Trp-49 of the family 3 beta-glucosidase from Aspergillus niger is important for its transglucosidic activity: creation of novel beta-glucosidases with low transglucosidic efficiencies

Family 3 beta-glucosidases from Aspergillus niger with substitutions for Trp-49 result in the accumulation of very small amounts of transglucosidic adducts, compared to the large amounts that accumulate with wild type enzyme. On the other hand, the amounts of the hydrolytic products that form is decreased by only small amounts. Kinetic studies showed that the main reason for the decreased accumulation of transglucosidic intermediates is a large decrease in binding capacity for Glc at site +1 and an increase in binding ability at site-1. The hydrolytic catalytic constants (kcat(h)) of the substituted enzymes were 3 to 4-fold smaller than those of wild type enzymes, while the Km(h) values were less than 2-fold smaller. The catalytic constants of the transglucosidic reactions (kcat(t) values) were essentially unchanged, but the Km(t) values of the substituted enzymes were about 25-fold larger than those of wild type enzymes. These changes mean that the efficiencies of hydrolytic reactions (kcat(h)/Km(h)) of beta-glucosidases created through substitutions for Trp-49 are less than 2-fold smaller than those of wild type beta-glucosidase, but the efficiencies of the transglucosidic reactions (kcat(t)/Km(t)) of the substituted enzymes are 25 to 30-fold smaller. This results in a significantly decreased formation of transglucosidic intermediates. In addition, the high hydrolytic efficiencies of the substituted enzymes, cause even the very small amounts of transglucosidic intermediates that form to be rapidly hydrolyzed. The overall effect is a very small accumulation of intermediates.     

Transglucosidic reactions of the Aspergillus niger family 3 beta-glucosidase: qualitative and quantitative analyses and evidence that the transglucosidic rate is independent of pH

The hydrolytic and transglucosidic reactions of the Aspergillus niger Family 3 beta-glucosidase were characterized. Michaelis-Menten plots of the rates of aglycone formation were normal (hyperbolic) at low [substrate]. However, at high [substrate] the rates decreased at pH below approximately 5.5 but increased at pH above approximately 5.5. Each decrease or increase took the form of a second hyperbola adjoining the first. Thin layer chromatography, gas-liquid chromatography, and NMR analyses indicated that the substrates became transglucosidic acceptors when present at high concentrations. When pNPGlc and cellobiose reacted as acceptors, the C6 hydroxyl of the non-reducing substrate component reacted to form beta-D-glucopyranosyl-(1-6)-beta-D-glucopyranosyl-p-nitrophenol and beta-D-glucopyranosyl-(1-6)-beta-D-glucopyranosyl-(1-4)-D-glucopyranose, respectively. The acceptor action accounted for the second adjoining hyperbolas. Rate equations were derived for the production of the aglycone and the transglucosidic intermediate, and these equations described the data very well. Hydrolytic Vmax {Vmax(h)}, hydrolytic Km {Km(h)}, transglucosidic Vmax {Vmax(t)}, and transglucosidic Km {Km(t)} values were obtained by non-linear regression analysis using these equations. Vmax(h) pH profiles were bell shaped with optima between pH 4 and 4.5 but the Vmax(t) values did not change substantially between pH 3 and 7. These differences in the pH profiles explain the decreasing and increasing adjoining hyperbolas since Vmax(t) is lower than Vmax(h) at pH less than approximately 5.5 but higher than Vmax(h) at pH greater than approximately 5.5. The reason for these pH effects is that the value of the hydrolytic rate constant (k3) decreases while the value of the transglucosidic rate constant (k4) does not change between pH 3 and 7. The study also showed that gentiobiose forms by an intermolecular reaction of the C6 hydroxyl of Glc rather than an intramolecular reaction and that an equatorial orientation of the C2 hydroxyl, the presence of a C6 primary hydroxyl and beta-linkages with oligosaccharide acceptors are important for acceptor reactivity.     

