alpha-cyclodextrin and maltohexaose

Cyclodextrin glycosyltransferases (CGTase) (EC 2.4.1.19) are extracellular bacterial enzymes that generate cyclodextrins from starch. All known CGTases produce mixtures of alpha, beta, and gamma-cyclodextrins. A maltononaose inhibitor bound to the active site of the CGTase from Bacillus circulans strain 251 revealed sugar binding subsites, distant from the catalytic residues, which have been proposed to be involved in the cyclodextrin size specificity of these enzymes. To probe the importance of these distant substrate binding subsites for the alpha, beta, and gamma-cyclodextrin product ratios of the various CGTases, we have constructed three single and one double mutant, Y89G, Y89D, S146P and Y89D/S146P, using site-directed mutagenesis. The mutations affected the cyclization, coupling; disproportionation and hydrolyzing reactions of the enzyme. The double mutant Y89D/S146P showed a twofold increase in the production of alpha-cyclodextrin from starch. This mutant protein was crystallized and its X-ray structure, in a complex with a maltohexaose inhibitor, was determined at 2.4 A resolution. The bound maltohexaose molecule displayed a binding different from the maltononaose inhibitor, allowing rationalization of the observed change in product specificity. Hydrogen bonds (S146) and hydrophobic contacts (Y89) appear to contribute strongly to the size of cyclodextrin products formed and thus to CGTase product specificity. Changes in sugar binding subsites -3 and -7 thus result in mutant proteins with changed cyclodextrin production specificity.

Topics: alpha-Cyclodextrins; Amino Acid Substitution; Bacillus; Crystallography, X-Ray; Cyclodextrins; Escherichia coli; Glucosyltransferases; Hydrolysis; Models, Molecular; Mutagenesis, Site-Directed; Oligosaccharides; Protein Conformation; Protein Engineering; Substrate Specificity

To study the effect of mechanical grinding on crystallinity changes of naproxen (NAP) in mixtures with alpha-cyclodextrin (alphaCd), amorphous alphaCd, and maltohexaose (M6); and the possible formation of a pseudo-inclusion complex between NAP and M6 in aqueous solution.. NAP-additive physical mixtures at 0.30, 0.18, and 0.10 mass fraction of drug were tested, after increasing grinding times, by differential scanning calorimetry (DSC) and X-ray powder diffractometry (XRD). Interaction in aqueous solution was examined by phase-solubility and fluorescence analyses supported by molecular modelling.. In the mixtures with each additive the fusion enthalpy per unit mass of NAP decreased and the half width at half maximum of selected X-ray diffraction peaks of NAP increased with the progress of grinding time following the loss of crystallinity of the samples. The mechanical treatment apparently did not affect the chemical integrity of the drug. Particularly active in the equimolar mixture was the best amorphizing agent, M6. Solution studies and molecular modelling confirmed M6 may have the feature of a supermolecule for NAP, which forms a 1:1 pseudo-inclusion complex that was as stable as the true inclusion complex with alphaCd.. The intrinsically amorphous linear analog of aCd might be a potential amorphism-inducing agent and solubilizer for scarcely water soluble drugs.

Topics: alpha-Cyclodextrins; Anti-Inflammatory Agents, Non-Steroidal; Calorimetry, Differential Scanning; Crystallography, X-Ray; Cyclodextrins; Drug Interactions; Models, Molecular; Naproxen; Oligosaccharides; Solubility; Temperature; Thermogravimetry; Water

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 maltononaose inhibitor bound to the enzyme in an extended conformation. An identical experiment with the CGTase from T. thermosulfurigenes EM1 resulted in a 2.6-A 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. Glu258 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 Glu258 in the CGTase from T. thermosulfurigenes EM1 were changed. Phe284 was replaced by Lys and Asn327 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 Glu258.

Topics: alpha-Cyclodextrins; Archaea; Archaeal Proteins; Binding Sites; Crystallography, X-Ray; Cyclodextrins; Enzyme Inhibitors; Enzyme Stability; Glucosyltransferases; Hydrogen-Ion Concentration; Models, Molecular; Molecular Conformation; Mutagenesis, Site-Directed; Oligosaccharides; Protein Binding; Protein Engineering; Starch

Metastable decay rates of alpha-cyclodextrin and maltohexaose coordinated to proton and alkali metal ions were determined from ions produced by liquid secondary ion mass spectrometry in an external source Fourier transform mass spectrometry instrument. For both oligosaccharide compounds the decay rates of the protonated species are faster than any alkali metal coordinated species. Decay rates of the metal cationized species decrease in the order Li+, Na+, K+, and Cs+. The anion of alpha-cyclodextrin has the slowest measurable decomposition rate. The relationships between cation affinities and rates are explored.

Topics: alpha-Cyclodextrins; Carbohydrate Sequence; Cations; Cyclodextrins; Models, Chemical; Molecular Sequence Data; Oligosaccharides