Residue Leu940 Has a Crucial Role in the Linkage and Reaction Specificity of the Glucansucrase GTF180 of the Probiotic Bacterium Lactobacillus reuteri 180

Highly conserved glycoside hydrolase family 70 glucansucrases are able to catalyze the synthesis of α-glucans with different structure from sucrose. The structural determinants of glucansucrase specificity have remained unclear. Residue Leu⁹⁴⁰ in domain B of GTF180, the glucansucrase of the probiotic bacterium Lactobacillus reuteri 180, was shown to vary in different glucansucrases and is close to the +1 glucosyl unit in the crystal structure of GTF180-ΔN in complex with maltose. Herein, we show that mutations in Leu⁹⁴⁰ of wild-type GTF180-ΔN all caused an increased percentage of (α1→6) linkages and a decreased percentage of (α1→3) linkages in the products. α-Glucans with potential different physicochemical properties (containing 67–100% of (α1→6) linkages) were produced by GTF180 and its Leu⁹⁴⁰ mutants. Mutant L940W was unable to form (α1→3) linkages and synthesized a smaller and linear glucan polysaccharide with only (α1→6) linkages. Docking studies revealed that the introduction of the large aromatic amino acid residue tryptophan at position 940 partially blocked the binding groove, preventing the isomalto-oligosaccharide acceptor to bind in an favorable orientation for the formation of (α1→3) linkages. Our data showed that the reaction specificity of GTF180 mutant was shifted either to increased polysaccharide synthesis (L940A, L940S, L940E, and L940F) or increased oligosaccharide synthesis (L940W). The L940W mutant is capable of producing a large amount of isomalto-oligosaccharides using released glucose from sucrose as acceptors. Thus, residue Leu⁹⁴⁰ in domain B is crucial for linkage and reaction specificity of GTF180. This study provides clear and novel insights into the structure-function relationships of glucansucrase enzymes.     

Characterization of the Different Dextransucrase Activities Excreted in Glucose, Fructose, or Sucrose Medium by Leuconostoc mesenteroides NRRL B-1299

When grown in glucose or fructose medium in the absence of sucrose, Leuconostoc mesenteroides NRRL B-1299 produces two distinct extracellular dextransucrases named glucose glucosyltransferase (GGT) and fructose glucosyltransferase (FGT). The production level of GGT and FGT is 10 to 20 times lower than that of the extracellular dextransucrase sucrose glucosyltransferase (SGT) produced on sucrose medium (traditional culture conditions). GGT and FGT were concentrated by ultrafiltration before sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Their molecular masses were 183 and 186 kDa, respectively, differing from the 195 kDa of SGT. The structural analysis of the dextran produced from sucrose and of the oligosaccharides synthesized by acceptor reaction in the presence of maltose showed that GGT and FGT are two different enzymes not previously described for this strain. The polymer synthesized by GGT contains 30% α(1→2) linkages, while FGT catalyzes the synthesis of a linear dextran only composed of α(1→6) linkages.     

Phosphorylated α(1→4)Glucans as Substrate for Potato Starch-Branching Enzyme I

The possible involvement of potato (Solanum tuberosum L.) starch-branching enzyme I (PSBE-I) in the in vivo synthesis of phosphorylated amylopectin was investigated in in vitro experiments with isolated PSBE-I using ³³P-labeled phosphorylated and ³H end-labeled nonphosphorylated α(1→4)glucans as the substrates. From these radiolabeled substrates PSBE-I was shown to catalyze the formation of dual-labeled (³H/³³P) phosphorylated branched polysaccharides with an average degree of polymerization of 80 to 85. The relatively high molecular mass indicated that the product was the result of multiple chain-transfer reactions. The presence of α(1→6) branch points was documented by isoamylase treatment and anion-exchange chromatography. Although the initial steps of the in vivo mechanism responsible for phosphorylation of potato starch remains elusive, the present study demonstrates that the enzyme machinery available in potato has the ability to incorporate phosphorylated α(1→4)glucans into neutral polysaccharides in an interchain catalytic reaction. Potato mini tubers synthesized phosphorylated starch from exogenously supplied ³³PO₄³⁻ and [U-¹⁴C]Glc at rates 4 times higher than those previously obtained using tubers from fully grown potato plants. This system was more reproducible compared with soil-grown tubers and was therefore used for preparation of ³³P-labeled phosphorylated α(1→4)glucan chains.     

