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M Schülein

Publications and source records attributed to M Schülein.

At least 37 records · Page 2Linked to original sources
PubMed1998

Structure of the Bacillus agaradherans family 5 endoglucanase at 1.6 A and its cellobiose complex at 2.0 A resolution.

The enzymatic degradation of cellulose, by cellulases, is not only industrially important in the food, paper, and textile industries but also a potentially useful method for the environmentally friendly recycling of municipal waste. An understanding of the structural and mechanistic requirements for the hydrolysis of the beta-1,4 glycosidic bonds of cellulose is an essential prerequisite for beneficial engineering of cellulases for these processes. Cellulases have been classified into 13 of the 62 glycoside hydrolase families [Henrissat, B., and Bairoch, A. (1996) Biochem J. 316, 695-696]. The structure of the catalytic core of the family 5 endoglucanase, Ce15A, from the alkalophilic Bacillus agaradherans has been solved by multiple isomorphous replacement at 1.6 A resolution. Ce15A has the (alpha/beta)8 barrel structure and signature structural features typical of the grouping of glycoside hydrolase families known as clan GH-A, with the catalytic acid/base Glu 139 and nucleophile Glu 228 on barrel strands beta 4 and beta 7 as expected. In addition to the native enzyme, the 2.0 A resolution structure of the cellobiose-bound form of the enzyme has also been determined. Cellobiose binds preferentially in the -2 and -3 subsites of the enzyme. Kinetic studies on the isolated catalytic core domain of Ce15A, using a series of reduced cellodextrins as substrates, suggest approximately five to six binding sites, consistent with the shape and size of the cleft observed by crystallography.

Bacillus↗
PubMed1998

Characterization of a cellobiose dehydrogenase from Humicola insolens.

The major cellobiose dehydrogenase (oxidase) (CBDH) secreted by the soft-rot thermophilic fungus Humicola insolens during growth on cellulose has been isolated and purified. It was shown to be a haemoflavoprotein with a molecular weight of 92 kDa and a pI of 4.0, capable of oxidizing the anomeric carbon of cellobiose, soluble cellooligosaccharides, lactose, xylobiose and maltose. Possible electron acceptors are 2,6-dichlorophenol-indophenol (DCPIP), Methylene Blue, 3,5-di-t-butyl-1,2-benzoquinone, potassium ferricyanide, cytochrome c and molecular oxygen. The oxidation of the prosthetic groups by oxygen was monitored at 449 nm for the flavin group and at 562 nm for the haem group. The curves were very similar to those of the cellobiose dehydrogenase from Phanerochaete chrysosporium, suggesting a similar mechanism. The pH-optima for the oxidation varied remarkably depending on the electron acceptor. For the organic electron acceptors, the pH-optima ranged from pH 4 for Methylene Blue to pH 7 for DCPIP and the benzoquinone. In the case of the FeIII-containing electron acceptors, the enzyme displayed alkaline pH-optima, in contrast to the properties of cellobiose dehydrogenases from Phanerochaete chrysosporium and Myceliophthora (Sporotrichum) thermophila. The enzyme has optimal activity at 65 degrees C.

Amino Acids↗
PubMed1997

Enzymatic properties of cellulases from Humicola insolens.

We present the analysis of the activities towards soluble and insoluble substrates of seven cellulases cloned from the saprophytic fungus Humicola insolens. The activity on the soluble polymer substrate carboxymethylcellulose (CMC) was used to determine the pH activity profiles of the five endoglucanases (EG), whereas cellotriose and reduced cellohexaose were used to determine the pH activity profiles of cellobiohydrolase I (CBH) and CBH II. All the EGs show optimal activity between pH 7 and 8.5, while CBH I and CBH II peak around pH 5.5 and 9, respectively. The catalytic activities of five of these cellulases were investigated under neutral and alkaline conditions using reduced cellohexaose as a substrate in a cellobiose oxidase coupled assay. EG I and CBH I both belong to family (7) according to a recent classification of glycosyl hydrolases. They both have activity against cellotriose. Therefore, they were studied using a coupled assay involving glucose oxidase. The activity on insoluble substrate (phosphoric-acid swollen cellulose) was assessed by the formation of reducing groups. The presence of a cellulose binding domain (CBD) lowers the apparent KM. This can be explained by the dispersing action of CBD. However, the CBD also reduces the apparent k(cat) probably by slowing down the mobility. EG I, EG II and EG III show similar activity towards CMC and amorphous cellulose, while EG V, EG VI, CBH I and CBH II have the highest catalytic rate on amorphous cellulose. In summary, Humicola insolens possesses a battery of cellulose-degrading enzymes which cooperate in the efficient hydrolysis of cellulose.

