3 and +2, the cyclodextrin binds in a manner different from linear sugars. Sequence comparisons and site-directed mutagenesis experiments support the conclusion that subsites -3 and +2 confer the cyclization activity in addition to subsite -6 and Tyr-195, On this basis, a role of the individual residues during the cyclization reaction cycle is proposed.

The starch-degrading enzymes alpha-amylase and cyclodextrin glycosyltransferase (CGTase) are functionally and structurally closely related, with CGTases containing two additional domains (called D and E) compared to the three domains of alpha-amylases (A, B and C). Amino acid residue 196 (Thermoanaerobacterium thermosulfurigenes EM1 CGTase numbering) occupies a dominant position in the active-site cleft. All alpha-amylases studied have a small residue at this position (Gly, Leu, Ser, Thr or Val), in contrast to CGTases which have a more bulky aromatic residue (Tyr or Phe) at this position, which is highly conserved. Characterization of the F196G mutant CGTase of T. thermosulfurigenes EM1 revealed that, for unknown reasons, apart from the F196G mutation, domain E as well as a part of domain D had become deleted [mutant F196G(Delta'DE)]. This, nevertheless, did not prevent the purification of a stable and active mutant CGTase protein (62 kDa). The mutant protein was more similar to an alpha-amylase protein in terms of the identity of residue 196, and in the domain structure containing, however some additional C-terminal structure. The mutant showed a strongly reduced temperature optimum. Due to a frameshift mutation in mutant F196G, a separate protein of 19 kDa with the DE domains was also produced. Mutant F196G(Delta'DE) displayed a strongly reduced raw-starch-binding capacity. similar to the situation in most alpha-amylases that lack a raw-starch-binding E domain. Compared to wild-type CGTase, cyclization, coupling and disproportionation activities had become drastically reduced in the mutant F196G(Delta'DE), but its saccharifying activity had doubled, reaching the highest level ever reported for a CGTase. Under industrial production process conditions, wild-type CGTase converted starch into 35% cyclodextrins and 11% linear oligosaccharides (glucose, maltose and maltotriose), whereas mutant F196G(Delta'DE) converted starch into 21% cyclodextrins and 18% into linear oligosaccharides. These biochemical characteristics indicate a clear shift from CGTase to alpha-amylase specificity.

The starch-degrading enzymes alpha-amylase and cyclodextrin glycosyltransferase (CGTase) are functionally and structurally closely related, with CGTases containing two additional domains (called D and E) compared to the three domains of alpha-amylases (A, B and C). Amino acid residue 196 (Thermoanaerobacterium thermosulfurigenes EM1 CGTase numbering) occupies a dominant position in the active-site cleft. All alpha-amylases studied have a small residue at this position (Gly, Leu, Ser, Thr or Val), in contrast to CGTases which have a more bulky aromatic residue (Tyr or Phe) at this position, which is highly conserved. Characterization of the F196G mutant CGTase of T. thermosulfurigenes EM1 revealed that, for unknown reasons, apart from the F196G mutation, domain E as well as a part of domain D had become deleted [mutant F196G(Delta'DE)]. This, nevertheless, did not prevent the purification of a stable and active mutant CGTase protein (62 kDa). The mutant protein was more similar to an alpha-amylase protein in terms of the identity of residue 196, and in the domain structure containing, however some additional C-terminal structure. The mutant showed a strongly reduced temperature optimum. Due to a frameshift mutation in mutant F196G, a separate protein of 19 kDa with the DE domains was also produced. Mutant F196G(Delta'DE) displayed a strongly reduced raw-starch-binding capacity. similar to the situation in most alpha-amylases that lack a raw-starch-binding E domain. Compared to wild-type CGTase, cyclization, coupling and disproportionation activities had become drastically reduced in the mutant F196G(Delta'DE), but its saccharifying activity had doubled, reaching the highest level ever reported for a CGTase. Under industrial production process conditions, wild-type CGTase converted starch into 35% cyclodextrins and 11% linear oligosaccharides (glucose, maltose and maltotriose), whereas mutant F196G(Delta'DE) converted starch into 21% cyclodextrins and 18% into linear oligosaccharides. These biochemical characteristics indicate a clear shift from CGTase to alpha-amylase specificity.

