Mapping the conformational itinerary of beta-glycosidases by X-ray crystallography.

The conformational agenda harnessed by different glycosidases along the reaction pathway has been mapped by X-ray crystallography. The transition state(s) formed during the enzymic hydrolysis of glycosides features strong oxocarbenium-ion-like character involving delocalization across the C-1-O-5 bond. This demands planarity of C-5, O-5, C-1 and C-2 at or near the transition state. It is widely, but incorrectly, assumed that the transition state must be (4)H(3) (half-chair). The transition-state geometry is equally well supported, for pyranosides, by both the (4)H(3) and (3)H(4) half-chair and (2,5)B and B(2,5) boat conformations. A number of retaining beta-glycosidases acting on gluco -configured substrates have been trapped in Michaelis and covalent intermediate complexes in (1)S(3) (skew-boat) and (4)C(1) (chair) conformations, respectively, pointing to a (4)H(3)-conformed transition state. Such a (4)H(3) conformation is consistent with the tight binding of (4)E- (envelope) and (4)H(3)-conformed transition-state mimics to these enzymes and with the solution structures of compounds bearing an sp (2) hybridized anomeric centre. Recent work reveals a (1)S(5) Michaelis complex for beta-mannanases which, together with the (0)S(2) covalent intermediate, strongly implicates a B(2,5) transition state for beta-mannanases, again consistent with the solution structures of manno -configured compounds bearing an sp (2) anomeric centre. Other enzymes may use different strategies. Xylanases in family GH-11 reveal a covalent intermediate structure in a (2,5)B conformation which would also suggest a similarly shaped transition state, while (2)S(0)-conformed substrate mimics spanning the active centre of inverting cellulases from family GH-6 may also be indicative of a (2,5)B transition-state conformation. Work in other laboratories on both retaining and inverting alpha-mannosidases also suggests non-(4)H(3) transition states for these medically important enzymes. Three-dimensional structures of enzyme complexes should now be able to drive the design of transition-state mimics that are specific for given enzymes, as opposed to being generic or merely fortuitous.

Atomic resolution structure of endoglucanase Cel5A in complex with methyl 4,4II,4III,4IV-tetrathio-alpha-cellopentoside highlights the alternative binding modes targeted by substrate mimics.

Many three-dimensional structures of retaining beta-D-glycoside hydrolases have been determined, yet oligosaccharide complexes in which the ligand spans the catalytic centre are rare. Those that have been reported so far have revealed two modes of binding: those in which the substrate adopts a distorted skew-boat or envelope conformation in the -1 subsite, reflecting the distortion observed during the catalytic cycle, and those which bypass the true catalytic centre and thus lie in a non-productive manner across the -1 subsite. The three-dimensional structure of a retaining endocellulase, Bacillus agaradhaerens Cel5A, in complex with methyl 4,4(II),4(III),4(IV)-tetrathio-alpha-cellopentoside falls into this latter category. The 1.1 A structure reveals the binding of five pyranosides, all in the (4)C(1) chair conformation, occupying the -3, -2, +1 and +2 subsites whilst evading the catalytic machinery located in the true -1 subsite. Such binding is in marked contrast to the structure of another retaining endocellulase, the Fusarium oxysporum Cel7B, the identical ligand in which displayed a distorted skew-boat conformation at the active centre. These two binding modes may reflect different steps in the binding and catalytic process.

Mixed-linkage cellooligosaccharides: a new class of glycoside hydrolase inhibitors.

A new class of inhibitors for beta-D-glycoside hydrolases, in which a single alpha-(1-->4)-glycosidic bond is incorporated into an otherwise all-beta-(1-->4)-linked oligosaccharide, is described. Such mixed beta/alpha-linkage cellooligosaccharides are not transition-state mimics, but instead are capable of utilising binding energy from numerous subsites, spanning either side of the catalytic centre, without the need for substrate distortion. This binding is significant; a mixed alpha/beta-D-tetrasaccharide acts competitively on a number of cellulases, displaying inhibition constants in the range of 40-300 microM. Using the Bacillus agaradhaerens enzyme Cel5A as a model system, one such mixed beta/alpha-cellooligosaccharide, methyl 4(II),4(III)-dithio-alpha-cellobiosyl-(1-->4)-beta-cellobioside, displays a K(i) value of 100 microM, an inhibition at least 150 times better than is observed with an equivalent all-beta-linked compound. The three-dimensional structure of B. agaradhaerens Cel5A in complex with methyl 4(II),4(III)-dithio-alpha-cellobiosyl-(1-->4)-beta-cellobioside has been determined at 1.8 A resolution. This confirms the expected mode of binding in which the ligand, with all four pyranosides in the (4)C(1) chair conformation, occupies the -3, -2 and +1 subsites whilst evading the catalytic (-1) subsite. Such "by-pass" compounds offer great scope for the development of a new class of beta-D-glycoside hydrolase inhibitors.

