Despite its relatively low pH and temperature optimum, the xylanase from Penicillium simplicissimum performs exceedingly well under conditions of paper bleaching. We have purified and characterized this enzyme, which belongs to family 10 of glycosyl hydrolases. Its gene was cloned, and the sequence of the protein was deduced from the nucleotide sequence. The xylanase was crystallized from ammonium sulfate at pH 8.4, and X-ray data were collected at cryo-temperature to a Crystallographic resolution of 1.75 A. The crystal structure was solved by molecular replacement using the catalytic domain of the Clostridium thermocellum xylanase as a search model, and refined to a residual of R = 20% (Rf,, = 23%) for data between 10 and 1.75 A. The xylanase folds in an ( ( Y / P )~ barrel (TIM-barrel), with additional helices and loops arranged at the "top" forming the active site cleft. In its overall shape, the R simplicissimum xylanase structure is similar to other family 10 xylanases, but its active site cleft is much shallower and wider. This probably accounts for the differences in catalysis and in the mode of action of this enzyme. Three glycerol molecules were observed to bind within the active site groove, one of which interacts directly with the catalytic glutamate residues. It appears that they occupy putative xylose binding subsites.

Bacterial cyclodextrin glycosyltransferases use starch to produce cyclic maltooligosaccharides (cyclodextrins) which are of interest in various applications. The cyclization reaction gives rise to a spectrum of ring sizes consisting of predominantly six to eight glucosyl units. Using the enzyme from Bacillus circulans strain no. 8, binding studies have been performed with several substrates and analogues. The observed binding modes differ in detail, but agree in general with data on homologous enzymes. Based on these binding studies, two mutations were designed that changed the production spectrum from the predominant product β‐cyclodextrin of the wild‐type enzyme towards γ‐cyclodextrin, which is of practical interest because it is rare and can encapsulate larger nonpolar compounds.

Crystals of the inactive mutant Glu257-->Ala of cyclodextrin glycosyltransferase were soaked with the cyclodextrin (CD) derivative S-(alpha-D-glucopyranosyl)-6-thio-beta-CD. The structural analysis showed its beta-CD moiety with no density indication for the exocyclic glucosyl unit. For steric reasons, however, the position of this unit is restricted to be at only two of the seven glucosyl groups of beta-CD. The analysis indicated that the enzyme can cyclize branched alpha-glucans. The ligated beta-CD moiety revealed how the enzyme binds its predominant cyclic product. The conformation of the ligated beta-CD was intermediate between the more symmetrical conformation in beta-CD dodecahydrate crystals and the conformation of a bound linear alpha-glucan chain. Its scissile bond was displaced by 2.8 A from the position in linear alpha-glucans. Accordingly, the complex represents the situation after the cyclization reaction but before diffusion into the solvent, where a more symmetrical conformation is assumed, or the equivalent state in the reverse reaction. Furthermore, a unifying nomenclature for oligosaccharide-binding subsites in proteins is proposed.

The synthesis and structural characterization of the hitherto unknown parent Co(bz) (bz=benzene) complex and several of its derivatives are described. Their synthesis starts either from a CoCO salt, or directly from Co (CO) and a Ag salt. Stability and solubility of these complexes was achieved by using the weakly coordinating anions (WCAs) [Al(OR ) ] and [F{Al(OR ) } ] {R =C(CF ) } and the solvent ortho-difluorobenzene (o-DFB). The magnetic properties of Co(bz) were measured and compared in the condensed and gas phases. The weakly bound Co(o-dfb) salts are of particular interest for the preparation of further Co salts, for example, the structurally characterized low-coordinate 12 valence electron Co(P Bu ) and Co(NHC) salts.