The starch-degrading enzymes alpha-amylase and cyclodextrin glycosyltransferase (CGTase) are functionally and structurally closely related, with CGTases containing two additional domains (called D and E) compared to the three domains of alpha-amylases (A, B and C). Amino acid residue 196 (Thermoanaerobacterium thermosulfurigenes EM1 CGTase numbering) occupies a dominant position in the active-site cleft. All alpha-amylases studied have a small residue at this position (Gly, Leu, Ser, Thr or Val), in contrast to CGTases which have a more bulky aromatic residue (Tyr or Phe) at this position, which is highly conserved. Characterization of the F196G mutant CGTase of T. thermosulfurigenes EM1 revealed that, for unknown reasons, apart from the F196G mutation, domain E as well as a part of domain D had become deleted [mutant F196G(delta'DE)]. This, nevertheless, did not prevent the purification of a stable and active mutant CGTase protein (62 kDa). The mutant protein was more similar to an alpha-amylase protein in terms of the identity of residue 196, and in the domain structure containing, however, some additional C-terminal structure. The mutant showed a strongly reduced temperature optimum. Due to a frameshift mutation in mutant F196G, a separate protein of 19 kDa with the DE domains was also produced. Mutant F196G(delta'DE) displayed a strongly reduced raw-starch-binding capacity, similar to the situation in most alpha-amylases that lack a raw-starch-binding E domain. Compared to wild-type CGTase, cyclization, coupling and disproportionation activities had become drastically reduced in the mutant F196G(delta'DE), but its saccharifying activity had doubled, reaching the highest level ever reported for a CGTase. Under industrial production process conditions, wild-type CGTase converted starch into 35% cyclodextrins and 11% linear oligosaccharides (glucose, maltose and maltotriose), whereas mutant F196G(delta'DE) converted starch into 21% cyclodextrins and 18% into linear oligosaccharides. These biochemical characteristics indicate a clear shift from CGTase to alpha-amylase specificity.

The crystal structure of endo-1,4-beta-xylanase I from Aspergillus niger has been solved by molecular replacement and was refined to 2.4 Angstrom resolution. The final R-factor for all data from 6 to 2.4 Angstrom is 17.9%. The A. niger xylanase has a characteristic fold which is unique for family G xylanases (root-mean-square deviation = 1.1 Angstrom to Trichoderma reesei xylanase I, which has 53% sequence identity). It consists of a single domain composed predominantly of beta-strands. Two beta-sheets are twisted around a deep, long cleft, which is lined with many aromatic amino acid residues and is large enough to accommodate at least four xylose residues. The two conserved glutamate residues, Glu79 and Glu170, which are likely to be involved in catalysis, reach into this cleft from opposite sides. A. niger xylanase I is of particular commercial interest because of its low pH optimum. A model is proposed which explains this low pH optimum compared to other members of xylanase family G. (C) 1996 Academic Press Limited

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.

The catalytic mechanism of cyclodextrin glycosyltransferase, a member of the a-amylase family, is reviewed. The focus is put on the bond cleavage mechanism, the nature of the transition state and of the covalent intermediate, and on the stereo-electronic and lateral protonation contributions to catalysis. The functions in catalysis of the absolutely conserved residues in this family are discussed. Finally, the fascinating capability of cyclodextrin glycosyltransferase to produce cyclodextrins from linear starch oligosaccharide chains is reviewed, together with protein engineering studies to modify the enzyme's product specificity. (C) 2002 Elsevier Science Inc. All rights reserved.

Many plants produce starch, a high molecular weight polymer of glucose, for storage as a carbon and energy source. These starch molecules are mostly found in seeds (e.g., wheats) or roots (e.g., potato) in the form of granules consisting of two types of glucan polymers: highly branched amylopectin and linear amylose. Many bacteria are able to use starch as a carbon and energy source for growth. For this purpose these micro-organisms convert starch molecules extracellularly into molecules suitable for uptake and further conversion by the cells. A whole range of starch-degrading enzymes with diierent reaction speci¢cities has evolved in these organisms yielding a wide variety of products. A number of these enzymes ¢nd application in the industrial processing of starch, either for modi¢cation of starch molecules or for the production of speci¢c degradation products. These enzymes are therefore studied extensively, leading to increasing knowledge of their reaction mechanisms and factors determining substrate and product speci¢city. A particularly interesting enzyme is cyclodextrin glycosyltransferase (CGTase), which has the unique capability of forming cyclodextrins from starch. This enzyme is a member of the K-amylase family (family 13) of glycosyl hydrolases. Current insights in the catalytic mechanism employed by these enzymes is discussed. Emphasis in this review is on structural and mechanistic features of CGTase determining cyclodextrin product speci¢city.