Allosteric regulation provides a molecular mechanism for preferential utilization of the fully assembled dolichol-linked oligosaccharide by the yeast oligosaccharyltransferase

The oligosaccharyltransferase (OST) preferentially utilizes the fully assembled dolichol-linked oligosaccharide Glc(3)Man(9)GlcNAc(2)-PP-Dol as the donor for N-linked glycosylation of asparagine residues in N-X-T/S consensus sites in newly synthesized proteins. A wide variety of assembly intermediates (Glc(0-2)Man(0-9)GlcNAc(2)-PP-Dol) can serve as the donor substrate for N-linked glycosylation of peptide acceptor substrates in vitro or of nascent glycoproteins in mutant cells that are defective in donor substrate assembly. A kinetic mechanism that can account for the selection of the fully assembled donor substrate from a complex mixture of dolichol-linked oligosaccharides (OS-PP-Dol) has not been elucidated. Here, the steady-state kinetic properties of the OST were reinvestigated using a proteoliposome assay system consisting of the purified yeast enzyme, near-homogeneous preparations of a dolichol-linked oligosaccharide (Glc(3)Man(9)GlcNAc(2)-PP-Dol or Man(9)GlcNAc(2)-PP-Dol) and an (125)I-labeled tripeptide as the acceptor substrate. The K(m) of the OST for the acceptor tripeptide was only slightly enhanced when Glc(3)Man(9)GlcNAc(2)-PP-Dol was the donor substrate relative to when Man(9)GlcNAc(2)-PP-Dol was the donor substrate. Evaluation of the kinetic data for both donor substrates showed deviations from typical Michaelis-Menten kinetics. Sigmoidal saturation curves, Lineweaver-Burk plots with upward curvature, and apparent Hill coefficients of about 1.4 suggested a substrate activation mechanism involving distinct regulatory (activator) and catalytic binding sites for OS-PP-Dol. Results of competition experiments using either oligosaccharide donor as an alternative substrate were also consistent with this hypothesis. We propose that binding of either donor substrate to the activator site substantially enhances Glc(3)Man(9)GlcNAc(2)-PP-Dol occupancy of the enzyme catalytic site via allosteric activation.     

Acceptor reactions of a novel transfructosylating enzyme from Bacillus sp.

Many different oligosaccharides were produced by transferring the fructose residue of sucrose to maltose, cellobiose, lactose and sucrose (self-transfer), where their yields of fructosylated acceptor products accounted for 26–30% (w/w). The maximum conversion yield (30%) was obtained in fructosyl cellobioside formation with 500 g sucrose l^–1 (substrate) and 200 g cellobiose l^–1 (acceptor). These four acceptors gave various products having DP (degree of polymerization) 2–7 by successive transfer reactions.     

Active-site mapping of a Populus xyloglucan endo-transglycosylase with a library of xylogluco-oligosaccharides

Restructuring the network of xyloglucan (XG) and cellulose during plant cell wall morphogenesis involves the action of xyloglucan endo-transglycosylases (XETs). They cleave the XG chains and transfer the enzyme-bound XG fragment to another XG molecule, thus allowing transient loosening of the cell wall and also incorporation of nascent XG during expansion. The substrate specificity of a XET from Populus (PttXET16-34) has been analyzed by mapping the enzyme binding site with a library of xylogluco-oligosaccharides as donor substrates using a labeled heptasaccharide as acceptor. The extended binding cleft of the enzyme is composed of four negative and three positive subsites (with the catalytic residues between subsites -1 and +1). Donor binding is dominated by the higher affinity of the XXXG moiety (G=Glcbeta(1-->4) and X=Xylalpha(1-->6)Glcbeta(1-->4)) of the substrate for positive subsites, whereas negative subsites have a more relaxed specificity, able to bind (and transfer to the acceptor) a cello-oligosaccharyl moiety of hybrid substrates such as GGGGXXXG. Subsite mapping with k(cat)/K(m) values for the donor substrates showed that a GG-unit on negative and -XXG on positive subsites are the minimal requirements for activity. Subsites -2 and -3 (for backbone Glc residues) and +2' (for Xyl substitution at Glc in subsite +2) have the largest contribution to transition state stabilization. GalGXXXGXXXG (Gal=Galbeta(1-->4)) is the best donor substrate with a "blocked" nonreducing end that prevents polymerization reactions and yields a single transglycosylation product. Its kinetics have unambiguously established that the enzyme operates by a ping-pong mechanism with competitive inhibition by the acceptor.     

