Degradation of endosulfan during substrate preparation and cultivation of Pleurotus pulmonarius

Summary Endosulfan is an insecticide used on many vegetable crops. In mushroom cultivation, vegetable materials used as a growth substrate may contain residues of endosulfan that may accumulate in the final mushroom biomass. After preparing the substrate, it is subjected to pasteurization and/or composting and then inoculated with the desired fungus. The purpose of this research was to determine the rate and extent of endosulfan reduction from a grass substrate that was either composted or sterilized by autoclaving. In addition, the rate and extent of removal of endosulfan from substrate colonized with Pleurotus pulmonarius was determined. The degradation of 65 mg/kg endosulfan was analyzed on both, the substrate preparation and the culture of P. pulmonarius on the grass Digitaria decumbens. During composting in presence of Ca(OH)₂ for 120 h, the concentrations of α and β endosulfan were reduced by 61.4 and 49.5% respectively, significantly higher compared with the control (without Ca(OH)_2,) in which the reduction was 38.5%. After sterilization the concentration of α and β endosulfan was reduced by 84.8 and 87.5% respectively. After the colonization of substrate by P. pulmonarius (15 days after spawning) α and β endosulfan were reduced by 96% and at the end of cultivation (35 days after spawning) were reduced by 99%. When carpophores were analyzed, residues of α and β endosulfan were observed between 0.019–0.084 mg/kg. The results showed that α and β endosulfan were partially removed during the preparation of substrate and entirely eliminated during fungal colonization on the substrate.     

Studies on solution NMR structure of brazzein

Brazzein is a sweet-tasting protein isolated from the fruit of West African plantPentadiplandra brazzeana Baillon. It is the smallest and the most water-soluble sweet protein discovered so far and is highly thermostable. The proton NMR study of brazzein at 600 MHz (pH 3.5, 300 K) is presented. The complete sequence specific assignments of the individual backbone and sidechain proton resonances were achieved using through-bond and through-space connectivities obtained from standard two-dimensional NMR techniques. The secondary structure of brazzein contains one α-helix (residues 21–29), one short 3₁₀-helix (residues 14–17), two strands of antiparallel β-sheet (residues 34–39, 44–50) and probably a third strand (residues 5–7) near the N-terminus. A comparative analysis found that brazzein shares a so-called ‘cysteine-stabilized alpha-beta’ (CSαβ) motif with scorpion neurotoxins, insect defensins and plant γ - thionins. The significance of this multi-function motif, the possible active sites and the structural basis of themostability were discussed.     

Proton relay system in the active site of maltodextrinphosphorylase via hydrogen bonds with large proton polarizability: an FT-IR difference spectroscopy study

Maltodextrinphosphorylase (MDP) was studied in the pH range 5.4–8.4 by Fourier transform infrared (FT-IR) spectroscopy. The pK _a value of the cofactor pyridoxalphosphate (PLP) was found between 6.5 and 7.0, which closely resembles the second pK _a of free PLP. FT-IR difference spectra of the binary complex of MDP+α-d-glucose-1-methylenephosphonate (Glc-1-MeP) minus native MDP were taken at pH 6.9. Following binary complex formation, two Lys residues, tentatively assigned to the active site residues Lys533 and Lys539, became deprotonated, and PLP as well as a carboxyl group, most likely of Glu637, protonated. A system of hydrogen bonds which shows large proton polarizability due to collective proton tunneling was observed connecting Lys533, PLP, and Glc-1-MeP. A comparison with model systems shows, furthermore, that this hydrogen bonded chain is highly sensitive to local electrical fields and specific interactions, respectively. In the binary complex the proton limiting structure with by far the highest probability is the one in which Glc-1-MeP is singly protonated. In a second hydrogen bonded chain the proton of Lys539 is shifted to Glu637. In the binary complex the proton remains located at Glu637. In the ternary complex composed of phosphorylase, glucose-1-phosphate (Glc-1-P), and the nonreducing end of a polysaccharide chain (primer), a second proton may be shifted to the phosphate group of Glc-1-P. In the doubly protonated phosphate group the loss of mesomeric stabilization of the phosphate ester makes the C₁–O₁ bond of Glc-1-P susceptible to bond cleavage. The arising glucosyl carbonium ion will be a substrate for nucleophilic attack by the nonreducing terminal glucose residue of the polysaccharide chain. Received: 15 June 1997 / Revised version: 15 October 1998 / Accepted: 15 October 1998     