The enzyme cyclodextrin glycosyltransferase is closely related to alpha-amylases but has the unique ability to produce cyclodextrins (circular alpha(1-->4)-linked glucoses) from starch. To characterize this specificity we determined a 1.8-Angstrom structure of an E257Q/D229N mutant cyclodextrin glycosyltransferase in complex with its product gamma-cyclodextrin, which reveals for the first time how cyclodextrin is competently bound. Across subsites -2, -1, and fl, the cyclodextrin ring binds in a twisted mode similar to linear sugars, giving rise to deformation of its circular symmetry. At subsites -3 and +2, the cyclodextrin binds in a manner different from linear sugars. Sequence comparisons and site-directed mutagenesis experiments support the conclusion that subsites -3 and +2 confer the cyclization activity in addition to subsite -6 and Tyr-195, On this basis, a role of the individual residues during the cyclization reaction cycle is proposed.

The product specificity and pH optimum of the thermostable cyclodextrin glycosyltransferase (CGTase) from Thermoanaerobacterium thermosulfurigenes EM1 was engineered using a combination of x-ray crystallography and site-directed mutagenesis. Previously, a crystal soaking experiment with the Bacillus circulans strain 251 beta-CGTase had revealed a maltohexaose inhibitor bound to the enzyme in an extended conformation. An identical experiment with the CGTase from T. thermosulfurigenes EM1 resulted in a 2.6-Angstrom resolution x-ray structure of a complex with a maltohexaose inhibitor, bound in a different conformation, We hypothesize that the new maltohexaose conformation is related to the enhanced alpha-cyclodextrin production of the CGTase.The detailed structural information subsequently allowed engineering of the cyclodextrin product specificity of the CGTase from T. thermosulfurigenes EM1 by site directed mutagenesis, Mutation D371R was aimed at hindering the maltohexaose conformation and resulted in enhanced production of larger size cyclodextrins (beta- and gamma-CD). Mutation D197H was aimed at stabilization of the new maltohexaose conformation and resulted in increased production of alpha-CD.Glu(258) is involved in catalysis in CGTases as well as alpha-amylases, and is the proton donor in the first step of the cyclization reaction. Amino acids close to Glu(258) in the CGTase from T. thermosulfurigenes EM1 were changed. Phe(284) was replaced by Lys and Asn(327) by Asp. The mutants showed changes in both the high and low pH slopes of the optimum curve for cyclization and hydrolysis when compared with the wild-type enzyme, This suggests that the pH optimum curve of CGTase is determined only by residue Glu(258).

Cyclodextrin glycosyltransferases (CGTases) catalyze the degradation of starch into linear or cyclic oligosaccharides via a glycosyl transfer reaction occurring with retention of anomeric configuration. They are also shown to catalyze the coupling of maltooligosaccharyl fluorides. Reaction is thought to proceed via a double-displacement mechanism involving a covalent glycosyl-enzyme intermediate. This intermediate can be trapped by use of 4-deoxymaltotriosyl alpha-fluoride (4DG3 alpha F). This substrate contains a good leaving group, fluoride, thus facilitating formation of the intermediate, but cannot undergo the transglycosylation step since the nucleophilic hydroxyl group at the 4-position is missing. When 4DG3 alpha F was reacted with wild-type CGTase (Bacillus circulans 251), it was found to be a slow substrate (k(cat) = 2 s(-1)) compared with the parent glycosyl fluoride, maltotriosyl alpha-fluoride (k(cat) = 275 s(-1)). Unfortunately, a competing hydrolysis reaction reduces the lifetime of the intermediate precluding its trapping and identification. However, when 4DG3 alpha F was used in the presence of the presumed acid/base catalyst mutant Glu257Gln, the intermediate could be trapped and analyzed because the first step remained fast while the second step was further slowed (k(cat) = 0.6 s(-1)). Two glycosylated peptides were identified in a proteolytic digest of the inhibited enzyme by means of neutral loss tandem mass spectrometry. Edman sequencing of these labeled peptides allowed identification of Asp229 as the catalytic nucleophile and provided evidence for a covalent intermediate in CGTase. Asp229 is found to be conserved in all members of the family 13 glycosyl transferases.

The effective conversion of 4-thio-alpha-maltosyl fluoride 1 into cyclothiomaltins 2, 3 and 4, using cyclodextrin glycosyltransferase enzymes from Bacillus circulans, is described.