Mechanisms in the glucansucrase synthesis of polysaccharides and oligosaccharides from sucrose.
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Amino Acid Sequence↗
Biomedical subjects
J F Robyt
Publications and source records attributed to J F Robyt.
Determination of the number of sucrose and acceptor binding sites for Leuconostoc mesenteroides B-512FM dextransucrase, and the confirmation of the two-site mechanism for dextran synthesis.
In previous studies on dextransucrase using pulse and chase experiments with [14C]sucrose, Robyt et al. [Arch. Biochem. Biophys. 165 (1974) 634-640] proposed a two-site insertion mechanism to explain the data for the synthesis of dextran. To further establish the validity of the two-site mechanism, the number of sucrose binding sites at the active site have been determined by using equilibrium dialysis with 6-deoxysucrose, a strong competitive inhibitor for dextransucrase. A ligand binding plot gave a straight line that indicated there were two sucrose binding sites at the active site. A similar experiment was performed using the acceptor, maltose. The ligand binding plot for maltose also gave a straight line and indicated that there was one acceptor binding site at the active site. These results corroborate the proposed two-site mechanism for dextran synthesis. To further test the two-site mechanism, dextransucrase was partially inactivated to varying extents by reaction with diethylpyrocarbonate, which chemically modifies essential active-site histidines. The various partially inactivated enzymes were assayed for dextran synthesis and for the synthesis of maltose acceptor products. A plot of the log of the relative percentage of dextran synthesized and acceptor products synthesized against varying degrees of enzyme inactivation showed that the synthesis of dextran decreased to a greater extent than did the decrease of the synthesis of acceptor product. The proposed mechanism requires two sucrose sites for the synthesis of dextran and only one sucrose site for the synthesis of acceptor product. When one site is modified, the synthesis of dextran stops, but the synthesis of acceptor products can continue at the other site. Thus, the greater loss of dextran synthesis in comparison with the lesser loss of acceptor product synthesis by enzymes modified to varying degrees, gives further evidence for the two-site mechanism for dextran synthesis.
Binding Sites↗
Production and selection of mutants of Leuconostoc mesenteroides constitutive for glucansucrases.
After chemical mutagenesis using ethyl methane sulfonate, we isolated mutants constitutive for glucansucrases from Leuconostoc mesenteroides NRRL B-512FM, B-1142, and B-1355. Those mutants produced glucansucrases when grown on D-glucose as well as on sucrose. They produced higher glucansucrase activities (3 to 22 times) when grown on D-glucose than the parent strains grown on sucrose. Glucansucrases from mutants B-1355C and B-1142C grown on glucose formed glucans that were highly resistant to Penicillium dextranase hydrolysis. Mutant B-512FMC dextransucrase formed the same kind of dextran as the parent strain; however, it showed higher thermal stability, even when dextran was absent.
Biotechnology↗
Properties of Leuconostoc mesenteroides B-512FMC constitutive dextransucrase.
Leuconostoc mesenteroides B-512FMC, a constitutive mutant for dextransucrase, was grown on glucose, fructose, or sucrose. The amount of cell-associated dextransucrase was about the same for the three sugars at different concentrations (0.6% and 3%). Enzyme produced in glucose medium was adsorbed on Sephadex G-100 and G-200, but much less enzyme was adsorbed when it was produced in sucrose medium. Sephadex adsorption decreased when the glucose-produced enzyme was preincubated with dextrans of molecular size greater than 10 kDa. The release of dextransucrase activity from Sephadex by buffer (20 mM acetate, pH 5.2) was the highest at 28 degrees-30 degrees C. The addition of dextran to the enzyme stimulated dextran synthesis but had very little effect on the temperature or pH stability. Dextransucrase purified by ammonium sulfate precipitation, hydroxyapatite chromatography, and Sephadex G-200 adsorption did not contain any carbohydrate, and it synthesized dextran, showing that primers are not necessary to initiate dextran synthesis. The purified enzyme had a molecular size of 184 kDa on SDS-PAGE. On standing at 4 degrees C for 30 days, the native enzyme was dissociated into three inactive proteins of 65, 62, and 57 kDa. However, two protein bands of 63 and 59 kDa were obtained on SDS-PAGE after heat denaturation of the 184-kDa active enzyme at 100 degrees C. The amount of 63-kDa protein was about twice that of 59-kDa protein. The native enzyme is believed to be a trimer of two 63-kDa and one 59-kDa monomers.
Biotechnology↗
Control of the synthesis of dextran and acceptor-products by Leuconostoc mesenteroides B-512FM dextransucrase.