Trp-999 of beta-galactosidase (Escherichia coli) is a key residue for binding,catalysis, and synthesis of allolactose,the natural lac operon inducer

Trp-999 is a key residue for the action of beta-galactosidases (Escherichia coli). Several site specific substitutions (Phe, Gly, Tyr, Leu) for Trp-999 were made. Each substitution caused greatly decreased affinities for substrates and inhibitors that bind in the "shallow" mode, while the affinities of inhibitors that bind in the "deep" mode were not decreased nearly as much. This shows that Trp-999 is important for binding in the shallow mode. The residue is also very important for binding glucose to galactosyl-beta-galactosidase (as a transgalactosidic acceptor). Substitution greatly diminished the affinity for glucose. Substitutions also changed the activation thermodynamics and, subsequently, the rates of the catalytic reactions. The enthalpies of activation of the glycolytic bond cleavage step (galactosylation, k(2)) became less favorable while the entropies of activation of that step became more favorable as a result of the substitutions. Differing magnitudes of these enthalpic and entropic effects with ONPG as compared to PNPG caused the k(2) values for ONPG to decrease but to increase for PNPG. The enthalpies of activation for the common hydrolytic step (degalactosylation, k(3)) increased while the entropies of activation for this step did not change much. As a result, k(3) became small and rate determining for each substituted enzyme. The substitutions caused the rate constant (k(4)) of the transgalactosidic acceptor reactions with glucose (for the formation of allolactose) to become much larger and of the same order of magnitude as the normally large rate constants for transgalactosidic acceptor reactions with small alcohols. This is probably because glucose can approach with less restriction in the absence of Trp-999. However, since glucose binds very poorly to the galactosyl-beta-galactosidases with substitutions for Trp-999, the proportion of lactose molecules converted to allolactose is small. Thus, Trp-999 is also important for ensuring that an appropriate proportion of lactose is converted to allolactose.     

Enzymatic characterization and substrate specificity of thermostable beta-glycosidase from hyperthermophilic archaea, Sulfolobus shibatae, expressed in E. coli

Enzymatic properties and substrate specificity of recombinant beta-glycosidases from a hyperthermophilic archaeon, Sulfolobus shibatae (rSSG), were analyzed. rSSG showed its optimum temperature and pH at 95 degrees C and pH 5.0, respectively. Thermal inactivation of rSSG showed that its half-life of enzymatic activity at 75 degrees C was 15 h whereas it drastically decreased to 3.9 min at 95 degrees C. The addition of 10 mM of MnCl2 enhanced the hydrolysis activity of rSSG up to 23% whereas most metal ions did not show any considerable effect. Dithiothreitol (DTT) and 2-mercaptoethanol exhibited significant influence on the increase of the hydrolysis activity of rSSG. rSSG apparently preferred laminaribiose (beta1-->3Glc), followed by sophorose (beta1-->2Glc), gentiobiose (beta1-->6Glc), and cellobiose (beta1--4Glc). Various intermolecular transfer products were formed by rSSG in the lactose reaction, indicating that rSSG prefers lactose as a good acceptor as well as a donor. The strong intermolecular transglycosylation activity of rSSG can be applied in making functional oligosaccharides.     

Synthetic reaction of Cellvibrio gilvus cellobiose phosphorylase.

The synthetic reactions of the cellobiose phosphorylase from Cellvibrio gilvus were investigated in detail. It was found that, besides D-glucose, some sugars having substitution or deletion of the hydroxyl group at C2 or C6 of the D-glucose molecule could serve as a glucosyl acceptor, though less effectively than D-glucose. The enzyme showed higher activity with beta-D-glucose than with the alpha-anomer as an acceptor. This result indicates that it recognizes the anomeric hydroxyl group not involved directly in the reaction. beta-D-Cellobiose was also phosphorolyzed faster than the alpha-anomer. Substrate inhibition was observed with D-glucose, 6-deoxy-D-glucose, or D-glucosamine as an acceptor, with D-glucose being most inhibiting. This inhibition was studied in detail and it was found that D-glucose competes with alpha-D-glucose-1-phosphate for its binding site. A model of competitive substrate inhibition was proposed, and the experimental data fit well to the theoretical values that were calculated in accordance with this model.     