Structure-Function Relationships of Glucansucrase and Fructansucrase Enzymes from Lactic Acid Bacteria

Lactic acid bacteria (LAB) employ sucrase-type enzymes to convert sucrose into homopolysaccharides consisting of either glucosyl units (glucans) or fructosyl units (fructans). The enzymes involved are labeled glucansucrases (GS) and fructansucrases (FS), respectively. The available molecular, biochemical, and structural information on sucrase genes and enzymes from various LAB and their fructan and α-glucan products is reviewed. The GSand FS enzymes are both glycoside hydrolase enzymes that act on the same substrate (sucrose) and catalyze (retaining) transglycosylation reactions that result in polysaccharide formation, but they possess completely different protein structures. GS enzymes (family GH70) are large multidomain proteins that occur exclusively in LAB. Their catalytic domain displays clear secondary-structure similarity with α-amylase enzymes (family GH13), with a predicted permuted (β/α)₈ barrel structure for which detailed structural and mechanistic information is available. Emphasis now is on identification of residues and regions important for GS enzyme activity and product specificity (synthesis of α-glucans differing in glycosidic linkage type, degree and type of branching, glucan molecular mass, and solubility). FS enzymes (family GH68) occur in both gram-negative and gram-positive bacteria and synthesize β-fructan polymers with either β-(2→6) (inulin) or β-(2→1) (levan) glycosidic bonds. Recently, the first high-resolution three-dimensional structures have become available for FS (levansucrase) proteins, revealing a rare five-bladed β-propeller structure with a deep, negatively charged central pocket. Although these structures have provided detailed mechanistic insights, the structural features in FS enzymes dictating the synthesis of either β-(2→6) or β-(2→1) linkages, degree and type of branching, and fructan molecular mass remain to be identified.     

Action of Endo-(1 → 6)-α-d-glucanases on the soluble dextrans produced by three extracellular α-d-glucosyltransferases of Streptococcus sobrinus

Four different α-d-glucosyltransferases (GTF) have been obtained from culture filtrates of Streptococcus sobrinus strains grown in the chemostat at pH 6·5 in complex medium supplemented with Tween 80. Three of the enzymes, GTF-S1, GTF-S3 and GTF-S4, converted sucrose into soluble glucans. Their limit of hydrolysis with endodextranase, the proportion of linear to branched oligosaccharides among the end products of enzymic degradation, and methylation analysis, all supported the view that the glucans were dextrans. The S1-dextrans were highly branched (32% of α-(1 → 3)-branch points), S3-dextrans were linear, and the branching of S4-dextrans was intermediate in value (9%). The enzymes that catalyze the synthesis of three such diverse dextrans were thus proved to be three different GTF, each with a characteristic specificity. Conditions of growth in the chemostat could be varied to provide maximum yields of either GTF-S1, -S3 or -S4.     

Purification and Characterization of Wheat beta(2-->1) Fructan:Fructan Fructosyl Transferase Activity

Fructans are the major storage carbohydrate in vegetative tissues of wheat (Triticum aestivum L.). Fructan:fructan fructosyl transferase (FFT) catalyzes fructosyl transfer between fructan molecules to elongate the fructan chain. The objective of this research was to isolate this activity in wheat. Wheat (cv Caldwell) plants grown at 25°C for 3 weeks were transferred to 10°C to induce fructan synthesis. From the leaf blades kept at 10°C for 4 days, fructosyl transferase activity was purified using salt precipitation and a series of chromatographic procedures including size exclusion, anion-exchange, and affinity chromatography. The transferase activity was free from invertase and other fructan-metabolizing activities. Fructosyl transferase had a broad pH spectrum with a peak activity at 6.5. The temperature optimum was 30°C. The activity was specific for fructosyl transfer from β(2→1)-linked 1-kestose or fructan to sucrose and β(2→1) fructosyl transfer to other fructans (1-FFT). Fructosyl transfer from oligofructans to sucrose was most efficient when 1-kestose was used as donor molecule and declined as the degree of polymerization of the donor increased from 3 to 5. 1-FFT catalyzed the in vitro synthesis of inulin tetra- and penta-saccharides from 1-kestose; however, formation of the tetrasaccharide was greatly reduced at high sucrose concentration. 6-Kestose could not act as donor molecule, but could accept a fructosyl moiety from 1-kestose to produce bifurcose and a tetrasaccharide having a β(2→1) fructose attached to the terminal fructose of 6-kestose. The role of this FFT activity in the synthesis of fructan in wheat is discussed.     

