The cyclization mechanism of cyclodextrin glycosyltransferase (CGTase) as revealed by a gamma-cyclodextrin-CGTase complex at 1.8-A resolution.
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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-A 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 +1, 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. (+info)
Cyclodextrin encapsulation to prevent the loss of l-menthol and its retention during drying.
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The taste and flavor of spray-dried powdered products are the most important quality factors. In the present study, molecular encapsulation in cyclodextrin was applied to prevent the loss of a hydrophobic flavor compound (l-menthol) during the drying of a droplet. beta-Cyclodextrin appeared to be a better encapsulant for menthol than alpha- and gamma-cyclodextrin. The retention of menthol increased with increasing concentration of both cyclodextrin and maltodextrin. A simple mathematical model is proposed for estimating the flavor retention. The theoretical results by this model estimated well the final retention of menthol encapsulated in a blend of beta-cyclodextrin and maltodextrin. (+info)
Mechanism of porcine pancreatic alpha-amylase. Inhibition of amylose and maltopentaose hydrolysis by alpha-, beta- and gamma-cyclodextrins.
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The effects of alpha-, beta- and gamma-cyclodextrins on the amylose and maltopentaose hydrolysis catalysed by porcine pancreatic alpha-amylase (PPA) were investigated. The results of the statistical analysis performed on the kinetic data using the general initial velocity equation of a one-substrate reaction in the presence of one inhibitor indicate that the type of inhibition involved depends on the substrate used: the inhibition of amylose hydrolysis by alpha-, beta- and gamma-cyclodextrin is of the competitive type, while the inhibition of maltopentaose hydrolysis is of the mixed noncompetitive type. Consistently, the Lineweaver-Burk plots intersect on the vertical axis when amylose is used as the substrate, while in the case of maltopentaose, the intersection occurs at a point located in the second quadrant. The inhibition of the hydrolysis therefore involves only one abortive complex, PPA-cyclodextrin, when amylose is used as the substrate, while two abortive complexes, PPA-cyclodextrin and PPA-maltopentaose-cyclodextrin, are involved with maltopentaose. The mixed noncompetitive inhibition thus shows the existence of one accessory binding site. In any case, only one molecule of inhibitor binds to PPA. In line with these findings, the difference spectra of PPA produced by alpha-, beta- and gamma-cyclodextrin indicate that binding occurs at a tryptophan and a tyrosine residue. The corresponding dissociation constants and the inhibition constants obtained using the kinetic approach are in the same range (1.2-7 mM). The results obtained here on the inhibition of maltopentaose hydrolysis by cyclodextrin are similar to those previously obtained with acarbose as the inhibitor [Alkazaz, M., Desseaux, V., Marchis-Mouren, G., Prodanov, E. & Santimone, M. (1998) Eur. J. Biochem. 252, 100-107], but differ from those obtained with amylose as the substrate and acarbose as inhibitor [Alkazaz, M., Desseaux, V., Marchis-Mouren, G., Payan, F., Forest, E. & Santimone, M. (1996) Eur. J. Biochem. 241, 787-796]. It is concluded that the hydrolysis of both long and short chain substrates requires at least one secondary binding site, including a tryptophan residue. (+info)
Dietary beta- and gamma-cyclodextrins stimulation of hepatic metallothionein gene expression in rats.
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This study investigated whether hepatic metallothionein gene expression is affected by dietary cyclodextrins. Young male Wistar rats were fed a basal diet or cyclodextrin-supplemented (50 g of cyclodextrin per kg diet) diets for 7 d. Copper content in the liver did not show any significant changes among rats fed the basal, beta- and gamma-cyclodextrin diets. There were no differences in liver or serum zinc among groups. Copper content in serum was markedly decreased in rats fed the gamma-cyclodextrin-supplemented diet. Liver metallothionein mRNA levels were significantly elevated in both beta- and gamma-cyclodextrins-fed rats, but not in alpha-cyclodextrin-fed rats. Thus, the increase in hepatic metallothionein mRNA levels might be due to this mechanism except for the contents of copper and zinc in the liver. (+info)
Characteristics of pyrene phospholipid/gamma-cyclodextrin complex.