Yeast surviving factor Svf1 as a new interacting partner, regulator and in vitro substrate of protein kinase CK2

Since Svf1 is phosphoprotein, we investigated whether it was a substrate for protein kinase CK2. According to the amino acid sequence Svf1 harbours 20 putative CK2 phosphorylation sites. Here, we have reported cloning, overexpression, purification and characterization of yeast Svf1 as a substrate for three forms of yeast CK2. Svf1 serves as a substrate for both the recombinant CK2α (K _m 0.35 μM) and CK2α′ (K _m 0.18 μM) as well as CK2 holoenzyme (K _m 1.1 μM). Different K _m values argue that CK2β(β′) subunit has an inhibitory effect on the activity of both CK2α and CK2α′ towards surviving factor Svf1. Reconstitution of α′₂ββ′ isoform of CK2 holoenzyme shows that β/β′ subunits have regulatory effect depending on the kind of CK2 catalytic subunit. This effect was not observed in the case of α₂ββ′ isoform, which may be due to interaction between Svf1 and regulatory CK2β subunit (shown by co-immunoprecipitation experiments). Interactions between CK2 subunits and Svf1 protein may have influence on ATP as well as ATP-competitive inhibitors (TBBt and TBBz) binding. CK2 phosphorylates up to six serine residues in highly acidic peptide K¹⁹⁹EVIPESDEEESSADEDDNEDEDEESGDSEEESGSEEESDSEEVEITYED²⁴⁸ of the Svf1 protein in vitro. Presented data may help to elucidate the role of protein kinase CK2 and Svf1 in the regulation of cell survival pathways.     

Structural and immunochemical characterization of β-1,2-linked mannobiosyl phosphate residue in the cell wall mannan of Candida glabrata

A mannan of Candida glabrata IFO 0622 digested by Arthrobacter exo-α-mannosidase and a β-1,2-linked mannobiose obtained from the parent mannan by acid treatment was analyzed using ¹³C nuclear magnetic resonance spectroscopy. The results show that the β-1,2-linked mannobiosyl residue is esterified to a phosphate group through position C-1 in the α-configuration, Manβ1– 2Manα1–HPO₃–. The results of immunochemical assays of these mannans using the commercial antigenic factor sera of the genus Candida (Candida Check, Iatron) indicate that the main recognition site of serum no. 6 in this kit is the mannotetraosyl side-chain Manβ1–2Manα1– 2Manα1–2Man in C. glabrata mannan and also suggest that the phosphate-containing unit (such as Manβ1– 2Manα1–HPO₃– in this mannan) behaves as one of the antigenic determinants of serum no. 6, but not of serum no. 5. Therefore, the present and previous findings indicate that serum no. 5 recognizes relatively longer β-1,2-linked oligomannosyl side-chains, Manβ1–[2Manβ1–]_n 2Man (n = 1–6), attached to the phosphate groups previously observed in the cell wall mannans of Candida albicans, Candida stellatoidea, and Candida tropicalis. Received: 18 March 1997 / Accepted: 16 September 1997     

Solution NMR structures reveal a distinct architecture and provide first structures for protein domain family PF04536

The protein family (Pfam) PF04536 is a broadly conserved domain family of unknown function (DUF477), with more than 1,350 members in prokaryotic and eukaryotic proteins. High-quality NMR structures of the N-terminal domain comprising residues 41–180 of the 684-residue protein CG2496 from Corynebacterium glutamicum and the N-terminal domain comprising residues 35–182 of the 435-residue protein PG0361 from Porphyromonas gingivalis both exhibit an α/β fold comprised of a four-stranded β-sheet, three α-helices packed against one side of the sheet, and a fourth α-helix attached to the other side. In spite of low sequence similarity (18%) assessed by structure-based sequence alignment, the two structures are globally quite similar. However, moderate structural differences are observed for the relative orientation of two of the four helices. Comparison with known protein structures reveals that the α/β architecture of CG2496(41–180) and PG0361(35–182) has previously not been characterized. Moreover, calculation of surface charge potential and identification of surface clefts indicate that the two domains very likely have different functions.     