Characterization of the Functional Roles of Amino Acid Residues in Acceptor-binding Subsite +1 in the Active Site of the Glucansucrase GTF180 from Lactobacillus reuteri 180

α-Glucans produced by glucansucrase enzymes hold strong potential for industrial applications. The exact determinants of the linkage specificity of glucansucrase enzymes have remained largely unknown, even with the recent elucidation of glucansucrase crystal structures. Guided by the crystal structure of glucansucrase GTF180-ΔN from Lactobacillus reuteri 180 in complex with the acceptor substrate maltose, we identified several residues (Asp-1028 and Asn-1029 from domain A, as well as Leu-938, Ala-978, and Leu-981 from domain B) near subsite +1 that may be critical for linkage specificity determination, and we investigated these by random site-directed mutagenesis. First, mutants of Ala-978 (to Leu, Pro, Phe, or Tyr) and Asp-1028 (to Tyr or Trp) with larger side chains showed reduced degrees of branching, likely due to the steric hindrance by these bulky residues. Second, Leu-938 mutants (except L938F) and Asp-1028 mutants showed altered linkage specificity, mostly with increased (α1→6) linkage synthesis. Third, mutation of Leu-981 and Asn-1029 significantly affected the transglycosylation reaction, indicating their essential roles in acceptor substrate binding. In conclusion, glucansucrase product specificity is determined by an interplay of domain A and B residues surrounding the acceptor substrate binding groove. Residues surrounding the +1 subsite thus are critical for activity and specificity of the GTF180 enzyme and play different roles in the enzyme functions. This study provides novel insights into the structure-function relationships of glucansucrase enzymes and clearly shows the potential of enzyme engineering to produce tailor-made α-glucans.     

Messenger RNAs from the Scutellum and Aleurone of Germinating Barley Encode (1-->3,1-->4)-beta-d-Glucanase, alpha-Amylase and Carboxypeptidase

Polyclonal antibodies raised against barley (1→3,1→4)-β-d-glucanase, α-amylase and carboxypeptidase were used to detect precursor polypeptides of these hydrolytic enzymes among the in vitro translation products of mRNA isolated from the scutellum and aleurone of germinating barley. In the scutellum, mRNA encoding carboxypeptidase appeared to be relatively more abundant than that encoding α-amylase or (1→3,1→4)-β-d-glucanase, while in the aleurone α-amylase and (1→3,1→4)-β-d-glucanase mRNAs predominated. The apparent molecular weights of the precursors for (1→3,1→4)-β-d-glucanase, α-amylase, and carboxypeptidase were 33,000, 44,000, and 35,000, respectively. In each case these are slightly higher (1,500-5,000) than molecular weights of the mature enzymes. Molecular weights of precursors immunoprecipitated from aleurone and scutellum mRNA translation products were identical for each enzyme.     

Highly Hydrolytic Reuteransucrase from Probiotic Lactobacillus reuteri Strain ATCC 55730

Lactobacillus reuteri strain ATCC 55730 (LB BIO) was isolated as a pure culture from a Reuteri tablet purchased from the BioGaia company. This probiotic strain produces a soluble glucan (reuteran), in which the majority of the linkages are of the α-(1→4) glucosidic type (~70%). This reuteran also contains α-(1→6)- linked glucosyl units and 4,6-disubstituted α-glucosyl units at the branching points. The LB BIO glucansucrase gene (gtfO) was cloned and expressed in Escherichia coli, and the GTFO enzyme was purified. The recombinant GTFO enzyme and the LB BIO culture supernatants synthesized identical glucan polymers with respect to linkage type and size distribution. GTFO thus is a reuteransucrase, responsible for synthesis of this reuteran polymer in LB BIO. The preference of GTFO for synthesizing α-(1→4) linkages is also evident from the oligosaccharides produced from sucrose with different acceptor substrates, e.g., isopanose from isomaltose. GTFO has a relatively high hydrolysis/transferase activity ratio. Complete conversion of 100 mM sucrose by GTFO nevertheless yielded large amounts of reuteran, although more than 50% of sucrose was converted into glucose. This is only the second example of the isolation and characterization of a reuteransucrase and its reuteran product, both found in different L. reuteri strains. GTFO synthesizes a reuteran with the highest amount of α-(1→4) linkages reported to date.     