In the maltose-acceptor reaction of Leuconostoc mesenteroides B-512FM dextransucrase, some of the D-glucose moieties of sucrose are diverted from the synthesis of dextran and are transferred to the nonreducing end of maltose to form panose. Glucose is also transferred to panose and to subsequent acceptor products to give a homologous series of isomaltosyl dextrins attached alpha-(1-->6) to maltose. Three experimental parameters were studied to obtain quantitative information about the yield and distribution of acceptor products and the yield of dextran: (a) the ratio of maltose to sucrose, (b) the concentration of maltose and sucrose, and (c) the amount of enzyme. The reactions were run with [14C]sucrose and the amount of each acceptor product and the amount of dextran synthesized were determined for (a), (b), and (c) by TLC separation and measurement of the radioactivity with a PhosphorImager. It was found that an increase in the ratio of maltose to sucrose increased the amount of acceptor products with a concomitant decrease in the synthesis of dextran. Further, as the ratio was increased, the number of acceptor-products decreased. When the concentrations of maltose and sucrose were increased and the ratio was maintained at 1:1, there also was a decrease in the amount of dextran and an increase in the amount of acceptor-products. In addition, there was a decrease in the amount of dextran and an increase in the amount and number of acceptor-products when the amount of enzyme was increased. The first acceptor-product can be exclusively obtained without the formation of any dextran, by using a specific ratio and concentration of maltose and sucrose and a specified amount of enzyme.
Autoradiography↗
Interpretation of dextransucrase inhibition at high sucrose concentrations.
When acceptor reactions were carried out at high sucrose concentrations (> or = 200 mM), dextran synthesis was inhibited and the acceptor reactions were increased. A model, based on the known mechanisms of dextran synthesis and acceptor reactions, is proposed to explain the inhibition of dextran synthesis and the increase in the acceptor products at high sucrose concentrations. According to the model, sucrose binds to a third, low-affinity binding site, allosterically changing the conformation of the active site so that dextran cannot be formed but acceptor products can be formed.
Binding Sites↗
Maltodextrin acceptor reactions of Streptococcus mutans 6715 glucosyltransferases.
The maltodextrin (maltose through maltoheptaose) acceptor reactions of two Streptococcus mutans 6715 glucosyltransferases (GTF-I and GTF-S) were studied. The acceptor product structures were determined by comparing them with the known structures of the acceptor products of Leuconostoc mesenteroides B-512FM dextransucrase (EC 2.4.1.5) and L. mesenteroides B-1355 alternansucrase (EC 2.4.1.140). When reacted with maltose (G2), both GTF-I and GTF-S transferred a D-glucopyranose from sucrose to the nonreducing glucosyl residue to give panose (6(2)-alpha-D-glucopyranosyl maltose). Panose then served as an acceptor to give two further acceptor products, 6(2)-alpha-isomaltosyl maltose and 6(2)-alpha-nigerosyl maltose. 6(2)-alpha-Isomaltosyl maltose then went on to serve as an acceptor to give a series of homologous acceptor products with isomaltodextrin chains attached to C-6 of the nonreducing-end residue of maltose, while 6(2)-alpha-nigerosyl maltose did not further react. When reacted with other maltodextrins (G3-G7), both GTF-I and GTF-S transferred a D-glucopyranose to C-6 of either the nonreducing-end or the reducing-end residues of the maltodextrins, forming alpha(1----6) linkages. When D-glucopyranose was transferred to the nonreducing-end residue by GTF-I or GTF-S, the first product was also an acceptor to give the second product, which then served as an acceptor to give the third product, etc., to give a homologous series of products. When D-glucopyranose was transferred to the reducing-end residue, the acceptor product that formed did not readily serve as an acceptor, or served only as a very poor acceptor, to give a small amount of the next homologue, as was the case for G7 with GTF-S. In addition, GTF-I also transferred D-glucopyranose to the reducing-end or to the nonreducing-end residue of maltotriose, forming alpha(1----3) linkages, to give 3(3)-alpha-D-glucopyranosyl maltotriose and 3(1)-alpha-D-glucopyranosyl maltotriose. Neither of these acceptor products further served as acceptors to give a homologous series. Under equivalent conditions of equimolar amounts of acceptor and sucrose, maltose and maltotriose are much better acceptors with GTF-I than they are with GTF-S, which is better than L. mesenteroides B-512FM dextransucrase. The three enzymes display significantly different efficiencies for the different maltodextrin acceptor reactions, GTF-I and GTF-S having much higher efficiencies than L. mesenteroides B-512FM dextransucrase.