Structure and function of Humicola insolens family 6 cellulases: structure of the endoglucanase, Cel6B, at 1.6 A resolution.

Cellulases are traditionally classified as either endoglucanases or cellobiohydrolases on the basis of their respective catalytic activities on crystalline cellulose, which is generally hydrolysed more efficiently only by the cellobiohydrolases. On the basis of the Trichoderma reesei cellobiohydrolase II structure, it was proposed that the active-site tunnel of cellobiohydrolases permitted the processive hydrolysis of cellulose, whereas the corresponding endoglucanases would display open active-site clefts [Rouvinen, Bergfors, Teeri, Knowles and Jones (1990) Science 249, 380-386]. Glycoside hydrolase family 6 contains both cellobiohydrolases and endoglucanases. The structure of the catalytic core of the family 6 endoglucanase Cel6B from Humicola insolens has been solved by molecular replacement with the known T. reesei cellobiohydrolase II as the search model. Strangely, at the sequence level, this enzyme exhibits the highest sequence similarity to family 6 cellobiohydrolases and displays just one of the loop deletions traditionally associated with endoglucanases in this family. However, this enzyme shows no activity on crystalline substrates but a high activity on soluble substrates, which is typical of an endoglucanase. The three-dimensional structure reveals that the deletion of just a single loop of the active site, coupled with the resultant conformational change in a second 'cellobiohydrolase-specific' loop, peels open the active-site tunnel to reveal a substrate-binding groove.

Insights into ligand-induced conformational change in Cel5A from Bacillus agaradhaerens revealed by a catalytically active crystal form.

Glycoside hydrolases are ubiquitous enzymes involved in a diverse array of biological processes, from the breakdown of biomass, through to viral invasion and cellular signalling. Endoglucanase Cel5A from Bacillus agaradhaerens, classified into glycoside hydrolase family 5, has been studied in a catalytically inactive crystal form at low pH conditions, in which native and complex structures revealed the importance of ring distortion during catalysis. Here, we present the structure of Cel5A in a new crystal form obtained at higher pH values in which the enzyme is active "in-crystal". Native, cellotriosyl-enzyme intermediate and beta-d-cellobiose structures were solved at 1.95, 1.75 and 2.1 A resolution, respectively. These structures reveal two classes of conformational change: those caused by crystal-packing and pH, with others induced upon substrate binding. At pH 7 a histidine residue, His206, implicated in substrate-binding and catalysis, but previously far removed from the substrate-binding cleft, moves over 10 A into the active site cleft in order to interact with the substrate in the +2 subsite. Occupation of the -1 subsite by substrate induces a loop closure to optimise protein-ligand interactions. Cel5A, along with the unrelated family 45 and family 6 cellulases, provides further evidence of substantial conformational change in response to ligand binding for this class of hydrolytic enzyme.

Structure of the specificity domain of the Dorsal homologue Gambif1 bound to DNA.

BACKGROUND: NF-kappa B/Rel transcription factors play important roles in immunity and development in mammals and insects. Their activity is regulated by their cellular localization, homo- and heterodimerization and association with other factors on their target gene promoters. Gambif1 from Anopheles gambiae is a member of the Rel family and a close homologue of the morphogen Dorsal, which establishes dorsoventral polarity in the Drosophila embryo. RESULTS: We present the crystal structure of the N-terminal specificity domain of Gambif1 bound to DNA. This first structure of an insect Rel protein-DNA complex shows that Gambif1 binds a GGG half-site element using a stack of three arginine sidechains. Differences in affinity to Dorsal binding sites in target gene promoters are predicted to arise from base changes in these GGG elements. An arginine that is conserved in class II Rel proteins (members of which contain a transcription activation domain) contacts the outermost guanines of the DNA site. This previously unseen specific contact contributes strongly to the DNA-binding affinity and might be responsible for differences in specificity between Rel proteins of class I and II. CONCLUSIONS: The Gambif1-DNA complex structure illustrates how differences in Dorsal affinity to binding sites in developmental gene promoters are achieved. Comparison with other Rel-DNA complex structures leads to a general model for DNA recognition by Rel proteins.

Structural changes of the active site tunnel of Humicola insolens cellobiohydrolase, Cel6A, upon oligosaccharide binding.