Physical and Kinetic Properties of the Family 3 β-Glucosidase from Aspergillus niger Which Is Important for Cellulose Breakdown

A beta-glucosidase (BGS) purified from Aspergillus niger cellulase powder (obtained from Sigma, St. Louis, MO, USA) was characterized. Electrophoresis, size exclusion chromatography, and dynamic light scattering indicated that the enzyme is a dimer of approximately 200 kDa. Five of the seven N-glycosylated oligosaccharides attached to BGS were composed of D-mannoses attached to a beta(1-4)-N-acetyl-glucosamine-beta-(1-4)-fucose-alpha-(1-6)-N-acetylglucosamine core. The other two were similar, but the cores of these did not have the D-fucose. The enzyme is a retaining glycosidase, and it also has a distinct preference for the beta-configuration at the reducing end of cellobiose. BGS is thermostable up to 65 degrees C but is sensitive to freezing and thawing. The extinction coefficient of BGS was found to be 1.8 cm(-1) mg(-1). All substrates assayed resulted in Eadie-Hofstee plots that were curved at high substrate concentrations. TLC of the reaction products showed that the substrates themselves act as acceptors when present at high concentrations. The transglucosidic activity rate is different from the hydrolytic activity rate and this causes the curvature at high substrate concentrations. The enzyme produces gentiobiose when D-glucose is the acceptor. pH optima of the Vmax(h) with pNPGlc, oNPGlc, and cellobiose were between pH 4 and 4.5, and the Km values decreased at pH values between 3 and 5. Inhibition experiments indicated that the enzyme is specific for glucosyl substrates and suggested that D-gluconolactone is a transition state analog. Studies with cello-oligosaccharides and 3,4-dinitrophenyl-cellobiose showed that BGS is an exo-hydrolase having at least five glucose subsites and that it cleaves from the nonreducing end. The properties of a family 3 beta-glucosidase (BG3) sequenced by Dan et al. [Dan, S., Marton, I., Dekel, M., Bravdo, B-A., He, S., Withers, S. G., and Shoseyov, O. (2000) J. Biol. Chem. 275: 4973-4980] was also studied and was shown to have very similar properties to those of BGS. Sequence analysis of a portion of BGS verified that these are the same enzymes.     

Studies of the cellulolytic system of Trichoderma reesei QM 9414. Reaction specificity and thermodynamics of interactions of small substrates and ligands with the 1,4-beta-glucan cellobiohydrolase II

The 1,4-beta-glucan cellobiohydrolase II (CBH II) from Trichoderma reesei QM 9414 catalyses the hydrolysis of the 4-methylumbelliferyl beta-D-glycosides derived from cellotriose, cellotetraose and cellopentaose [MeUmb(Glc)n; n = 3 - 5]. The reaction has been followed by quantitative high-performance liquid chromatography. Specific activity for cellobiose removal at apparent substrate saturation were determined as (0.8 +/- 0.2) min-1 for MeUmb(Glc)3 and (9 +/- 2) min-1 for MeUmb(Glc)4. The enzyme showed a deviant specificity with MeUmb(Glc)5 as substrate. Two chromophoric products were formed simultaneously [MeUmb(Glc)3 and MeUmb(Glc)2] with turn-over numbers (17 +/- 4) min-1 and (21 +/- 6) min-1, respectively. Methylumbelliferyl beta-glucoside (MeUmbGlc) and the corresponding cellobioside [MeUmb(Glc)2] were used in equilibrium binding experiments. Both ligands yielded one binding site per molecule of Mr = 54000 upon forced flow dialysis (diafiltration). The association constants found were in fair agreement with those determined from MeUmb fluorescence quenching titrations. Quenching was total at all temperatures investigated for MeUmb(Glc)2, whereas for MeUmbGlc it increased from 80% to 100% between 2 degrees C and 20 degrees C. The association constants fitted linear van't Hoff plots in both cases. MeUmb(Glc)2 and MeUmbGlc were also used as indicator ligands to determine the association constants and thermodynamic parameters of several non-chromophoric ligands of CBH II. The binding of glucose increased the affinity for MeUmb(Glc)2 whereas it displaced MeUmbGlc from its complex. A putative binding site of the CBH II containing four subsites can be proposed. The thermodynamic data for methyl beta-D-glucopyranoside and cellobiose as ligands also point at an extended binding site.     

