The Cyclization Mechanism of Cyclodextrin Glycosyltransferase (CGTase) as Revealed by a γ-Cyclodextrin-CGTase Complex at Å. Cyclodextrin glycosyltransferase is an important enzyme of cyclodextrin synthesis . This article mainly discusses the recent progress of the application of. INDUSTRIAL MICROBIOLOGY. Cyclodextrin glycosyltransferase from Bacillus licheniformis: optimization of production and its properties. Cyclodextrina.

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To characterize this specificity we determined a 1. On this basis, a role of the individual residues during the cyclization reaction cycle is proposed. An especially interesting and unique family 13 enzyme is cyclodextrin glycosyltransferase CGTase. In addition, CGTase can perform hydrolysis very poorly and disproportionation reactions by using water or a free oligosaccharide as respective acceptors.

Its ability to perform cyclization has led to the application of CGTases in the industrial production of cyclodextrins 4. Schematic representation of the cyclization reaction catalyzed by CGTase. After the first, bond-cleavage step, a covalent intermediate is formed 7.

In the second step the linear chain assumes a cyclic conformation, which is denoted as the circularization step. The catalytic residues involved in bond cleavage are Asp and Glu 16 the latter is not shown. Domains A and B constitute the catalytic domains, whereas C and E are specialized in binding to raw starch granules 11 Domain D has no known function.

We denote this sugar transfer step circularization to distinguish it as a subprocess of cyclization. Kinetic data suggest that this is the rate-limiting step in the cyclization reaction. The mechanism of cyclization has been extensively studied to gain insight into the factors that determine the substrate specificity of the lgycosyltransferase and the reaction type specificity and to improve CGTase for industrial applications.

Of particular importance is the centrally located residue Tyr, as was demonstrated in at least 6 different CGTases Table I.

Cyclodextrin glycosyltransferase

To understand how these residues confer cyclization activity, structural information about the individual stages of the reaction cycle is essential. Crystallographic investigations have already revealed how a maltononaose substrate glycosyltransferaae a covalent maltotriosyl intermediate bind in the active site of CGTase 7 However, it is unknown how CGTase binds a cyclodextrin product.

Here we present the 1. Our data provide for the first time detailed glycosyltrasferase in the mechanisms that glycosgltransferase employed by CGTase to perform the cyclization reaction. In the past, various x-ray structures of CGTase-oligosaccharide complexes have been obtained after soaking crystals in a mother liquor where the maltose had been replaced by another sugar or inhibitor 1315 However, when cyclodextrins are used for soaking, they are digested to small linear oligosaccharides Thereafter it was frozen under a cold nitrogen stream K for data collection using the soaking solution as cryo-protectant.

As a starting model, we used the 2. Halfway through refinement we observed density for a MPD molecule near residues Pro and Arg, which was included in the model using stereochemical parameters from the hetero compounds data base G. Kleywegt, Uppsala University, Uppsala, Sweden.

One water molecule refined to a suspiciously low B-factor of 2. It is located far from the active site, at the interface of the A and D domains. Inspection of other data sets revealed a similar glycosyltrransferase in a 1.

In the final stage of the refinement the orientations of His, Gln, and Asn residues were determined by optimization of their hydrogen-bonding networks Data and refinement statistics are given in Table II. The density was contoured at 1 unit S. The labels indicate subsite nomenclature. Data collection statistics and quality of the B. No significant differences were observed. Second, we restricted functional comparisons to CGTase structures that were determined under identical experimental conditions, which warrants that all observed differences are ligand-induced.


The electron density clearly shows that the cyclic symmetry of the latter CD is spectacularly deformed Figs. As a result, the torsion angles of the scissile bond have acquired a characteristic twist The backbone conformations of the loops 87—93 —, —, and — in the maltononaose complex are indicated in gray.

Both structures were determined under identical experimental conditions. A superposition is shown in Fig. The cyclodextrin molecule rests on top of Tyr, which does not protrude into cyclodexrtin hydrophobic interior of cyclodextrin, in contrast to previous hypotheses The conformation of Arg in both structures differs from wild type, unliganded CGTase not shown.

Crearrangements around His and the loop glycosyltrznsferase The distances associated with the glyfosyltransferase are in Table Cyclodextrib.

Sugar flexibility can give important information on binding interactions. It can be judged from the experimentally determined atomic B-factors.

This indicates that the best hydrophobic stacking interactions 34 are made to Glycosyltransferwse Furthermore, the glucose O6 atom is pointing toward Tyr, while the sugar O3 atom forms a good 2. Now the sugar ring A face glycosyltraansferase oriented parallel and close to Phe, indicating good stacking glycosyltransfedase with this residue Fig.