Novel Nociceptin Analogues: Synthesis and Biological Activity

Syntheses are described of the nociceptin (1–13) amide [NC(1–13)-NH₂] and of several analogues in which either one or both the phenylalanine residues (positions 1 and 4), the arginine residues (positions 8 and 12) and the alanine residues (positions 7 and 11) have been replaced by N-benzyl-glycine, N-(3-guanidino-propyl)-glycine and β-alanine, respectively. The preparation is also described of NC(1–13)-NH₂ analogues in which either galactose or N-acetyl-galactosamine are β-O-glycosidically linked to Thr⁵ and/or to Ser¹⁰. Preliminary pharmacological experiments on mouse vas deferens preparations showed that Phe⁴, Thr⁵, Ala⁷ and Arg⁸ are crucial residues for OP₄ receptor activation. Manipulation of Phe¹ yielded peptides endowed with antagonist activity but [Nphe¹] NC(1–13)-NH₂ acted as an antagonist still possessing weak agonist activity. Introduction of the βAla residue either in position 7 or 11 of the [Nphe¹] NC(1–13)-NH₂ sequence, abolished any residual agonist activity and [Nphe¹, βAla⁷] NC(1–13)-NH₂ and [Nphe¹, βAla¹¹] NC(1–13)-NH₂ acted as competitive antagonists only. Modification of both Ala⁷ and Ala¹¹ abolished the antagonist activity of [Nphe¹]NC(1–13)-NH₂ probably by hindering receptor binding. Changes at positions 10 and 11 gave analogues still possessing agonist activity. [Ser(βGal)¹⁰] NC(1–13)-NH₂ displayed an activity comparable with that of NC(1–13)-NH₂, [Ser(βGalNAc)¹⁰] NC(1–13)-NH₂ and [βAla¹¹] NC(1–13)-NH₂ were five and 10 times less active, respectively.The α-amino acid residues are of the l-configuration. Standard abbreviations for amino acid derivatives and peptides are according to the suggestions of the IUPAC-IUB Commission on Biochemical Nomeclature (1984), Eur. J. Biochem. 138, 9–37. Abbreviations listed in the guide published in (2003), J. Peptide Sci. 9, 1–8 are used without explanation.     

Characterization of a novel ginsenoside-hydrolyzing α-l-arabinofuranosidase, AbfA, from Rhodanobacter ginsenosidimutans Gsoil 3054T

The gene encoding an α-l-arabinofuranosidase that could biotransform ginsenoside Rc {3-O-[β-d-glucopyranosyl-(1–2)-β-d-glucopyranosyl]-20-O-[α-l-arabinofuranosyl-(1–6)-β-d-glucopyranosyl]-20(S)-protopanaxadiol} to ginsenoside Rd {3-O-[β-d-glucopyranosyl-(1–2)-β-d-glucopyranosyl]-20-O-β-d-glucopyranosyl-20(S)-protopanaxadiol} was cloned from a soil bacterium, Rhodanobacter ginsenosidimutans strain Gsoil 3054^T, and the recombinant enzyme was characterized. The enzyme (AbfA) hydrolyzed the arabinofuranosyl moiety from ginsenoside Rc and was classified as a family 51 glycoside hydrolase based on amino acid sequence analysis. Recombinant AbfA expressed in Escherichia coli hydrolyzed non-reducing arabinofuranoside moieties with apparent K _m values of 0.53 ± 0.07 and 0.30 ± 0.07 mM and V _max values of 27.1 ± 1.7 and 49.6 ± 4.1 μmol min⁻¹ mg⁻¹ of protein for p-nitrophenyl-α-l-arabinofuranoside and ginsenoside Rc, respectively. The enzyme exhibited preferential substrate specificity of the exo-type mode of action towards polyarabinosides or oligoarabinosides. AbfA demonstrated substrate-specific activity for the bioconversion of ginsenosides, as it hydrolyzed only arabinofuranoside moieties from ginsenoside Rc and its derivatives, and not other sugar groups. These results are the first report of a glycoside hydrolase family 51 α-l-arabinofuranosidase that can transform ginsenoside Rc to Rd.     

