Water-soluble and water-insoluble glucans produced by Escherichia coli recombinant dextransucrases from Leuconostoc mesenteroides NRRL B-512F.

Two dextransucrase genes, dsrS and dsrT5, from Leuconostoc mesenteroides NRRL B-512F were expressed in Escherichia coli, and recombinant dsrT5 dextransucrase was shown to produce a water-insoluble glucan. In contrast, native dextran from L. mesenteroides B-512F is water-soluble. The water-insoluble glucan was shown by 13C NMR and glycosyl-linkage composition analysis to contain about 50% 6-linked Glcp and 40% 3-linked Glcp. The 'primitive' B-512F strain is suggested to have produced water-insoluble glucan containing 3-linked Glcp. The glucans produced by dextransucrases expressed in E. coli contained 4-linked Glcp, as shown by glycosyl-linkage composition analysis. The amount of 4-linked Glcp was increased when the truncated, water-insoluble, glucan-producing dextransucrase, which does not have C-terminal repeating units, was added to the water-soluble, glucan-producing dextransucrase. Trace amounts of 4-linked Glcp were also detected in the dextran obtained from the B-512F culture supernatant, in dextran produced by dextransucrase purified from the B-512F strain culture supernatant, and in clinical dextran. The results of glycosyl-linkage composition analysis suggest that dextransucrases produce 4-linked Glcp as well as 6- and 3-linked Glcp.

Characterization of an isoform of rice starch branching enzyme, RBE4, in developing seeds.

cDNA clones encoding an isoform of starch branching enzyme, RBE4, have been identified from a developing rice seed cDNA library, using a synthetic oligonucleotide probe corresponding to the N-terminal amino acid sequence of RBE4. The cDNA-derived amino acid sequence indicated that RBE4 is initially produced as a precursor protein of 841 amino acids, including a 53-residue transit peptide at the N-terminus. The mature form of RBE4 shared a high degree of sequence identity (80%) with mature RBE3, and possessed an N-terminal extra sequence, as found in RBE3. Northern blot analysis demonstrated that the RBE4 gene is expressed in both leaves and developing seeds. The RBE4 gene was distinguished from the RBE1 and RBE3 genes by expression at the earlier stages of seed development. To examine enzymatic functions of RBE4, recombinant proteins were produced in Escherichia coli cells, and purified by two chromatographic separations. The branched alpha-glucans produced by the recombinant enzymes from potato amylose revealed the different patterns of oligosaccharide chain transfer. The peak of major branches of the products by RBE3 or RBE4 was 6 glucose units, whereas the peaks of major branches of the products by RBE1 were 6 and 11 glucose units. The similar property between RBE3 and RBE4 is supported by high similarity of the amino acid sequences between them.

Gene encoding a dextransucrase-like protein in Leuconostoc mesenteroides NRRL B-512F.

A gene, dsrT, encoding a dextransucrase-like protein was isolated from the genomic DNA libraries of Leuconostoc mesenteroides NRRL B-512F dextransucrase-like gene. The gene was similar to the intact open reading frames of the dextransucrase gene dsrS of L. mesenteroides NRRL B-512F, dextransucrase genes of strain NRRL B-1299 and streptococcal glucosyltransferase genes, but was truncated after the catalytic domain, apparently by the deletion of five nucleotides. dsrT mRNA was produced in this strain L. mesenteroides when cells were grown in a sucrose medum, but at a level of 20% of that of dsrS mRNA. The molecular weight of the dsrT gene product was 150,000 by SDS-PAGE. The product did not synthesize dextran, but had weak sucrose cleaving activity. The insertion of five nucleotides at the putative deletion point in dsrT resulted in an enzyme with a molecular weight of 210,000 and with dextransucrase activity.

Analysis of essential histidine residues of maize branching enzymes by chemical modification and site-directed mutagenesis.

Incubation of maize branching enzyme, mBEI and mBEII, with 100 microM diethylpyrocarbonate (DEPC) rapidly inactivated the enzymes. Treatment of the DEPC-inactivated enzymes with 100500 mM hydroxylamine restored the enzyme activities. Spectroscopic data indicated that the inactivation of BE with DEPC was the result of histidine modification. The addition of the substrate amylose or amylopectin retarded the enzyme inactivation by DEPC, suggesting that the histidine residues are important for substrate binding. In maize BEII, conserved histidine residues are in catalytic regions 1 (His320) and 4 (His508). His320 and His508 were individually replaced by Ala via site-directed mutagenesis to probe their role in catalysis. Expression of these mutants in E. coli showed a significant decrease of the activity and the mutant enzymes had Km values 10 times higher than the wild type. Therefore, residues His320 and His508 do play an important role in substrate binding.

Glucan binding regions of dextransucrase from Leuconostoc mesenteroides NRRL B-512F.

We isolated glucan-binding peptides of a dextransucrase from Leuconostoc mesenteroides B-512F. The dextransucrase was bound to DEAE-Sephadex A-50, Sephadex G-100, and mutan from Streptococcus mutans. Mild trypsin digestion dissociated the enzyme and glucan binding. In the presence of ammonium sulfate, several peptides were bound to glucan after trypsin digestion. Four main mutan-binding peptides were obtained by this method, and those amino acid sequences were analyzed. One of them was identical with the dextran-binding peptide that contains lysine, which was previously isolated by differential chemical modification with o-phthalaldehyde. We also found mutan-binding peptides in sucrose- and dextran-binding regions and a lysine-rich region. Also, there was a peptide similar in sequence to glucan-binding A-repeat of streptococcal glucosyltransferases.

Mold Pectinase Modified with Dialdehyde Derivatives of Dextran and Cellulose.