Purification and Properties of beta-Glucosidase from Aspergillus terreus

A beta-glucosidase (EC 3.2.1.21) from the fungus Aspergillus terreus was purified to homogeneity as indicated by disc acrylamide gel electrophoresis. Optimal activity was observed at pH 4.8 and 50 degrees C. The beta-glucosidase had K(m) values of 0.78 and 0.40 mM for p-nitrophenyl-beta-d-glucopyranoside and cellobiose, respectively. Glucose was a competitive inhibitor, with a K(i) of 3.5 mM when p-nitrophenyl-beta-d-glucopyranoside was used as the substrate. The specific activity of the enzyme was found to be 210 IU and 215 U per mg of protein on p-nitrophenyl-beta-d-glucopyranoside and cellobiose substrates, respectively. Cations, proteases, and enzyme inhibitors had little or no effect on the enzyme activity. The beta-glucosidase was found to be a glycoprotein containing 65% carbohydrate by weight. It had a Stokes radius of 5.9 nm and an approximate molecular weight of 275,000. The affinity and specific activity that the isolated beta-glucosidase exhibited for cellobiose compared favorably with the values obtained for beta-glucosidases from other organisms being studied for use in industrial cellulose saccharification.     

Functions of amino acid residues in the active site of Escherichia coli pyrroloquinoline quinone-containing quinoprotein glucose dehydrogenase

Several mutants of quinoprotein glucose dehydrogenase (GDH) in Escherichia coli, located around its cofactor pyrroloquinoline quinone (PQQ), were constructed by site-specific mutagenesis and characterized by enzymatic and kinetic analyses. Of these, critical mutants were further characterized after purification or by different amino acid substitutions. H262A mutant showed reduced affinities both for glucose and PQQ without significant effect on glucose oxidase activity, indicating that His-262 occurs very close to PQQ and glucose, but is not the electron acceptor from PQQH(2). W404A and W404F showed pronounced reductions of affinity for PQQ, and the latter rather than the former had equivalent glucose oxidase activity to the wild type, suggesting that Trp-404 may be a support for PQQ and important for the positioning of PQQ. D466N, D466E, and K493A showed very low glucose oxidase activities without influence on the affinity for PQQ. Judging from the enzyme activities of D466E and K493A, as well as their absorption spectra of PQQ during glucose oxidation, we conclude that Asp-466 initiates glucose oxidation reaction by abstraction of a proton from glucose and Lys-493 is involved in electron transfer from PQQH(2).     

Enzymatic characteristics of cellobiose phosphorylase from Ruminococcus albus NE1 and kinetic mechanism of unusual substrate inhibition in reverse phosphorolysis

Cellobiose phosphorylase (CBP) catalyzes the reversible phosphorolysis of cellobiose to produce α-D-glucopyranosyl phosphate (Glc1P) and D-glucose. It is an essential enzyme for the metabolism of cello-oligosaccharides in a ruminal bacterium, Ruminococcus albus. In this study, recombinant R. albus CBP (RaCBP) produced in Escherichia coli was characterized. It showed highest activity at pH 6.2 at 50 °C, and was stable in a pH range of 5.5-8.8 and at below 40 °C. It phosphorolyzed only cellobiose efficiently, and the reaction proceeded through a random-ordered bi bi mechanism, by which inorganic phosphate and cellobiose bind in random order and D-glucose is released before Glc1P. In the synthetic reaction, RaCBP showed highest activity to D-glucose, followed by 6-deoxy-D-glucose. D-Mannose, 2-deoxy-D-glucose, D-glucosamine, D-xylose, 1,5-anhydro-D-glucitol, and gentiobiose also served as acceptors, although the activities for them were much lower than for D-glucose. D-Glucose acted as a competitive-uncompetitive inhibitor of the reverse synthetic reaction, which bound not only the Glc1P site (competitive) but also the ternary enzyme-Glc1P-D-glucose complex (uncompetitive).     

Structure-function studies on bacteriorhodopsin. IX. Substitutions of tryptophan residues affect protein-retinal interactions in bacteriorhodopsin