Top-down approach in protein RDC data analysis: <Emphasis Type="Italic">de novo</Emphasis> estimation of the alignment tensor

In solution NMR spectroscopy the residual dipolar coupling (RDC) is invaluable in improving both the precision and accuracy of NMR structures during their structural refinement. The RDC also provides a potential to determine protein structure de novo. These procedures are only effective when an accurate estimate of the alignment tensor has already been made. Here we present a top–down approach, starting from the secondary structure elements and finishing at the residue level, for RDC data analysis in order to obtain a better estimate of the alignment tensor. Using only the RDCs from N–H bonds of residues in α-helices and CA–CO bonds in β-strands, we are able to determine the offset and the approximate amplitude of the RDC modulation-curve for each secondary structure element, which are subsequently used as targets for global minimization. The alignment order parameters and the orientation of the major principal axis of individual helix or strand, with respect to the alignment frame, can be determined in each of the eight quadrants of a sphere. The following minimization against RDC of all residues within the helix or strand segment can be carried out with fixed alignment order parameters to improve the accuracy of the orientation. For a helical protein Bax, the three components A _xx , A _yy and A _zz , of the alignment order can be determined with this method in average to within 2.3% deviation from the values calculated with the available atomic coordinates. Similarly for β-sheet protein Ubiquitin they agree in average to within 8.5%. The larger discrepancy in β-strand parameters comes from both the diversity of the β-sheet structure and the lower precision of CA–CO RDCs. This top-down approach is a robust method for alignment tensor estimation and also holds a promise for providing a protein topological fold using limited sets of RDCs.     

Identification of Phosphate Binding Residues of <Emphasis Type="Italic">Escherichia coli</Emphasis> ATP Synthase

Four positively-charged residues, namely βLys-155, βArg-182, βArg-246, and αArg-376 have been identified as Pi binding residues in Escherichia coli ATP synthase. They form a triangular Pi binding site in catalytic site βE where substrate Pi initially binds for ATP synthesis in oxidative phosphorylation. Positive electrostatic charge in the vicinity of βArg-246 is shown to be one important component of Pi binding.     

Effect of Auxiliary Substances on Complexation Efficiency and Intrinsic Dissolution Rate of Gemfibrozil–β-CD Complexes

The studies reported in this work are aimed to elucidate the ternary inclusion complex formation of gemfibrozil (GFZ), a poorly water-soluble drug, with β-cyclodextrin (β-CD) with the aid of auxiliary substances like different grades of povidone(s) (viz. PVP K-29/32, PVP K-40, Plasdone S-630, and Polyplasdone XL), organic base (viz. triethanolamine), and metal ion (viz. MgCl₂·6H₂O), by investigating their interactions in solution and solid state. Phase solubility studies were carried out to evaluate the solubilizing power of β-cyclodextrin, in association with various auxiliary substances, to determine the apparent stability constant (K _C) and complexation efficiency (CE) of complexes. Improvement in K _C values for ternary complexes clearly proves the benefit of the addition of auxiliary substances to promote CE. Of all the approaches used, the use of polymer Plasdone S-630 was found to be the most promising approach in terms of optimum CE and K _C. GFZ–β-CD (1:1) binary and ternary systems were prepared by kneading and lyophilization methods. The ternary systems clearly signified superiority over binary systems in terms of CE, solubility, K _C, and reduction in the formulation bulk. Optimized ternary system of GFZ–β-CD–Plasdone S-630 prepared by using lyophilization method indicated a significant improvement in intrinsic dissolution rate when compared with ternary kneaded system. Differential scanning calorimetry, X-ray diffraction, Fourier transform infrared, scanning electron microscopy, and proton nuclear magnetic resonance were carried out to characterize the binary and optimized ternary complex. The results suggested the formation of new solid phases, eliciting strong evidences of ternary inclusion complex formation between GFZ, β-CD, and Plasdone S-630, particularly for lyophilized products.     