Cellulase↗
PubMed1997

Oligosaccharide specificity of a family 7 endoglucanase: insertion of potential sugar-binding subsites.

Family 7 of the glycosyl hydrolases contains both endoglucanases and cellobiohydrolases. In addition to their different catalytic activities on crystalline substrates, the cellobiohydrolases differ from the endoglucanases in their activity on longer soluble substrates, indicative of a greater number of subsites on the enzyme. A double mutant (S37W, P39W) of the Humicola insolens endoglucanase I (EG I) has been constructed in order to mimic aspects of the subsite structure of the corresponding family 7 cellobiohydrolase, cellobiohydrolase-I (CBH I). The 3-D crystal structure of the double mutant has been solved and refined to a crystallographic R-factor of 0.17 at a resolution of 2.2 A (1 A = 0.1 nm). The two mutant tryptophans are clearly visible in the electron density and are in the same orientation as those found in the substrate binding groove of CBH I. In addition to the substitutions, the C-terminal amino acids (399QELQ), disordered in the native enzyme structure, are clearly visible and there are a small number of minor loop movements associated with differences in crystal packing. Kinetic determinations show that the S37W, P39W mutant EG I has almost identical activity, compared to native EG I, on small soluble cellodextrins. On phosphoric acid swollen cellulose there is a small (30%), but significant, decrease in the apparent KM indicating that the double mutant may indeed exhibit stronger binding to longer polymeric substrates.

Binding Sites↗
PubMed1997

Identification of the catalytic nucleophile of endoglucanase I from Fusarium oxysporum by mass spectrometry.

The endoglucanase EG I from Fusarium oxysporum catalyzes the hydrolysis of cellulose via a double-displacement mechanism involving the formation and hydrolysis of a glycosyl-enzyme intermediate. Treatment of EG I with 2',4'-dinitrophenyl-2-deoxy-2-fluoro-beta-D-cellobioside results in the time-dependent inactivation of the enzyme (k(i) = 1.36 min(-1), Ki = 0.88 mM) via trapping of a covalent 2-deoxy-2-fluorocellobiosyl-enzyme intermediate. This intermediate is, however, catalytically competent undergoing transglycosylation, thus reactivation, in the presence of D-cellobiose. Analysis of a peptic digest of the inactivated enzyme by HPLC/ESMS/MS in the neutral loss mode allowed identification of a 2-fluorocellobiosyl-labeled peptide containing Glu197. This was confirmed by comparative mapping studies and subsequent Edman degradation analysis. This residue is completely conserved in glycosidase family 7, to which EG I belongs, consistent with its key role as the catalytic nucleophile.

Amino Acid Sequence↗
PubMed1997

Structure of the endoglucanase I from Fusarium oxysporum: native, cellobiose, and 3,4-epoxybutyl beta-D-cellobioside-inhibited forms, at 2.3 A resolution.

The mechanisms involved in the enzymatic degradation of cellulose are of great ecological and commercial importance. The breakdown of cellulose by fungal species is performed by a consortium of free enzymes, known as cellobiohydrolases and endoglucanases, which are found in many of the 57 glycosyl hydrolase families. The structure of the endoglucanase I (EG I), found in glycosyl hydrolase family 7, from the thermophilic fungus Fusarium oxysporum has been solved at 2.3 A resolution. In addition to the native enzyme, structures have also been determined with both the affinity label, 3,4-epoxybutyl beta-D-cellobioside, and the reaction product cellobiose. The affinity label is covalently bound, as expected, to the catalytic nucleophile, Glu197, with clear evidence for binding of both the R and S stereoisomers. Cellobiose is found bound to the -2 and -1 subsites of the enzyme. In marked contrast to the structure of EG I with a nonhydrolyzable thiosaccharide analog, which spanned the -2, -1, and +1 subsites and which had a skew-boat conformation for the -1 subsite sugar [Sulzenbacher, G., et al. (1996) Biochemistry 35, 15280-15287], the cellobiose complex shows no pyranoside ring distortion in the -1 subsite, implying that strain is induced primarily by the additional +1 subsite interactions and that the product is found, as expected, in its unstrained conformation.