Replicative DNA Polymerase δ but Not ε Proofreads Errors in Cis and in Trans

It is now well established that in yeast, and likely most eukaryotic organisms, initial DNA replication of the leading strand is by DNA polymerase ε and of the lagging strand by DNA polymerase δ. However, the role of Pol δ in replication of the leading strand is uncertain. In this work, we use a reporter system in Saccharomyces cerevisiae to measure mutation rates at specific base pairs in order to determine the effect of heterozygous or homozygous proofreading-defective mutants of either Pol ε or Pol δ in diploid strains. We find that wild-type Pol ε molecules cannot proofread errors created by proofreading-defective Pol ε molecules, whereas Pol δ can not only proofread errors created by proofreading-defective Pol δ molecules, but can also proofread errors created by Pol ε-defective molecules. These results suggest that any interruption in DNA synthesis on the leading strand is likely to result in completion by Pol δ and also explain the higher mutation rates observed in Pol δ-proofreading mutants compared to Pol ε-proofreading defective mutants. For strains reverting via AT→GC, TA→GC, CG→AT, and GC→AT mutations, we find in addition a strong effect of gene orientation on mutation rate in proofreading-defective strains and demonstrate that much of this orientation dependence is due to differential efficiencies of mispair elongation. We also find that a 3′-terminal 8 oxoG, unlike a 3′-terminal G, is efficiently extended opposite an A and is not subject to proofreading. Proofreading mutations have been shown to result in tumor formation in both mice and humans; the results presented here can help explain the properties exhibited by those proofreading mutants.     

Two β-Galactosidases from the Human Isolate Bifidobacterium breve DSM 20213: Molecular Cloning and Expression,Biochemical Characterization and Synthesis of Galacto-Oligosaccharides

Two β-galactosidases, β-gal I and β-gal II, from Bifidobacterium breve DSM 20213, which was isolated from the intestine of an infant, were overexpressed in Escherichia coli with co-expression of the chaperones GroEL/GroES, purified to electrophoretic homogeneity and biochemically characterized. Both β-gal I and β-gal II belong to glycoside hydrolase family 2 and are homodimers with native molecular masses of 220 and 211 kDa, respectively. The optimum pH and temperature for hydrolysis of the two substrates o-nitrophenyl-β-D-galactopyranoside (oNPG) and lactose were determined at pH 7.0 and 50°C for β-gal I, and at pH 6.5 and 55°C for β-gal II, respectively. The k _cat/K _m values for oNPG and lactose hydrolysis are 722 and 7.4 mM⁻¹s⁻¹ for β-gal I, and 543 and 25 mM⁻¹s⁻¹ for β-gal II. Both β-gal I and β-gal II are only moderately inhibited by their reaction products D-galactose and D-glucose. Both enzymes were found to be very well suited for the production of galacto-oligosaccharides with total GOS yields of 33% and 44% of total sugars obtained with β-gal I and β-gal II, respectively. The predominant transgalactosylation products are β-D-Galp-(1→6)-D-Glc (allolactose) and β-D-Galp-(1→3)-D-Lac, accounting together for more than 75% and 65% of the GOS formed by transgalactosylation by β-gal I and β-gal II, respectively, indicating that both enzymes have a propensity to synthesize β-(1→6) and β-(1→3)-linked GOS. The resulting GOS mixtures contained relatively high fractions of allolactose, which results from the fact that glucose is a far better acceptor for galactosyl transfer than galactose and lactose, and intramolecular transgalactosylation contributes significantly to the formation of this disaccharide.     

Characterization of the oligosaccharides from lipid-linked oligosaccharides of mung bean seedlings