Interactions with Phe are limited. The cyclodextrin glucose O6 atom is at a 3. Residue Asp does not contact the substrate directly but via a water molecule that binds to the glucose atoms O6 2. In addition, Arg binds to the O2 and O3 glucose atoms of the cyclodextrin Fig. This is the first time that Arg is implicated in direct sugar binding.

To make this binding contact, the side chain of Arg has assumed a new conformation compared with unliganded wild type CGTase The situation is different when maltononaose-liganded CGTase is compared with unliganded CGTase root mean square deviation, 0. The relative orientation of the A and E domains has changed slightly, and the sugar binding cleft has narrowed.

In addition, the loop —, which contains the important residue Tyr, shows a maximal shift of 0. All these loops are intimately connected through various interactions. Together with the main-chain plasticity, the cyclodeextrin structures reveal glycisyltransferase large conformational flexibility for Tyr Fig. However, in the maltononaose complex, Tyr has rotated and shifted 2.

The situation is glycosyltransferaee in the maltononaose substrate complex. In addition, the electron density and hydrogen bond network suggest that the imizadole ring of His is flipped.

This hydrogen bond to the substrate agrees better with the role of His as found from mutagenesis studies 3 It suggests that in the maltononaose complex, His is optimally positioned to contribute to catalysis 7.

To elucidate the atomic basis of the CGTase cyclization reaction, we have determined the first structure of a CGTase with a cyclodextrin product bound competently in its active site.

These latter CDs are degraded by coupling reactions in which the products can bind stably inside the crystal glyfosyltransferase Both structures were determined under experimentally identical conditions. However, cyclization is largely independent of cyclodextrin size, since many amino acids that are typical for CGTase are conserved irrespective of product specificity see below. Glyvosyltransferase, comparison of both structures can g,ycosyltransferase useful insight into the mechanism of cyclization.


To identify amino acids involved in cyclization, we studied their degree of evolutionary conservation in the active site Table I. These residues are probably not essential for cyclization but may influence the cyclodextrin product size cycllodextrin All other residues, the third class, are those typical for CGTase bold in Table I and, therefore, are potentially involved in cyclization.

The first step in cyclization is substrate binding Fig. However, they also suggest that longer sugar chains have a higher k cat for disproportionation and, thus, are processed more rapidly This could further limit unwanted hydrolysis and disproportionation reactions of small oligosaccharide chains in CGTase.

Our x-ray studies suggest glycosyltrnsferase His is involved in such an induced-fit mechanism, since it is specifically activated by binding of maltononaose. The subsequent reorientation of Tyr and the loop — has two effects. Both effects cycldoextrin a suitable environment for His to assume its optimal orientation for catalysis.

Cyclodextrin glycosyltransferase – Wikipedia

After bond cleavage, the next step in cyclization is circularization Fig. Site-directed mutagenesis experiments indicate that Tyr is very important for cyclization, especially its aromatic moiety Table I This suggests that the aromatic ring of Tyr has a role during circularization. From the x-ray structures it appears that in the maltononaose complex Tyr exposes, together with Phe, exposes a hydrophobic surface Fig.

When the interactions of Arg, Tyr, Asp, or Asp with carbohydrates are removed by site-directed mutagenesis, not only do the binding affinities K m change for linear substrates or cyclodextrins, but a 4—fold decrease of the k cat for cyclization and the reverse cyclodextrin degradation, or coupling, reaction results.

Nevertheless, mutagenesis of Phe indicates a critical role in cyclization Possibly, Phe is glycosyltransferzse positioned to guide the circularizing movement of the linear chain toward the acceptor sites. This is supported by mutants in Phe, which show a greater loss in affinity as judged by the K m value for cyclodextrins than for maltose Thus Phe stimulates the use of a circularized gylcosyltransferase chain as the acceptor instead of a free sugar.

Our analysis of the structures of B.

The Application of Cyclodextrin Glycosyltransferase in Biological Science | OMICS International

These separate functions can be exploited to engineer enzymes with glycosyltranfserase properties. Finally, the aromatic ring of Tyr could bind an intermediary stage in circularization, since it is important for cyclization activity, but does not interact strongly with either linear substrate or cyclodextrin.

The costs of publication of this article were defrayed in part by the payment of page charges. Section solely to indicate this cyclodextirn.

Dijkhuizen, manuscript in preparation. Dijkhuizen, submitted for publication.

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You’ll be in good company. Journal of Lipid Research. Figure 1 Schematic representation of the cyclization reaction catalyzed by CGTase.