Bacteriorhodopsin contains 8 tryptophan residues distributed across the membrane-embedded helices. To study their possible functions, we have replaced them one at a time by phenylalanine; in addition, Trp-137 and -138 have been replaced by cysteine. The mutants were prepared by cassette mutagenesis of the synthetic bacterio-opsin gene, expression and purification of the mutant apoproteins, renaturation, and chromophore regeneration. The replacement of Trp-10, Trp-12 (helix A), Trp-80 (helix C), and Trp-138 (helix E) by phenylalanine and of Trp-137 and Trp-138 by cysteine did not significantly alter the absorption spectra or affect their proton pumping. However, substitution of the remaining tryptophans by phenylalanine had the following effects. 1) Substitution of Trp-86 (helix C) and Trp-137 gave chromophores blue-shifted by 20 nm and resulted in reduced proton pumping to about 30%. 2) As also reported previously (Hackett, N. R., Stern, L. J., Chao, B. H., Kronis, K. A., and Khorana, H. G. (1987) J. Biol. Chem. 262, 9277-9284), substitution of Trp-182 and Trp-189 (helix F) caused large blue shifts (70 and 40 nm, respectively) in the chromophore and affected proton pumping. 3) The substitution of Trp-86 and Trp-182 by phenylalanine conferred acid instability on these mutants. The spectral shifts indicate that Trp-86, Trp-182, Trp-189, and possibly Trp-137 interact with retinal. It is proposed that these tryptophans, probably along with Tyr-57 (helix B) and Tyr-185 (helix F), form a retinal binding pocket. We discuss the role of tryptophan residues that are conserved in bacteriorhodopsin, halorhodopsin, and the related family of opsin proteins.     

alpha-Lactalbumin (LA) stimulates milk beta-1,4-galactosyltransferase I (beta 4Gal-T1) to transfer glucose from UDP-glucose to N-acetylglucosamine. Crystal structure of beta 4Gal-T1 x LA complex with UDP-Glc

beta-1,4-Galactosyltransferase 1 (Gal-T1) transfers galactose (Gal) from UDP-Gal to N-acetylglucosamine (GlcNAc), which constitutes its normal galactosyltransferase (Gal-T) activity. In the presence of alpha-lactalbumin (LA), it transfers Gal to Glc, which is its lactose synthase (LS) activity. It also transfers glucose (Glc) from UDP-Glc to GlcNAc, constituting the glucosyltransferase (Glc-T) activity, albeit at an efficiency of only 0.3-0.4% of Gal-T activity. In the present study, we show that LA increases this activity almost 30-fold. It also enhances the Glc-T activity toward various N-acyl substituted glucosamine acceptors. Steady state kinetic studies of Glc-T reaction show that the K(m) for the donor and acceptor substrates are high in the absence of LA. In the presence of LA, the K(m) for the acceptor substrate is reduced 30-fold, whereas for UDP-Glc it is reduced only 5-fold. In order to understand this property, we have determined the crystal structures of the Gal-T1.LA complex with UDP-Glc x Mn(2+) and with N-butanoyl-glucosamine (N-butanoyl-GlcN), a preferred sugar acceptor in the Glc-T activity. The crystal structures reveal that although the binding of UDP-Glc is quite similar to UDP-Gal, there are few significant differences observed in the hydrogen bonding interactions between UDP-Glc and Gal-T1. Based on the present kinetic and crystal structural studies, a possible explanation for the role of LA in the Glc-T activity has been proposed.     

Regulation of Flagellar Morphogenesis by Temperature: Involvement of the Bacterial Cell Surface in the Synthesis of Flagellin and the Flagellum

The induction of beta-glucosidases (EC 3.2.1.21) was studied in Neurospora crassa. Cellobiase was induced by cellobiose, but other inducers had little effect on this enzyme. Cellobiase activity was very low in all stages of the vegetative life cycle in the absence of di-beta-glucoside inducer. Aryl-beta-glucosidase was semiconstitutive at late stages of culture growth prior to conidiation. At early stages, aryl-beta-glucosidase was induced by cellobiose, laminaribiose, and gentiobiose, and weakly induced by galactose, amino sugars, and aryl-beta-glucosides. The induction properties of the beta-glucosidases are compared with those of the other disaccharidases of Neurospora. The induction of beta-glucosidases was inhibited by glucose, 2-deoxy-d-glucose, and sodium acetate. Sodium phosphate concentrations between 0.01 and 0.1 M stimulated induction of both enzymes, while concentrations above 0.1 M were inhibitory. The optimal condition for induction of both beta-glucosidases was pH 6.0. Cellobiase induction was relatively more inhibited than aryl-beta-glucosidase in the range of pH 6.0 to 8.0.     