Binding Sites↗
PubMed1997

A bifunctionalized fluorogenic tetrasaccharide as a substrate to study cellulases.

Cellulases are usually classified as endoglucanases and cellobiohydrolases, but the heterogeneity of cellulose, in terms of particle size and crystallinity, has always represented a problem for the biochemical characterization of the enzymes. The synthesis of a bifunctionalized tetrasaccharide substrate suitable for measuring cellulase activity by resonance energy transfer is described. The substrate, which carries a 5-(2-aminoethylamino)-1-naphthalenesulfonate group on the non-reducing end and an indolethyl group on the reducing end, was prepared from beta-lactosyl fluoride and indolethyl beta-cellobioside by a chemoenzymatic approach using the transglycosylating activity of endoglucanase I of Humicola insolens as the key step. The bifunctionalized substrate has been used for the determination of the catalytic constants of H. insolens endoglucanase I and cellobiohydrolases I and II; this substrate could be of general use to measure the kinetic constants of cellulases able to act on oligomers of degree of polymerization <5. The data also provide evidence that cellobiohydrolases I and II are able to degrade an oligosaccharide substrate carrying non-carbohydrate substituents at both ends.

Carbohydrate Conformation↗
PubMed1996

Structure of the Fusarium oxysporum endoglucanase I with a nonhydrolyzable substrate analogue: substrate distortion gives rise to the preferred axial orientation for the leaving group.

Endoglucanase I (EG I) is a cellulase, from glycosyl hydrolase family 7, which cleaves the beta-1,4 linkages of cellulose with overall retention of configuration. The structure of the EG I from Fusarium oxysproum, complexed to a nonhydrolyzable thiooligosaccharide substrate analogue, has been determined by X-ray crystallography at a resolution of 2.7 A utilizing the 4-fold noncrystallographic symmetry present in the asymmetric unit. The electron density map clearly reveals the presence of three glucosyl units of the inhibitor, consistent with the known number of sugar-binding subsites, located at the active site of the enzyme in the -2, -1, and +1 subsites, i.e., actually spanning the point of enzymatic cleavage. The pyranose ring at the point of potential enzymatic cleavage is clearly distorted from the standard 4C1 chair as was originally suggested for beta-retaining enzymes by Phillips [Ford, L.O., Johnson, L.N., Machin, P. A., Phillips, D.C., & Tijan, T. (1974) J. Mol. Biol, 88, 349-371]. The distortion observed goes beyond the "sofa" conformation observed in previous studies and results in a conformation whose salient feature is the resulting quasi-axial orientation for the glycosidic bond and leaving group, as predicted by stereoelectronic theory. An almost identical conformation has recently been observed in a complex of chitobiase with its unhydrolyzed substrate [Tews, I., Perrakis, A., Oppenheim, A., Dauter, Z., Wilson, K. S., & Vorgias, C. E. (1996) Nat. Struct. Biol. 3, 638-648]. The striking similarity between these two complexes extends beyond the almost identical pyranose ring distortion. The overlap of the two respective sugars places the enzymatic nucleophile of endoglucanase I in coincidence with the C2 acetamido oxygen of N-acetylglucosamine in the catalytic site of the chitobiase, substantiating the involvement of this group in the catalytic mechanism of chitobiase and related chitinolytic enzymes. The endoglucanase I complex with the thiosaccharide substrate analogue clearly illustrates the potential of nonhydrolyzable sulfur-linked oligosaccharides in the elucidation of substrate binding and catalysis by glycosyl hydrolases.

Cellulase↗
PubMed1996

Structure determination and refinement of the Humicola insolens endoglucanase V at 1.5 A resolution.