Lipid-linked oligosaccharides were synthesized with the particulate enzyme preparation from mung bean (Phaseolus aureus) seedlings in the presence of GDP-[¹⁴C] mannose. The oligosaccharides were released from the lipids by mild acid hydrolysis and purified by several passages on Biogel P-4 columns. Five different oligosaccharides were purified in this way. Based on their relative elution constants (K_d) compared to a variety of standard oligosaccharides, they were sized as (mannose-acetylglucosamine) Man₇GlcNAc₂, Man₅GlcNAc₂, Man₃GlcNAc₂, Man₂GlcNAc₂, and ManGlcNAc₂. These oligosaccharides were treated with endoglucosaminidase H and α- and β-mannosidase, and the products were examined on Biogel P-4 columns. They also were subjected to a number of chemical treatments including analysis of the reducing sugar by NaB³H₄ reduction, methylation analysis, and in some cases acetolysis. From these data, the likely structures of these oligosaccharides are as follows: E, Manβ-GlcNAc-GlcNAc; D, Manα1→3Manβ-GlcNAc-GlcNAc; C, Manα1→2Manα1→3Manβ-GlcNAc-GlcNAc; B, Manα1→2Manα1→2Manα1→ 3(Manα1→6)Manβ-GlcNAc-GlcNAc; and A, Manα1→2Manα1→ 2Manα1→3(Manα1→ [Manα1→6]Manα1→6) Manβ-GlcNAc-GlcNAc. The synthesis of the Man₇GlcNAc₂ was greatly diminished when tunicamycin (10 μg/ml) was added to the incubation mixtures.     

Cell Walls of Germinating Uredospores: II. Carbohydrate Polymers

Polymeric carbohydrates in ¹⁴C-labeled germ tube and uredospore walls of Uromyces phaseoli var. typica were studied by permethylation and by enzymatic hydrolysis. The native structure of the uredospore wall limited the effectiveness of both techniques with this wall, but evidence for two distinct polysaccharides was obtained. A linear (1→3) glucan, containing minor quantities of (1→6) linkages, may account for most of the glucose in the uredospore wall. A second uredospore polymer was a glucomannan similar to one reported for other rust fungi in that it consisted of approximately equal numbers of β(1→3) and β(1→4) mannosidic linkages with glucose as a minor component at the nonreducing end. Branching, most likely by (1→6) mannose links, was low. In contrast to uredospore wall, considerably more germ tube polysaccharide was accessible to enzymes and to methylation. Methylation studies indicate that (1→3) glucose and mannose bonds occur predominantly. Evidence from hydrolysis with exo- (β)-(1→3) glucanase suggests distinct wall regions of β(1→3) glycan, highly branched by (1→6) bonds, as well as wall regions of a glucomannan with alternating (1→3) glucose and (1→3) mannose residues. Polymer heterogeneity was indicated by differences in the proportions of mannose, glucose, and galactose as reducing end groups in different solubility fractions. In germ tube walls, but not in uredospore walls, glucosamine apparently existed as part of chitin polymer as evidenced by the isolation of N,N-diacetylchitobiose from chitinase digestion.     

Enzymatic Synthesis and Characterization of Fructooligosaccharides and Novel Maltosylfructosides by Inulosucrase from Lactobacillus gasseri DSM 20604

The ability of an inulosucrase (IS) from Lactobacillus gasseri DSM 20604 to synthesize fructooligosaccharides (FOS) and maltosylfructosides (MFOS) in the presence of sucrose and sucrose-maltose mixtures was investigated after optimization of synthesis conditions, including enzyme concentration, temperature, pH, and reaction time. The maximum formation of FOS, which consist of β-2,1-linked fructose to sucrose, was 45% (in weight with respect to the initial amount of sucrose) and was obtained after 24 h of reaction at 55°C in the presence of sucrose (300 g liter⁻¹) and 1.6 U ml⁻¹ of IS–25 mM sodium acetate buffer–1 mM CaCl₂ (pH 5.2). The production of MFOS was also studied as a function of the initial ratios of sucrose to maltose (10:50, 20:40, 30:30, and 40:20, expressed in g 100 ml⁻¹). The highest yield in total MFOS was attained after 24 to 32 h of reaction time and ranged from 13% (10:50 sucrose/maltose) to 52% (30:30 sucrose/maltose) in weight with respect to the initial amount of maltose. Nuclear magnetic resonance (NMR) structural characterization indicated that IS from L. gasseri specifically transferred fructose moieties of sucrose to either C-1 of the reducing end or C-6 of the nonreducing end of maltose. Thus, the trisaccharide erlose [α-d-glucopyranosyl-(1→4)-α-d-glucopyranosyl-(1→2)-β-d-fructofuranoside] was the main synthesized MFOS followed by neo-erlose [β-d-fructofuranosyl-(2→6)-α-d-glucopyranosyl-(1→4)-α-d-glucopyranose]. The formation of MFOS with a higher degree of polymerization was also demonstrated by the transfer of additional fructose residues to C-1 of either the β-2,1-linked fructose or the β-2,6-linked fructose to maltose, revealing the capacity of MFOS to serve as acceptors.     

