Insights into the fine architecture of the active site of chicory fructan 1-exohydrolase: 1-kestose as substrate vs sucrose as inhibitor.

* Invertases and fructan exohydrolases (FEHs) fulfil important physiological functions in plants. Sucrose is the typical substrate for invertases and bacterial levansucrases but not for plant FEHs, which are usually inhibited by sucrose. * Here we report on complexes between chicory (Cichorium intybus) 1-FEH IIa with the substrate 1-kestose and the inhibitors sucrose, fructose and 2,5 dideoxy-2,5-imino-D-mannitol. Comparisons with other family GH32 and 68 enzyme-substrate complexes revealed that sucrose can bind as a substrate (invertase/levansucrase) or as an inhibitor (1-FEH IIa). * Sucrose acts as inhibitor because the O2 of the glucose moiety forms an H-linkage with the acid-base catalyst E201, inhibiting catalysis. By contrast, the homologous O3 of the internal fructose in the substrate 1-kestose forms an intramolecular H-linkage and does not interfere with the catalytic process. Mutagenesis showed that W82 and S101 are important for binding sucrose as inhibitor. * The physiological implications of the essential differences in the active sites of FEHs and invertases/levansucrases are discussed. Sucrose-inhibited FEHs show a K(i) (inhibition constant) well below physiological sucrose concentrations and could be rapidly activated under carbon deprivation.

X-ray diffraction structure of a cell-wall invertase from Arabidopsis thaliana.

Cell-wall invertases play crucial roles during plant development. They hydrolyse sucrose into its fructose and glucose subunits by cleavage of the alpha1-beta2 glycosidic bond. Here, the structure of the Arabidopsis thaliana cell-wall invertase 1 (AtcwINV1; gene accession code At3g13790) is described at a resolution of 2.15 A. The structure comprises an N-terminal fivefold beta-propeller domain followed by a C-terminal domain formed by two beta-sheets. The active site is positioned in the fivefold beta-propeller domain, containing the nucleophile Asp23 and the acid/base catalyst Glu203 of the double-displacement enzymatic reaction. The function of the C-terminal domain remains unknown. Unlike in other GH 32 family enzyme structures known to date, in AtcwINV1 the cleft formed between both domains is blocked by Asn299-linked carbohydrates. A preliminary site-directed mutagenesis experiment (Asn299Asp) removed the glycosyl chain but did not alter the activity profile of the enzyme.

His374 of wheat endoxylanase inhibitor TAXI-I stabilizes complex formation with glycoside hydrolase family 11 endoxylanases.

Wheat endoxylanase inhibitor TAXI-I inhibits microbial glycoside hydrolase family 11 endoxylanases. Crystallographic data of an Aspergillus niger endoxylanase-TAXI-I complex showed His374 of TAXI-I to be a key residue in endoxylanase inhibition. Its role in enzyme-inhibitor interaction was further investigated by site-directed mutagenesis of His374 into alanine, glutamine or lysine. Binding kinetics and affinities of the molecular interactions between A. niger, Bacillus subtilis, Trichoderma longibrachiatumendoxylanases and wild-type TAXI-I and TAXI-I His374 mutants were determined by surface plasmon resonance analysis. Enzyme-inhibitor binding was in accordance with a simple 1 : 1 binding model. Association and dissociation rate constants of wild-type TAXI-I towards the endoxylanases were in the range between 1.96 and 36.1 x 10(4)m(-1) x s(-1) and 0.72-3.60 x 10(-4) x s(-1), respectively, resulting in equilibrium dissociation constants in the low nanomolar range. Mutation of TAXI-I His374 to a variable degree reduced the inhibition capacity of the inhibitor mainly due to higher complex dissociation rate constants (three- to 80-fold increase). The association rate constants were affected to a smaller extent (up to eightfold decrease). Substitution of TAXI-I His374 therefore strongly affects the affinity of the inhibitor for the enzymes. In addition, the results show that His374 plays a critical role in the stabilization of the endoxylanase-TAXI-I complex rather than in the docking of inhibitor onto enzyme.

Structural analysis of the jacalin-related lectin MornigaM from the black mulberry (Morus nigra) in complex with mannose.