The structure of the catalytic core of the endoglucanase V (EGV) from Humicola insolens has been determined by the method of multiple isomorphous replacement at 1.5 A resolution. The final model, refined with X-PLOR and PROLSQ, has a crystallographic R factor of 0.163 (R(free) = 0.240) with deviations from stereochemical target values of 0.012 A and 0.037 degrees for bonds and angles, respectively. The model was further refined with SHELXL, including anisotropic modelling of the protein-atom temperature factors, to give a final model with an R factor of 0.105 and an R(free) of 0.154. The initial isomorphous replacement electron-density map was poor and uninterpretable but was improved by the use of synchrotron data collected at a wavelength chosen so as to optimize the f" contribution of the anomalous scattering from the heavy atoms. The structure of H. insolens EGV consists of a six-stranded beta-barrel domain, similar to that found in a family of plant defence proteins, linked by a number of disulfide-bonded loop regions. A long open groove runs across the surface of the enzyme either side of which lie the catalytic aspartate residues. The 9 A separation of the catalytic carboxylate groups is consistent with the observation that EGV catalyzes the hydrolysis of the cellulose, beta(1-->4) links with inversion of configuration at the anomeric C1 atom. This structure is the first representative from the glycosyl hydrolase family 45.

Journal Article↗
PubMed1996

Electron microscopic investigation of the diffusion of Bacillus licheniformis alpha-amylase into corn starch granules.

A method for the direct electron microscopic observation of amylases in interaction with starch granules is presented. The technique involves immuno-gold labeling of the enzymes and cross-sectioning of hydrated starch granules. This approach allows the analysis of the internal degradation of starch with a concomitant visualization of enzymes at the sites of hydrolysis. The visualization of enzymes at the surface, inside the channel and inside the core of the degraded granules shows that the alpha-amylase molecules first proceed from the surface toward the center (centripetal hydrolysis). Then the core is completely degraded from within by erosion of its periphery (centrifugal hydrolysis). In the first case (centripetal hydrolysis), the enzymes act by progressing along the polysaccharide chains. By contrast, the centrifugal hydrolysis leads to even erosion, indicative of a more diffusive motion of the enzymes.

Bacillus↗
PubMed1995

Structures of oligosaccharide-bound forms of the endoglucanase V from Humicola insolens at 1.9 A resolution.

Cellulose, a polymer of beta-1,4-linked glucose residues, is the major polysaccharide component of plant cell walls and the most abundant biopolymer. The underlying mechanisms of the enzymatic degradation of cellulose are of increasing commercial and ecological significance. Endoglucanase V, from the cellulolytic soil hyphomycete Humicola insolens, is an endocellulase, the catalytic core of which consists of 210 amino acids and is known to hydrolyze the beta-1,4 links with inversion of configuration at the anomeric carbon. The major products of cellulose hydrolysis are cellobiose and cellotriose. The crystal structures of the endoglucanase V (EGV) from H. insolens, in native, product (cellobiose), inactive mutant (D10N), and oligosaccharide-bound [(D10N)-cellohexaose] forms, have been determined at resolutions of 1.9 A or better. EGV consists of a six-stranded beta-barrel domain with long interconnecting loops. A 40 A groove exists along the surface of the enzyme, and this contains the catalytic residues, Asp 10 and Asp 121. The two catalytic aspartates sit to either side of the substrate binding groove in an ideal conformation for facilitating cleavage by inversion, their carboxyl groups being separated by approximately 8.5 A. The complex between substrate and inactive mutant reveals excellent density for an oligosaccharide in six of the enzyme's seven substrate binding subsites. No sugar moiety, however, is seen bound to the -1 subsite at the point of cleavage. The geometry of the cleavage site suggests that the enzyme would favor the binding of sugars with an elongated glycosidic bond, as found in the transition state, as opposed to the binding of substrate. The oligosaccharide complexes reveal solvent water suitably placed for participation in a single displacement reaction as first suggested by Koshland in 1953 [Koshland, D. E. (1953) Biol. Rev. 28, 416-436]. A large conformational change takes place upon substrate binding. This "lid flipping" has the effect of increasing the hydrophobic environment of the catalytic proton donor, enclosing the active site at the point of cleavage, and bringing a third aspartate (Asp 114) in close proximity to the substrate. Site-directed mutagenesis of the catalytic residues has been used to confirm their significance in catalysis.

Amino Acid Sequence↗
PubMed1995

Dynamic light scattering study of the two-domain structure of Humicola insolens endoglucanase V.