Intermediates in the recycling of 5-methylthioribose to methionine in fruits

The recycling of 5-methylthioribose (MTR) to methionine in avocado (Persea americana Mill, cv Hass) and tomato (Lycopersicum esculentum Mill, cv unknown) was examined. [¹⁴CH₃]MTR was not metabolized in cell free extract from avocado fruit. Either [¹⁴CH₃]MTR plus ATP or [¹⁴CH₃]5-methylthioribose-1-phosphate (MTR-1-P) alone, however, were metabolized to two new products by these extracts. MTR kinase activity has previously been detected in these fruit extracts. These data indicate that MTR must be converted to MTR-1-P by MTR kinase before further metabolism can occur. The products of MTR-1-P metabolism were tentatively identified as α-keto-γ-methylthiobutyric acid (α-KMB) and α-hydroxy-γ-methylthiobutyric acid (α-HMB) by chromatography in several solvent systems. [³⁵S]α-KMB was found to be further metabolized to methionine and α-HMB by these extracts, whereas α-HMB was not. However, α-HMB inhibited the conversion of α-KMB to methionine. Both [U-¹⁴C]α-KMB and [U-¹⁴C]methionine, but not [U-¹⁴C]α-HMB, were converted to ethylene in tomato pericarp tissue. In addition, aminoethoxyvinylglycine inhibited the conversion of α-KMB to ethylene. These data suggest that the recycling pathway leading to ethylene is MTR → MTR-1-P → α-KMB → methionine → S-adenosylmethionine → 1-aminocyclopropane-1-carboxylic acid → ethylene.     

Structure of the linear, low molecular weight dextran synthesized by a d-glucosyltransferase (GTF-S3) of Streptococcus sobrinus

Four extracellular α-d-glucosyltransferases (GTF) have been separated from cultures of Streptococcus sobrinus strains grown in continuous culture. Three of the GTF synthesized soluble dextrans from sucrose, and one of these enzymes, GTF-S3, catalysed the production of a low molecular weight, linear dextran. Methylation analysis and high field proton NMR spectroscopy on the intact S3 glucans confirmed that these dextrans were small (dp 20–30) and linear, with the majority of chains terminated with a sucrosyl moiety. Enzymic hydrolysis, followed by analytical and semi-preparative HPLC, led to the isolation of only linear oligosaccharides, one of which was identified as 6^G-glucosylsucrose.The results are accommodated by the two-site insertion mechanism for dextran synthesis proposed by Robyt et al. (1974) (Arch. Biochem. Biophys., 165, 634).     

DEMONSTRATION OF A FIBRILLAR COMPONENT IN THE CELL WALL OF THE YEAST SACCHAROMYCES CEREVISIAE AND ITS CHEMICAL NATURE

The ultrastructure of isolated cell walls of Saccharomyces cerevisiae from the log and stationary phases of growth was studied after treatment with the following enzymes: purified endo-β-(1 → 3)-glucanase and endo-β-(1 → 6)-glucanase produced by Bacillus circulans; purified exo-β-glucanase and endo-β-(1 → 3)-glucanase produced by Schizosaccharomyces versatilis; commercial Pronase. While exo-β-glucanase from S. versatilis had no electron microscopically detectable effect on the walls, Pronase removed part of the external amorphous wall material disclosing an amorphous wall layer in which fibrils were indistinctly visible. Amorphous wall material was completely removed by the effect of either endo-β-(1 → 3)- or endo-β-(1 → 6)-glucanase of B. circulans or by a mixture of the two enzymes. As a result of these treatments a continuous fibrillar component appeared, composed of densely interwoven microfibrils resisting further action by both of the B. circulans enzymes. The fibrillar wall component was also demonstrated in untreated cell walls by electron microscopy after negative staining. Because of the complete disappearance of the fibrils following treatment with the S. versatilis endo-β-(1 → 3)-glucanase it can be concluded that this fibrillar component is composed of β-(1 → 3)-linked glucan. Bud scars were the only wall structures resistant to the effect of the latter enzyme.     