Purification and characterization of two beta-glucosidases from a thermo-tolerant yeast Pichia etchellsii

The thermo-tolerant yeast Pichia etchellsii produced two cell-wall-bound inducible beta-glucosidases, BGLI (molecular mass 186 kDa) and BGLII (molecular mass 340 kDa), which were purified by a simple, three-step method, comprising ammonium sulfate precipitation, ion-exchange and hydroxyapatite chromatography. The two enzymes exhibited a similar pH and temperature optima, inhibitory effect by glucose and gluconolactone, and stability in the pH range of 3.0-9.0. Placed in family 3 of glycosylhydrolase families, BGLI was more active on salicin, p-nitrophenyl beta-D-glucopyranoside and alkyl beta-D-glucosides whereas BGLII was most active on cellobiose. k(cat) and K(M) values were determined for a number of substrates and, for BGLI, it was established that the deglycosylation step was equally effective on aryl- and alkyl-glucosides while the glycosylation step varied depending on the substrate used. This information was used to synthesize alkyl-glucosides (up to a chain length of C(10)) using dimethyl sulfoxide stabilized single-phase reaction microenvironment. About 12% molar yield of octyl-glucoside was calculated based on a simple spectrophotometric method developed for its estimation. Further, detailed comparison of properties of the enzymes indicated these to be different from the previously cloned beta-glucosidases from this yeast.     

Structural features of the ligand-binding domain of the serotonin 5HT3 receptor

The nicotinic acetylcholine receptor (AChR) and the serotonin type 3 receptor (5HT3R) are members of the ligand-gated ion channel gene family. Both receptors are inhibited by nanomolar concentrations of d-tubocurarine (curare) in a competitive fashion. Chemical labeling studies on the AChR have identified tryptophan residues on the gamma (gammaTrp-55) and delta (deltaTrp-57) subunits that interact with curare. Comparison of the sequences of these two subunits with the 5HT3R shows that a tryptophan residue is found in the homologous position in the 5HT3R (Trp-89), suggesting that this residue may be involved in curare-5HT3R interactions. Site-directed mutagenesis at position Trp-89 markedly reduces the affinity of the 5HT3R for the antagonists curare and granisetron but has little effect on the affinity for the agonist serotonin. To further examine the role of this region of the receptor in ligand-receptor interactions, alanine-scanning mutagenesis analysis of the region centered on Trp-89 (Thr-85 to Trp-94) was carried out, and the ligand binding properties of the mutant receptors were determined. Within this region of the receptor, curare affinity is reduced by substitution only at Trp-89, whereas serotonin affinity is reduced only by substitution at Arg-91. On the other hand, granisetron affinity is reduced by substitutions at Trp-89, Arg-91, and Tyr-93. This differential effect of substitutions on ligand affinity suggests that different ligands may have different points of interaction within the ligand-binding pocket. In addition, the every-other-residue periodicity of the effects on granisetron affinity strongly suggests that this region of the ligand-binding site of the 5HT3R (and by inference, other members of the ligand-gated ion channel family) is in a beta-strand conformation.     

Kinetics and mathematical model of hydrolysis and transglycosylation catalysed by cellobiase

The kinetics of hydrolysis and transglycosylation reactions catalysed by cellobiase (β-d-glucoside glucohydrolase, EC 3.2.1.21) from Aspergillus foetidus in the cellobiose-d-glucose reaction system have been studied. The formation of transglycosylation products was observed at cellobiose concentrations $\text{>10}{}^{\mathrm{?2}}\text{m}$, whereas at lower substrate concentrations the only reaction product was d-glucose. In the cellobiase-catalysed transglycosylation a (1→6)-β-linkage was formed after the transfer of a d-glucose residue to acceptor molecule. The basic transglycosylation products were isocellotriose and gentiobiose. A small amount of oligosaccharides with a higher degree of polymerization was also formed. The maximum content of transglycosylation products amounted to 25–30% of the total saccharide content in the system at the initial cellobiose concentration (0.1–0.3 m). The processes in the reaction system were inhibited by the substrate and product (d-glucose). A general scheme for cellobiose hydrolysis has been proposed and validated, allowing for the inhibition and transglycosylation effects. Based on this scheme, a mathematical model for cellobiose hydrolysis has been suggested to describe the kinetics of substrate consumption and product (d-glucose) accumulation, as well as the kinetics of formation and consumption of transglycosylation products throughout the course of enzymatic reaction with various initial amounts of cellobiose, starting from low concentrations up to 0.2–0.3 m (7–11% bv weight).