The structures of MornigaM and the MornigaM-mannose complex have been determined at 1.8 A and 2.0 A resolution, respectively. Both structures adopt the typical beta-prism motif found in other jacalin-related lectins and their tetrameric assembly closely resembles that of jacalin. The carbohydrate-binding cavity of MornigaM readily binds mannose. No major structural rearrangements can be observed in MornigaM upon binding of mannose. These results allow corroboration of the structure-function relationships within the small group of Moraceae lectins.

X-ray diffraction structure of a plant glycosyl hydrolase family 32 protein: fructan 1-exohydrolase IIa of Cichorium intybus.

Fructan 1-exohydrolase, an enzyme involved in fructan degradation, belongs to the glycosyl hydrolase family 32. The structure of isoenzyme 1-FEH IIa from Cichorium intybus is described at a resolution of 2.35 A. The structure consists of an N-terminal fivefold beta-propeller domain connected to two C-terminal beta-sheets. The putative active site is located entirely in the beta-propeller domain and is formed by amino acids which are highly conserved within glycosyl hydrolase family 32. The fructan-binding site is thought to be in the cleft formed between the two domains. The 1-FEH IIa structure is compared with the structures of two homologous but functionally different enzymes: a levansucrase from Bacillus subtilis (glycosyl hydrolase family 68) and an invertase from Thermotoga maritima (glycosyl hydrolase family 32).

Crystallization and preliminary X-ray diffraction study of a cell-wall invertase from Arabidopsis thaliana.

Cell-wall invertase 1 (AtcwINV1), a plant protein from Arabidopsis thaliana which is involved in the breakdown of sucrose, has been crystallized in two different crystal forms. Crystal form I grows in space group P3(1) or P3(2), whereas crystal form II grows in space group C222(1). Data sets were collected for crystal forms I and II to resolution limits of 2.40 and 2.15 A, respectively.

How calcium inhibits the magnesium-dependent enzyme human phosphoserine phosphatase.

The structure of the Mg(2+)-dependent enzyme human phosphoserine phosphatase (HPSP) was exploited to examine the structural and functional role of the divalent cation in the active site of phosphatases. Most interesting is the biochemical observation that a Ca(2+) ion inhibits the activity of HPSP, even in the presence of added Mg(2+). The sixfold coordinated Mg(2+) ion present in the active site of HPSP under normal physiological conditions, was replaced by a Ca(2+) ion by using a crystallization condition with high concentration of CaCl(2) (0.7 m). The resulting HPSP structure now shows a sevenfold coordinated Ca(2+) ion in the active site that might explain the inhibitory effect of Ca(2+) on the enzyme. Indeed, the Ca(2+) ion in the active site captures both side-chain oxygen atoms of the catalytic Asp20 as a ligand, while a Mg(2+) ion ligates only one oxygen atom of this Asp residue. The bidentate character of Asp20 towards Ca(2+) hampers the nucleophilic attack of one of the Asp20 side chain oxygen atoms on the phosphorus atom of the substrate phosphoserine.

Azospirillum irakense pectate lyase displays a toroidal fold.

The three-dimensional structure of Azospirillum irakense pectate lyase (PelA) has been determined at a resolution of 2.65 A. The crystals are hexagonal, belonging to space group P6(5)22, with unit-cell parameters a = b = 85.37, c = 231.32 angstroms. Phase information was derived from a multiple-wavelength anomalous dispersion (MAD) experiment using a Hg derivative. Refinement of the model converged to Rcryst = 20.08% and Rfree = 25.87%. The overall structure of PelA does not adopt the characteristic parallel beta-helix fold displayed by pectate lyases from polysaccharide lyase (PL) families PL1, PL3 and PL9. Instead, it displays a predominantly alpha-helical structure with irregular coils and short beta-strands, similar to the recently reported structure of the catalytic module of the Cellvibrio japonicus pectate lyase Pel10Acm. The topologies of the two structures have been compared. They show two 'domains' with the interface between them being a wide-open central groove in which the active site is located. The active sites of the crystal structures are also compared and their similarities and differences are discussed.

Structural basis for inhibition of Aspergillus niger xylanase by triticum aestivum xylanase inhibitor-I.