Functional and structural characterization of α-(1->2) branching sucrase derived from DSR-E glucansucrase

ΔN₁₂₃-glucan-binding domain-catalytic domain 2 (ΔN₁₂₃-GBD-CD2) is a truncated form of the bifunctional glucansucrase DSR-E from Leuconostoc mesenteroides NRRL B-1299. It was constructed by rational truncation of GBD-CD2, which harbors the second catalytic domain of DSR-E. Like GBD-CD2, this variant displays α-(1→2) branching activity when incubated with sucrose as glucosyl donor and (oligo-)dextran as acceptor, transferring glucosyl residues to the acceptor via a ping-pong bi-bi mechanism. This allows the formation of prebiotic molecules containing controlled amounts of α-(1→2) linkages. The crystal structure of the apo α-(1→2) branching sucrase ΔN₁₂₃-GBD-CD2 was solved at 1.90 Å resolution. The protein adopts the unusual U-shape fold organized in five distinct domains, also found in GTF180-ΔN and GTF-SI glucansucrases of glycoside hydrolase family 70. Residues forming subsite −1, involved in binding the glucosyl residue of sucrose and catalysis, are strictly conserved in both GTF180-ΔN and ΔN₁₂₃-GBD-CD2. Subsite +1 analysis revealed three residues (Ala-2249, Gly-2250, and Phe-2214) that are specific to ΔN₁₂₃-GBD-CD2. Mutation of these residues to the corresponding residues found in GTF180-ΔN showed that Ala-2249 and Gly-2250 are not directly involved in substrate binding and regiospecificity. In contrast, mutant F2214N had lost its ability to branch dextran, although it was still active on sucrose alone. Furthermore, three loops belonging to domains A and B at the upper part of the catalytic gorge are also specific to ΔN₁₂₃-GBD-CD2. These distinguishing features are also proposed to be involved in the correct positioning of dextran acceptor molecules allowing the formation of α-(1→2) branches.     

Chromosomal Location of Genes Encoding Barley (1-->3, 1-->4)-beta-Glucan 4-Glucanohydrolases

Preparations of DNA from wheat (Triticum aestivum, cv Chinese Spring), barley (Hordeum vulgare, cv Betzes) and six euplasmic wheat-barley addition lines were digested to completion with restriction endonucleases and the products probed by Southern blot analysis using a cDNA-encoding barley (1→3, 1→4)-β-glucanase isoenzyme II. It is shown that one of the barley (1→3, 1→4)-β-glucanase genes is located on chromosome 1.     

Molecular Mechanisms and Kinetic Effects of FXYD1 and Phosphomimetic Mutants on Purified Human Na,K-ATPase

Phospholemman (FXYD1) is a single-transmembrane protein regulator of Na,K-ATPase, expressed strongly in heart, skeletal muscle, and brain and phosphorylated by protein kinases A and C at Ser-68 and Ser-63, respectively. Binding of FXYD1 reduces Na,K-ATPase activity, and phosphorylation at Ser-68 or Ser-63 relieves the inhibition. Despite the accumulated information on physiological effects, whole cell studies provide only limited information on molecular mechanisms. As a complementary approach, we utilized purified human Na,K-ATPase (α1β1 and α2β1) reconstituted with FXYD1 or mutants S63E, S68E, and S63E,S68E that mimic phosphorylation at Ser-63 and Ser-68. Compared with control α1β1, FXYD1 reduces V_max and turnover rate and raises K_0.5Na. The phosphomimetic mutants reverse these effects and reduce K_0.5Na below control K_0.5Na. Effects on α2β1 are similar but smaller. Experiments in proteoliposomes reconstituted with α1β1 show analogous effects of FXYD1 on K_0.5Na, which are abolished by phosphomimetic mutants and also by increasing mole fractions of DOPS in the proteoliposomes. Stopped-flow experiments using the dye RH421 show that FXYD1 slows the conformational transition E₂(2K)ATP → E₁(3Na)ATP but does not affect 3NaE₁P → E₂P3Na. This regulatory effect is explained simply by molecular modeling, which indicates that a cytoplasmic helix (residues 60–70) docks between the αN and αP domains in the E₂ conformation, but docking is weaker in E₁ (also for phosphomimetic mutants). Taken together with previous work showing that FXYD1 also raises binding affinity for the Na⁺-selective site III, these results provide a rather comprehensive picture of the regulatory mechanism of FXYD1 that complements the physiological studies.