The Hormonal Control of Uterine Luminal Fluid Secretion and Absorption

GABA_A receptors composed of α, β and γ subunits display a significantly higher single-channel conductance than receptors comprised of only α and β subunits. The pore of GABA_A receptors is lined by the second transmembrane region from each of its five subunits and includes conserved threonines at the 6′, 10′ and 13′ positions. At the 2′ position, however, a polar residue is present in the γ subunit but not the α or β subunits. As residues at the 2′, 6′ and 10′ positions are exposed in the open channel and as such polar channel-lining residues may interact with permeant ions by substituting for water interactions, we compared both the single-channel conductance and the kinetic properties of wild-type α1β1 and α1β1γ2S receptors with two mutant receptors, αβγ(S2′A) and αβγ(S2′V). We found that the single-channel conductance of both mutant αβγ receptors was significantly decreased with respect to wild-type αβγ, with the presence of the larger valine side chain having the greatest effect. However, the conductance of the mutant αβγ receptors remained larger than wild-type αβ channels. This reduction in the conductance of mutant αβγ receptors was observed at depolarized potentials only (E_Cl = −1.8 mV), which revealed an asymmetry in the ion conduction pathway mediated by the γ2′ residue. The substitutions at the γ2′ serine residue also altered the gating properties of the channel in addition to the effects on the conductance with the open probability of the mutant channels being decreased while the mean open time increased. The data presented in this study show that residues at the 2′ position in M2 of the γ subunit affects both single-channel conductance and receptor kinetics.     

Glycoconjugate histochemistry of the digestive tract of <Emphasis Type="Italic">Triturus carnifex</Emphasis> (Amphibia,Caudata)

Summary In this study, the variety of sugar residues in the gut glycoconjugates of Triturus carnifex (Amphibia, Caudata) are investigated by carbohydrate conventional histochemistry and lectin histochemistry. The oesophageal surface mucous cells contained acidic glycoconjugates, with residues of GalNAc, Gal β1,3 GalNAc and (GlcNAc β1,4) _n oligomers. The gastric surface cells mainly produced neutral glycoproteins with residues of fucose, Gal β1-3 GalNAc, Gal-αGal, and (GlcNAc β1,4) _n oligomers in N- and O-linked glycans, as the glandular mucous neck cells, with residues of mannose/glucose, GalNAc, Gal β1,3 GalNAc, (GlcNAc β1,4) _n oligomers and fucose linked α1,6 or terminal α1,3 or α1,4 in O-linked glycans. The oxynticopeptic tubulo-vesicular system contained neutral glycoproteins with N- and O-linked glycans with residues of Gal-αGal, Gal β1-3 GalNAc and (GlcNAc β1,4) _n oligomers; Fuc linked α1,2 to Gal, α1,3 to GlcNAc in (poly)lactosamine chains and α1,6 to GlcNAc in N-linked glycans. Most of these glycoproteins probably corresponds to the H⁺K⁺-ATPase β-subunit. The intestinal goblet cells contained acidic glycoconjugates, with residues of GalNAc, mannose/ glucose, (GlcNAc β1,4) _n oligomers and fucose linked α1,2 to Gal in O-linked oligosaccharides. The different composition of the mucus in the digestive tracts may be correlated with its different functions. In fact the presence of abundant sulphation of glycoconjugates, mainly in the oesophagus and intestine, probably confers resistance to bacterial enzymatic degradation of the mucus barrier.     