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Recently, it was demonstrated that gamma-cyclodextrins (gamma-CDs) greatly accelerates transfer of hydrophobic pyrene-labeled and other fluorescent phospholipid derivatives from vesicles to cells in culture (). To understand better the characteristics of this process, we studied the interaction of gamma-CD with pyrene-labeled phosphatidylcholines (PyrPCs) using a variety of physical methods. Either one or both of the acyl chains of PC was labeled with a pyrene moiety (monoPyrPCs and diPyrPCs, respectively), and the length of the labeled chain(s) varied from 4 to 14 carbons. Fluorescent binding assays showed that the association constant decreases strongly with increasing acyl chain length. PyrPC/gamma-CD stoichiometry was 1:2 for the shorter chain species, but changed to 1:3 when the acyl chain length exceeded 8 (diPyrPCs) or 10 (monoPyrPCs) carbons. The activation energy for the formation of diPyr(10)PC/gamma-CD complex was high, i.e., +92 kJ/mol, indicating that the phospholipid molecule has to fully emerge from the bilayer before complex formation can take place. The free energy, enthalpy, and entropy of transfer of monoPyrPC from bilayer to gamma-CD complex were close to zero. The absorption, Fourier transform infrared, and fluorescence spectral measurements and lifetime analysis indicated that the pyrene moiety lies inside the CD cavity and is conformationally restricted, particularly when the labeled chain is short. The acyl chains of a PyrPC molecule seem to share a CD cavity rather than occupy different ones. The present data provide strong evidence that the ability of gamma-CD to enhance intermembrane transfer of pyrene-labeled phospholipids is based on the formation of stoichiometric complexes in the aqueous phase. This information should help in designing CD derivatives that are more efficient lipid carriers then those available at present. (+info)
Pharmacological in vitro evaluation of new substance P-cyclodextrin derivatives designed to drug targeting towards NK1-receptor bearing cells.
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Some biological properties of new bifunctional conjugates designed for drug targeting were evaluated through in vitro experiments. Eight peptidylcyclodextrin compounds were used, which correspond to modified beta- or gamma-cyclodextrin (CD) grafted on neuropeptide substance P (SP) or a shorter derivative (SP(4-11)). Using anti-SP and anti-CD antibodies as molecular probes, we showed that the main structural features of the two moieties of these adducts were preserved. Binding experiments, using CHO cells expressing the human SP-specific NK1 receptor, demonstrated the functionality of all peptidylcyclodextrin derivatives, which exhibited IC50 values in a 10(-9)-10(-7) M range. All compounds were able to induce a pharmacological response, triggering phosphatidylinositol turnover with EC50 values in the same range as the natural ligand. Moreover, autoradiography analysis of rat spinal corn sections proved that [125I]SP binding was dose-dependently displaced by one selected compound (a gamma-CD-SP), showing a similar affinity of this adduct for the rat neurokinin 1 receptor. Our observations demonstrate that these peptidylcyclodextrins efficiently target NK1 receptor-expressing cells. (+info)
Fluorescent molecular sensory system based on bis pyrene-modified gamma-cyclodextrin dimer for steroids and endocrine disruptors.
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A gamma-cyclodextrin dimer modified with two pyrene moieties, 6-(2-pyrenebutylate-aminoethyl)pyrenebutylate-amino-6-deoxy-bis-gamma-cyclodextri n, has been synthesized in the presence of N,N'-dicyclohexycarbodiimide from gamma-cyciodextrin dimer linked with ethylenediamine at an upper rim. The sensing ability and binding property of the titled dimer were investigated for bile acids and endocrine disruptors. This cyclodextrin dimer showed both monomer and excimer fluorescence; the guest-induced emissions were observed as increases or decreases depending on the guest. The values deltaI(m)/I0(m) and deltaI(ex)/I0(ex), where I0(m) and I(m) are fluorescence intensities of monomer emission in the absence and presence of a guest and I0(ex) and I(ex) are those of excimer emission and deltaI(m) and deltaI(ex) were I(m) - I0(m) and I(ex) - I0(ex), respectively, were used as a parameter of sensitivity. This host exhibited highly sensitive molecular recognition ability for bile acids and endocrine disruptors, in which the sensing parameters obtained from monomer emission were plus or minus values, whereas the parameters obtained as excimer emission were minus ones. The behavior of the appended moieties of the host during a host-guest complexation was studied by induced circular dichroism (ICD) and fluorescence spectra. The ICD intensities of the titled dimer were decreased upon an addition of a guest. The guest-induced variations in the fluorescence and ICD intensity suggest that the appended moieties move by altering the spatial relationship in the hydrophobic space between two linked cyclodextrins. (+info)
Biochemical and genetic analyses of a novel gamma-cyclodextrin glucanotransferase from an alkalophilic Bacillus clarkii 7364.
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On screening for microorganisms in soil obtained in Japan that produce large amounts of gamma-cyclodextrin (gamma-CD), we identified a novel alkalophilic bacterium, Bacillus clarkii 7364. The cyclodextrin glucanotransferase (CGTase) secreted into the culture medium by this bacterium was purified by affinity chromatography on a gamma-CD-immobilized column, followed by chromatography on a gel filtration column. The enzyme converted 13.7% of pre-gelatinized potato starch (10% w/w per reaction mixture) into CDs, and the majority (79%) of the product CDs was of the gamma form. This property is quite unique among known CGTases and thus we named this enzyme gamma-CGTase. The N-terminal and internal amino acid sequences of gamma-CGTase were determined and used to design PCR primers for amplification of the nucleotide sequence that encodes the gamma-CGTase gene. The entire gene sequence amplified by PCR was determined and then cloned into E. coli. The recombinant enzyme synthesized by E. coli retained biochemical properties quite similar to those of the original one. Comparison of the deduced amino acid sequence of gamma-CGTase with those of other known CGTases that have different product specificities revealed the importance of subsites -3 and -7 for the preferential gamma-cyclization activity. (+info)