Plants developed a diverse battery of defense mechanisms in response to continual challenges by a broad spectrum of pathogenic microorganisms. Their defense arsenal includes inhibitors of cell wall-degrading enzymes, which hinder a possible invasion and colonization by antagonists. The structure of Triticum aestivum xylanase inhibitor-I (TAXI-I), a first member of potent TAXI-type inhibitors of fungal and bacterial family 11 xylanases, has been determined to 1.7-A resolution. Surprisingly, TAXI-I displays structural homology with the pepsin-like family of aspartic proteases but is proteolytically nonfunctional, because one or more residues of the essential catalytical triad are absent. The structure of the TAXI-I. Aspergillus niger xylanase I complex, at a resolution of 1.8 A, illustrates the ability of tight binding and inhibition with subnanomolar affinity and indicates the importance of the C-terminal end for the differences in xylanase specificity among different TAXI-type inhibitors.

Crystallization and preliminary X-ray diffraction study of fructan 1-exohydrolase IIa from Cichorium intybus.

Fructan 1-exohydrolase IIa (1-FEH IIa), a plant enzyme involved in fructan breakdown, has been crystallized using the hanging-drop vapour-diffusion method at 277 K. The crystals are tetragonal, belonging to space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = 139.83, b = 139.83, c = 181.94 A. Calculation of the Matthews coefficient indicates there to be two or three molecules in the asymmetric unit. Synchrotron radiation was used to collect a complete native data set to a resolution of 2.35 A.

Crystallization and preliminary X-ray diffraction study of two complexes of a TAXI-type xylanase inhibitor with glycoside hydrolase family 11 xylanases from Aspergillus niger and Bacillus subtilis.

Endo-beta-1,4-xylanases hydrolyze arabinoxylan, a major constituent of cereal cell walls, and are nowadays widely used in biotechnological processes. Purified complexes of family 11 xylanases from Aspergillus niger and Bacillus subtilis with TAXI I, a TAXI-type xylanase inhibitor from Triticum aestivum L., were prepared. In both cases the complex was crystallized using the hanging-drop vapour-diffusion method. The needle-like crystals of TAXI I in complex with A. niger xylanase belong to the trigonal space group P3(1) or P3(2), with unit-cell parameters a = b = 88.43, c = 128.99 A, and diffract to 1.8 A resolution. TAXI I in complex with B. subtilis xylanase crystallizes in the monoclinic space group C2, with a = 107.89, b = 95.33, c = 66.31 A, beta = 122.24 degrees. Complete data sets were collected for both crystal types using synchrotron radiation.

High-resolution structure of human phosphoserine phosphatase in open conformation.

The crystal structure of human phosphoserine phosphatase (HPSP) in the open conformation has been determined at a resolution of 1.53 A. The crystals are orthorhombic, belonging to space group C222(1), with unit-cell parameters a = 49.03, b = 130.25, c = 157.29 A. The asymmetric unit contains two molecules. Phase information was derived from a multiwavelength anomalous dispersion (MAD) experiment conducted at three wavelengths using a selenomethionine-derivative crystal of HPSP. The structure was refined using CNS to a final crystallographic R value of 21.6% (R(free) = 23.4%). HPSP is a dimeric enzyme responsible for the third and final step of the l-serine biosynthesis pathway. It catalyses the Mg2+-dependent hydrolysis of l-phosphoserine. Recently, the structure of HPSP in complex with an inhibitor bound to the active site has been reported to be the open conformation of the enzyme. Here, the structure of HPSP is reported in the absence of substrate in the active site. Evidence is presented that HPSP in an uncomplexed form is in an even more open conformation than in the inhibitor complex. In this state, the enzyme is partially unfolded to allow the substrate to enter the active site. Binding of the substrate causes HPSP to shift to the closed conformation by stabilizing the partially unfolded region. In the present structure a Ca2+ ion is bound to the active site and an explanation is given why HPSP is not active when in the active site Mg2+ is replaced by a Ca2+ ion.

Previtamin D3 with a trans-fused decalin CD-ring has pronounced genomic activity.