Purification and characterization of the sunflower seed (Helianthus annuus L.) major aminopeptidase

The sunflower seed (Helianthus annuus L.) major peptidase was purified to molecular homogeneity. It is an 80 kDa enzyme with pI of 4.6 and optimal activity at pH 7.5–8.0 and 45–50°C. It is a thiol-dependent aminopeptidase hydrolyzing peptides in a step-by-step manner as cleaving after the N-terminal amino acid residue of the substrate. It requires substrate acyl parts with a free amino group in either α- or β-position and l-configuration of the adjacent carbon atom. The enzyme prefers amino acid residues with bulky hydrophobic side chains at P₁-position and its catalytic efficacy is affected by the structure of both P₁ and P₁′ parts of the substrate.     

A novel xyloglucan-specific endo-β-1,4-glucanase: biochemical properties and inhibition studies

A novel xyloglucan-specific endo-β-1,4-glucanase gene (xeg5A) was isolated, cloned, and expressed in Esherichia coli. The enzyme XEG5A consisted of a C-terminal catalytic domain and N-terminal sequence of ~90 amino acid residues with unknown function. The catalytic domain assumed an (α/β)₈-fold typical of glycoside hydrolase (GH) family 5, with the two catalytic residues Glu240 and Glu362 located on opposite sides of the surface groove of the molecule. The recombinant enzyme showed high specificity towards tamarind xyloglucan and decreasing activity towards xyloglucan oligosaccharide (HDP-XGO), carboxymethyl cellulose, and lichenan. Tamarind xyloglucan was hydrolyzed to three major fragments, XXXG, XXLG/XLXG, and XLLG. The hydrolysis followed the Michaelis–Menten kinetics, yielding K _m and V _max of 3.61 ± 0.23 mg/ml and 0.30 ± 0.01 mg/ml/min, respectively. However, the hydrolysis of HDP-XGO showed a decrease in the rate at high concentrations suggesting appearance of excess substrate inhibition. The addition of XXXG resulted in linear noncompetitive inhibition on the hydrolysis of tamarind xyloglucan giving a K _i of 1.46 ± 0.13 mM. The enzyme was devoid of transglycosylase activities.     

Isoforms of human O-GlcNAcase show distinct catalytic efficiencies

O-GlcNAcase (OGA) is a family 84 glycoside hydrolase catalyzing the hydrolytic cleavage of O-linked β-N-acetylglucosamine (O-GlcNAc) from serine and threonine residues of proteins. Thus far, three forms of OGA have been identified in humans. Here we optimized the expression of these isoforms in E. coli and characterized their kinetic properties. Using Geno 3D, we predicted that N-terminal amino acids 63–342 form the catalytic site for O-GlcNAc removal and characterized it. Large differences are observed in the K _m value and catalytic efficiency (k _cat/K _m) for the three OGA variants, though all of them displayed O-GlcNAc hydrolase activity. The full-length OGA had the lowest K _m value of 0.26 mM and the highest catalytic efficiency of 3.51·10³. These results reveal that the N-terminal region (a.a. 1–350) of OGA contains the catalytic site for glycoside hydrolase and the C-terminal region of the coding sequence has the ability to stabilize the native three-dimensional structure and further affect substrate affinity.     

Surface plasmon resonance imaging for real-time, label-free analysis of protein interactions with carbohydrate microarrays

Plant lectin recognition of glycans was evaluated by SPR imaging using a model array of N-biotinylated aminoethyl glycosides of β-d-glucose (negative control), α-d-mannose (conA-responsive), β-d-galactose (RCA₁₂₀-responsive) and N-acetyl-β-d-glucosamine (WGA-responsive) printed onto neutravidin-coated gold chips. Selective recognition of the cognate ligand was observed when RCA₁₂₀ was passed over the array surface. Limited or no binding was observed for the non-cognate ligands. SPR imaging of an array of 40 sialylated and unsialylated glycans established the binding preference of hSiglec7 for α2-8-linked disialic acid structures over α2-6-sialyl-LacNAcs, which in turn were recognized and bound with greater affinity than α2-3-sialyl-LacNAcs. Affinity binding data could be obtained with as little as 10–20 μg of lectin per experiment. The SPR imaging technique was also able to establish selective binding to the preferred glycan ligand when analyzing crude culture supernatant containing 10–20 μg of recombinant hSiglec7-Fc. Our results show that SPR imaging provides results that are in agreement with those obtained from fluorescence based carbohydrate arrays but with the added advantage of label-free analysis.     