The mechanisms of crystalline cellulose degradation by cellulases are of paramount importance for the exploitation of these enzymes in applied processes, such as biomass conversion. Cellulases have traditionally been classified into cellobiohydrolases, which are effective in the degradation of crystalline materials, and endoglucanases, which appear to act on "soluble" regions of the substrate. Humicola insolensCel6A (CBH II) is a cellobiohydrolase from glycoside hydrolase family 6 whose native structure has been determined at 1.9 A resolution [Varrot, A., Hastrup, S., Schülein, M., and Davies, G. J. (1999) Biochem. J. 337, 297-304]. Here we present the structure of the catalytic core domain of Humicola insolens cellobiohydrolase II Cel6A in complex with glucose/cellotetraose at 1.7 A resolution. Crystals of Cel6A, grown in the presence of cellobiose, reveal six binding subsites, with a single glucose moiety bound in the -2 subsite and cellotetraose in the +1 to +4 subsites. The complex structure is strongly supportive of the assignment of Asp 226 as the catalytic acid and consistent with proposals that Asp 405 acts as the catalytic base. The structure undergoes several conformational changes upon substrate binding, the most significant of which is a closing of the two active site loops (residues 174-196 and 397-435) with main-chain movements of up to 4.5 A observed. This complex not only defines the polysaccharide-enzyme interactions but also provides the first three-dimensional demonstration of conformational change in this class of enzymes.

Crystallization and preliminary X-ray analysis of the 6-phospho-alpha-glucosidase from Bacillus subtilis.

6-Phospho-alpha-glucosidase (GlvA) is the protein involved in the dissimilation of alpha-glycosides accumulated via a phosphoenolpyruvate-dependent maltose phosphotransferase system (PEP-PTS) in Bacillus subtilis. The purified enzyme has been crystallized in a form suitable for X-ray diffraction analysis. Thin rod-like crystals have been grown by the hanging-drop method in the presence of manganese and NAD. They diffract beyond 2.2 A using synchrotron radiation and belong to the space group I222 (or its enantiomorph) with unit-cell dimensions a = 83.26, b = 102.56, c = 145.31 A and contain a single molecule of GlvA in the asymmetric unit.

Crystal structure of the catalytic core domain of the family 6 cellobiohydrolase II, Cel6A, from Humicola insolens, at 1.92 A resolution.

The three-dimensional structure of the catalytic core of the family 6 cellobiohydrolase II, Cel6A (CBH II), from Humicola insolens has been determined by X-ray crystallography at a resolution of 1.92 A. The structure was solved by molecular replacement using the homologous Trichoderma reesei CBH II as a search model. The H. insolens enzyme displays a high degree of structural similarity with its T. reesei equivalent. The structure features both O- (alpha-linked mannose) and N-linked glycosylation and a hexa-co-ordinate Mg2+ ion. The active-site residues are located within the enclosed tunnel that is typical for cellobiohydrolase enzymes and which may permit a processive hydrolysis of the cellulose substrate. The close structural similarity between the two enzymes implies that kinetics and chain-end specificity experiments performed on the H. insolens enzyme are likely to be applicable to the homologous T. reesei enzyme. These cast doubt on the description of cellobiohydrolases as exo-enzymes since they demonstrated that Cel6A (CBH II) shows no requirement for non-reducing chain-ends, as had been presumed. There is no crystallographic evidence in the present structure to support a mechanism involving loop opening, yet preliminary modelling experiments suggest that the active-site tunnel of Cel6A (CBH II) is too narrow to permit entry of a fluorescenyl-derivatized substrate, known to be a viable substrate for this enzyme.

Snapshots along an enzymatic reaction coordinate: analysis of a retaining beta-glycoside hydrolase.

The enzymatic hydrolysis of O-glycosidic linkages is one of the most diverse and widespread reactions in nature and involves a classic "textbook" enzyme mechanism. A multidisciplinary analysis of a beta-glycoside hydrolase, the Cel5A from Bacillus agaradhaerens, is presented in which the structures of each of the native, substrate, covalent-intermediate, and product complexes have been determined and their interconversions analyzed kinetically, providing unprecedented insights into the mechanism of this enzyme class. Substrate is bound in a distorted 1S3 skew-boat conformation, thereby presenting the anomeric carbon appropriately for nucleophilic attack as well as satisfying the stereoelectronic requirements for an incipient oxocarbenium ion. Leaving group departure results in the trapping of a covalent alpha-glycosyl-enzyme intermediate in which the sugar adopts an undistorted 4C1 conformation. Finally, hydrolysis of this intermediate yields a product complex in which the sugar is bound in a partially disordered mode, consistent with unfavorable interactions and low product affinity.