Deletion of C19 in the structure of 1 alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] does not substantially alter the biological potency but prevents the conversion between the vitamin and the previtamin form. Hence, this modification allows the study of locked previtamin and vitamin forms. The locked 19-nor-1,25(OH)2-previtamin D3 analog (19-nor-previtamin D) had a low biological activity and was a rather weak activator of the genomic signal transduction pathway. 19-Nor-trans-decalin-1,25(OH)2-vitamin D3 (19-nor-TD-vitamin D), characterized by the presence of a trans-fused decalin CD-ring system, was 10-fold more potent than the parent compound and was a potent activator of the genomic signal transduction pathway. Surprisingly, the previtamin, 19-nor-trans-decalin-1,25(OH)2-previtamin D3 (19-nor-TD-previtamin D), was as potent as 1,25(OH)2D3 in inhibiting cell proliferation and inducing cell differentiation and represents the first previtamin structure with pronounced vitamin D-like activity. Furthermore, this compound interacted as efficiently as 1,25(OH)2D3 with the vitamin D receptor (VDR), retinoid X receptor (RXR), coactivators, and DNA, which illustrated its potent ability to activate the genomic signal transduction pathway. Analysis of the transactivation potency of 12 VDR point mutants after stimulation with 19-nor-TD-previtamin D revealed that this analog used the same contact points within the receptor as did 1,25(OH)2D3. This could be confirmed by modeling analysis of this compound in the ligand binding pocket of VDR. In conclusion, a previtamin D3 analog is presented with genomic activities equivalent to 1,25(OH)2D3.

Crystallization and preliminary X-ray diffraction study of a wheat (Triticum aestivum L.) TAXI-type endoxylanase inhibitor.

A TAXI-type endoxylanase inhibitor from T. aestivum L. wheat flour has been crystallized using the hanging-drop vapour-diffusion method. The needle-like crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 49.92, b = 66.45, c = 106.42 A. From these crystals, a native data set and a gold-derivative data set were collected to 2.25 and 1.75 A resolution, respectively. The heavy-atom derivative of this crystal form was obtained by the soaking method and allowed determination of the initial phases.

Actin-DBP: the perfect structural fit?

The multifunctional vitamin D binding protein (DBP) is an actin-sequestering protein present in blood. The crystal structure of the actin-DBP complex was determined at 2.4 A resolution. DBP binds to actin subdomains 1 and 3 and occludes the cleft at the interface between these subdomains. Most remarkably, DBP demonstrates an unusually large actin-binding interface, far exceeding the binding-interface areas reported for other actin-binding proteins such as profilin, DNase I and gelsolin. The fast-growing side of actin monomers is blocked completely through a perfect structural fit with DBP, demonstrating how DBP effectively interferes with actin-filament formation. It establishes DBP as the hitherto best actin-sequestering protein and highlights its key role in suppressing and preventing extracellular actin polymerization.

A structural basis for the unique binding features of the human vitamin D-binding protein.

The human serum vitamin D-binding protein (DBP) has many physiologically important functions, ranging from transporting vitamin D3 metabolites, binding and sequestering globular actin and binding fatty acids to functioning in the immune system. Here we report the 2.3 A crystal structure of DBP in complex with 25-hydroxyvitamin D3, a vitamin D3 metabolite, which reveals the vitamin D-binding site in the N-terminal part of domain I. To more explicitly explore this, we also studied the structure of DBP in complex with a vitamin D3 analog. Comparisons with the structure of human serum albumin, another family member, reveal a similar topology but also significant differences in overall, as well as local, folding. These observed structural differences explain the unique vitamin D3-binding property of DBP.

Crystallization and preliminary X-ray diffraction analysis of a novel pectate lyase from Azospirillum irakense.

The PelA gene from the N(2)-fixing plant-associated bacterium Azospirillum irakense encodes a pectate lyase. Analysis of the corresponding amino-acid sequence revealed no homology to other bacterial, plant and fungal pectinases of known published structure, resulting in the classification of the enzyme in a new pectate lyase family. The A. irakense PelA has been crystallized using the hanging-drop vapour-diffusion method at 277 K. The crystals are hexagonal, with unit-cell parameters a = b = 85.55, c = 230.13 A, gamma = 120 degrees, and belong to space group P6(5)22 or P6(1)22, having one molecule per asymmetric unit. Diffraction data to a resolution of 1.97 A were collected at synchrotron facilities, as well as a three-wavelength MAD data set from an Hg-derivative crystal to a resolution of 2.6 A.

Purification, crystallization and preliminary X-ray diffraction analysis of human phosphoserine phosphatase.

Phosphoserine phosphatase (PSP), a human enzyme involved in the L-serine biosynthesis pathway, has been crystallized using the hanging-drop vapour-diffusion method at 277 K. The crystals are orthorhombic, belonging to space group C222(1), with unit-cell parameters a = 49.03 A, b = 130.25 A, c = 157.29 A. Calculation of the Matthews coefficient indicates that there are two molecules in the asymmetric unit. A complete native data set to a resolution of 1.53 A has been collected at 100 K using synchrotron radiation.