Better prediction of protein contact number using a support vector regression analysis of amino acid sequence

Background  

Protein tertiary structure can be partly characterized via each amino acid's contact number measuring how residues are spatially arranged. The contact number of a residue in a folded protein is a measure of its exposure to the local environment, and is defined as the number of C _β atoms in other residues within a sphere around the C _β atom of the residue of interest. Contact number is partly conserved between protein folds and thus is useful for protein fold and structure prediction. In turn, each residue's contact number can be partially predicted from primary amino acid sequence, assisting tertiary fold analysis from sequence data. In this study, we provide a more accurate contact number prediction method from protein primary sequence.     

Dissimilatory sulfite reductase from Archaeoglobus profundus and Desulfotomaculum thermocisternum: phylogenetic and structural implications from gene sequences

The genes encoding the α- and β-subunits of dissimilatory sulfite reductase, dsrAB, from the hyper-thermophilic archaeon Archaeoglobus profundus and the thermophilic gram-positive bacterium Desulfotomaculum thermocisternum were cloned and sequenced. The dsrAB genes are contiguous, and most probably comprise an operon also including a dsrD homolog, a conserved gene of unknown function located downstream of dsrAB in all four sulfate reducers so far sequenced. Sequence comparison confirms that dissimilatory sulfite reductase, Dsr, is a highly conserved enzyme. A phylogenetic analysis using the available Dsr sequences, including Dsr-like proteins from nonsulfate reducers, suggests a paralogous origin of the α- and β-subunits. Furthermore, the Dsr from sulfate reducers forms a separate cluster, with Dsr from the bacterial sulfate reducers Desulfotomaculum thermocisternum and Desulfovibrio vulgaris branching together, next to Dsr from Archaeoglobus profundus and Archaeoglobus fulgidus. Based on an alignment with the assimilatory sulfite reductase from Escherichia coli, the amino acid residues involved in binding of sulfite, siroheme, and [Fe₄S₄]-clusters have been tentatively identified, which is consistent with the binding of two sirohemes and four [Fe₄S₄]-clusters per α₂β₂ structure. The evolution of Dsr and the structural basis for the binding of substrate and cofactors are discussed. Received: May 1, 1998 / Accepted: August 10, 1998     

Critical roles of Asp270 and Trp273 in the α-repeat of the carbohydrate-binding module of endo-1,3-β-glucanase for laminarin-binding avidity

A carbohydrate-binding module from family 13 (CBM13), appended to the catalytic domain of endo-1,3-β-glucanase from Cellulosimicrobium cellulans, was overexpressed in E. coli, and its interactions with β-glucans, laminarin and laminarioligosaccharides, were analyzed using surface plasmon resonance biosensor and isothermal titration calorimetry. The association constants for laminarin and laminarioligosaccharides were determined to be approximately 10⁶ M⁻¹ and 10⁴ M⁻¹, respectively, indicating that 2 or 3 binding sites in the α-, β-, and γ-repeats of CBM13 are involved in laminarin binding in a cooperative manner. The binding avidity is approximately 2-orders higher than the monovalent binding affinity. Mutational analysis of the conserved Asp residues in the respective repeats showed that the α-repeat primarily contributes to β-glucan binding. A Trp residue is predicted to be exposed to the solvent only in the α-repeat and would contribute to β-glucan binding. The α-repeat bound β-glucan with an affinity of approximately 10⁴ M⁻¹, and the other repeats additionally bound laminarin, resulting in the increased binding avidity. This binding is unique compared to the recognition mode of another CBM13 from Streptomyces lividans xylanase.