Chemical modification of mold pectinase with dextran- and cellulose-dialdehydes was examined to improve the enzyme characteristics. The modified pectinase with dextran-dialdehyde retained about 50% of the original activity, and more than 80% of the total amino groups were modified. HPLC gel filtration analysis showed an increase in molecular weight of the reaction product. Reaction with cellulose-dialdehyde provided an immobilized form of pectinase. The immobilized pectinase was resistant to both acidic and alkaline pHs, and also acquired heat stability at 60°C. The optimum pH of the modified enzyme shifted from pH 4.5 to 5.0-5.5, and this enzyme had higher activity at neutral pH regions than the native enzyme. A rather low recovery of immobilized enzyme (14.5%) should be improved by the combination with various methods hitherto established.

Inhibition of dextran and mutan synthesis by cycloisomaltooligosaccharides.

Novel cyclic isomaltooligosaccharides, cyclodextran, strongly inhibited the dextransucrase reaction. The inhibition was dependent on the cyclodextran concentration and greatly enhanced by the first incubation at 30 degrees for 30 min. Cyclodextran-heptaose and -octaose were competitive inhibitors for sucrose yielding Ki's of 0.25 and 0.64 mM, respectively. Both reducing sugar and dextran producing activities of dextransucrase were almost equally inhibited by the cyclodextrans. Although gamma-cyclodextrin, palatinose, sucrose-monocaprate, and maltitol gave 5-35% inhibition, cyclodextran-heptaose gave 95% inhibition. Moreover, water-insoluble glucan (mutan) synthesis by the glucosyltransferase from Streptococcus mutans was significantly repressed by the addition of cyclodextran.

Aggregated form of dextransucrases from Leuconostoc mesenteroides NRRL B-512F and its constitutive mutant.

Purified dextransucrases [EC 2.4.1.5], DSW-D and DSW-G, from Leuconostoc mesenteroides B-512F were obtained from affinity chromatography with DEAE-Sephadex A-50 by elution with clinical dextran and guanidine-HCl, respectively. DSM-G was purified from the B-512F mutant strain SH 3002, which produces dextransucrase constitutively. Although the sugar contents of the purified enzymes were different, their molecular masses by SDS-PAGE were all 170 kDa. DSW-D and DSW-G were highly aggregated and the all the activities were eluted at the void volume (V0) on Sepharose 6B, while the DSM-G was eluted at 1.2 x V0 volume. On rechromatography, DSM-G was separated into three peaks corresponding to the aggregated form, monomeric form, and partially digested form, respectively. The aggregation of Leuconostoc dextransucrase was looser than that of streptococcal glucosyltransferases, but the structures of these enzymes had high homology with each other.

An active-site peptide containing the second essential carboxyl group of dextransucrase from Leuconostoc mesenteroides by chemical modifications.

The treatment of Leuconostoc mesenteroides B-512F dextransucrase with 10 mM 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) and glycine ethyl ester (GEE) inactivated the enzyme almost completely within 24 min where the modification of one carboxyl group/mol of the enzyme by EDC was attained. Though 30 mM diethyl pyrocarbonate (DEP) also inactivated the enzyme, about 35% of the activity remained during a 36-min incubation. When 10 mol of imidazole residues/mol of the enzyme was modified by DEP, 50% of the activity was still retained. The addition of the substrate sucrose greatly retarded the enzyme inactivation by EDC. However, the addition of dextran slightly protected the inactivation of the glucosyl-transferring activity and accelerated the inactivation of the sucrose-cleaving activity. In the case of DEP, the addition of sucrose or dextran gave no influence on the inactivation of the enzyme. Therefore, the carboxyl group seemed to play a more important role in the substrate binding and in the catalytic activity of the dextransucrase than the imidazolium group. Differential labeling of Leuconostoc dextransucrase by EDC was conducted in the presence of a sucrose analog, sucrose monocaprate. The fluorescent probe N-(1-naphthyl)ethylenediamine (EDAN) was used as the nucleophile instead of GEE. A fluorescent labeled peptide was isolated from a trypsin digest of the EDC-EDAN modified enzyme. The amino acid sequence of the isolated peptide was Leu-Gln-Glu-Asp-Asn-Ser-Asn-Val-Val-Val-Glu-Ala.(ABSTRACT TRUNCATED AT 250 WORDS)

Structural studies of the contractile tail sheath protein of bacteriophage T4. 2. Structural analyses of the tail sheath protein, Gp18, by limited proteolysis, immunoblotting, and immunoelectron microscopy.

The molecular structure of the T4 phage tail sheath protein, gp18, was studied by limited proteolysis, immunoblotting, and immunoelectron microscopy. Gp18 is extremely resistant to proteolysis in the assembled form of either extended or contracted sheaths, but it is readily cleaved by proteases in the monomeric form, giving rise to stable protease-resistant fragments. Limited proteolysis with trypsin gave rise to a trypsin-resistant fragment, Ala82-Lys316, with a molecular weight of 27K. Chymotrypsin- and thermolysin-resistant fragments were also mapped close to the trypsin-resistant region. The time course of trypsin digestion of the monomeric gp18 as monitored by SDS-polyacrylamide gel electrophoresis and immunoblotting of the gel revealed that the polypeptide chain consisting of 658 amino acid residues is sequentially cleaved at several positions from the C terminus. The N-terminal portion, Thr1-Arg81, was then removed to form the trypsin-resistant fragment. Immunoelectron microscopy revealed that the polyclonal antibodies against the trypsin-resistant fragment bound to the tail sheath. This supported the idea that at least part of the protease-resistant region of gp18 constitutes the protruding part of the sheath protein as previously revealed with three-dimensional image reconstruction from electron micrographs by Amos and Klug [Amos, L. A., & Klug, A. (1975) J. Mol. Biol. 99, 51-73].