Endoglucanase V (EG V) of HUmicola insolens is composed of a catalytic domain and of a cellulose-binding domain linked by a 33 amino acid long peptide rich in Ser, Thr and Pro residues. This work describes the dynamic behavior of the two-domain structure of EG V as revealed by quasi-elastic light scattering experiments. For both the full-length and the isolated catalytic domain, the autocorrelation function is essentially described by a single relaxation mode. The equivalent hydrodynamic radius of the catalytic domain was found to correspond precisely to the dimensions measured from the previously determined three-dimensional structure. The results obtained with the full-length protein allow a description of the two domain structure of EG V similar to that resulting from earlier studies using small angle X-ray scattering on cellulases from Trichoderma reesei. The hydrodynamic dimensions of the entire enzyme can be approximated as an ellipsoid with dimensions of 42 x 133.6 A.

Binding Sites↗
PubMed1994

Structure determination of glucose isomerase from Streptomyces murinus at 2.6 A resolution.

Glucose isomerase from Streptomyces murinus has been crystallized in space group P4(1)2(1)2, cell dimensions a = b = 137.65 and c = 132.20 A. One dimer of the tetrametric molecule is found per asymmetric unit. An initial structure solution was obtained by the molecular replacement method. The crystallographic refinement was performed using molecular dynamics techniques with X-ray restraints. The final crystallographic R value is 21.4% at 2.6 A resolution including 3023 non-H atoms, two metal ions and two water molecules per monomer.

Journal Article↗
PubMed1993

Stereochemistry, specificity and kinetics of the hydrolysis of reduced cellodextrins by nine cellulases.

The catalytic activity of nine enzymes (endoglucanases I-III, V, VI and cellobiohydrolases I and II from Humicola insolens; endoglucanases A and C from Bacillus lautus), representative of cellulase families A-C, H, J and K, has been investigated using a series of reduced cellooligosaccharides (cellotriitol to cellohexaitol) as substrates. For each enzyme, the specificity of cleavage was determined by analytical HPLC while the kinetic constants were obtained from a kinetic assay involving a cellobiose dehydrogenase purified from H. insolens as a coupled enzyme using 2,6-dichloroindophenol as the electron acceptor. These data were used to estimate the number of subsites in the enzymes. The stereochemical course of hydrolysis by seven enzymes, representing the six different families, was assessed using 1H-NMR. The enzymes belonging to families which had already been investigated (A-C), showed results in agreement with previous studies. The three other families (H, J and K), for which no mechanistic data was previously available, gave results which indicated that enzymes in group H had retaining-type activity and enzymes in groups J and K had inverting-type activity. The retaining endoglucanases I and III displayed a high glycosyl-transferase activity under the conditions used during the NMR experiments resulting in precipitates of higher oligomers.

Bacillus↗
PubMed1993

Structure and function of endoglucanase V.

Cellulose is the major polysaccharide component of plant cell walls and is the most abundant organic compound on the planet. A number of bacterial and fungal organisms can use cellulose as a food source, possessing cellulases (cellobiohydrolases and endoglucanases) that can catalyse the hydrolysis of the beta-(1,4) glycosidic bonds. They can be classified into seven distinct families. The three-dimensional structures of members of two of these families are known. Here we report the structure of a third cellulase, endoglucanase V, whose sequence is not represented in any of the above families. The enzyme is structurally distinct from the previously determined cellulases but is similar to a recently characterized plant defence protein. The active site region resembles that of lysozyme, despite the lack of structural similarity between these two enzymes.

Binding Sites↗
PubMed1992

Crystallization and preliminary X-ray analysis of a fungal endoglucanase I.

An endoglucanase I (EG1) from a fungal source (Humicola insolens) has been crystallized in a number of forms suitable for X-ray diffraction analysis. Four crystal forms have been grown from various precipitants using vapour phase diffusion methods in hanging drops. Three of these crystal forms diffract to beyond 2.5 A resolution. Two forms, obtained from ammonium sulphate at pH 5.4, or 8.0, grow as tetragonal bipyramids in space group P4(1)22 or P4(3)22, with approximate cell dimensions a = b = 102 A, c = 282 A. The other crystal forms were grown from polyethylene glycol 8000 at pH 8.0. One grows as monoclinic plates, space group P2(1), with cell dimensions a = 66.9 A, b = 75.2 A, c = 86.9 A and beta = 102.9 degrees and the other as long hexagonal rods in space group P6(1)22 or P6(5)22, with cell dimensions a = b = 119 A, c = 83 A.

Cellulose 1,4-beta-Cellobiosidase↗