Carbohydrate Conformation↗
Miniaturization of three carbohydrate analyses using a microsample plate reader.
Three carbohydrate analyses (reducing value by copper-bicinchoninate, total carbohydrate by phenol-sulfuric acid, and D-glucose by glucose oxidase) have been miniaturized using a microsample plate reader. The use of the reducing-value procedure to measure the hydrolysis of starch by alpha-amylase and the use of the glucose oxidase method to measure the hydrolysis of lactose by lactase are illustrated.
Carbohydrates↗
Specificity of acceptor binding to Leuconostoc mesenteroides B-512F dextransucrase: binding and acceptor-product structure of alpha-methyl-D-glucopyranoside analogs modified at C-2, C-3, and C-4 by inversion of the hydroxyl and by replacement of the hydroxyl with hydrogen.
The specificity of acceptor binding to the active site of dextransucrase was studied by using alpha-methyl-D-glucopyranoside analogs modified at C-2, C-3, and C-4 positions by (a) inversion of the hydroxyl group and (b) replacement of the hydroxyl group with hydrogen. 2-Deoxy-alpha-methyl-D-glucopyranoside was synthesized from 2-deoxyglucose; 3- and 4-deoxy-alpha-methyl-D-glucopyranosides were synthesized from alpha-methyl-D-glucopyranoside; and alpha-methyl-D-allopyranoside was synthesized from D-glucose. The analogs were incubated with [14C]sucrose and dextransucrase, and the products were separated by thin-layer chromatography and quantitated by liquid scintillation spectrometry. Structures of the acceptor products were determined by methylation analyses and optical rotation. The relative effectiveness of the acceptor analogs in decreasing order were 2-deoxy, 2-inverted, 3-deoxy, 3-inverted, 4-inverted, and 4-deoxy. The enzyme transfers D-glucopyranose to the C-6 hydroxyl of analogs modified at C-2 and C-3, to the C-4 hydroxyl of 4-inverted, and to the C-3 hydroxyl of 4-deoxy analogs of alpha-methyl-D-glucopyranoside. The data indicate that the hydroxyl group at C-2 is not as important for acceptor binding as the hydroxyl groups at C-3 and C-4. The hydroxyl group at C-4 is particularly important as it determines the binding orientation of the alpha-methyl-D-glucopyranoside ring.
Glucosyltransferases↗
Acceptor reactions of maltodextrins with Leuconostoc mesenteroides B-512FM dextransucrase.
The acceptor products of maltose with Leuconostoc mesenteroides B-512FM dextransucrase are panose (6(2)-alpha-D-glucopyranosyl maltose) and a homologous series of 6(2)-isomaltodextrinosyl maltoses. The structures of the acceptor products of dextransucrase with other maltodextrins, maltotriose to maltooctaose (G3-G8), were determined by using the known specificities of alpha-glucosidase and porcine pancreatic alpha-amylase, and by methylation analysis. It has been found that dextransucrase transfers a D-glucopyranosyl residue to C-6 of either the nonreducing end or the reducing end residues of the maltodextrins, G3-G8, forming an alpha(1----6) linkage. When a D-glucose was transferred to the nonreducing residue, the first product was also an acceptor to give the second product, which served as an acceptor to give the third product, etc. to give a homologous series. When D-glucose was transferred to the reducing residue, the first product did not readily serve as an acceptor to give products or it served only as a very poor acceptor to give a small amount of the next homologue. The effectiveness of maltodextrins as acceptors decreased as the size of the maltodextrin chain increased. Maltotriose was 40% as effective as maltose and maltooctaose was only 6% as effective.
Autoradiography↗
A facile purification of Leuconostoc mesenteroides B-512FM dextransucrase.
Leuconostoc mesenteroides NRRL B-512F has been mutated by treatment with N-nitrosoguanidine. The resulting mutant (designated as B-512FM) produces 300 times as much enzyme as the parent strain. B-512FM dextransucrase was treated extensively with Sigma crude dextranase, followed by column chromatography on Bio-Gel A-5m. The purified dextransucrase had a specific activity of 84 IU/mg, a 100-fold purification with 42% yield, and was shown by SDS-PAGE to have a single protein of molecular weight of 158,000 with dextransucrase activity. The procedure has been used to produce purified enzyme for sequencing. The molecular weight of 158,000 agrees with that calculated from its amino acid sequence.
Chromatography, Gel↗
Detection of a covalent intermediate in the mechanism of action of porcine pancreatic alpha-amylase by using 13C nuclear magnetic resonance.
The catalytic mechanism of porcine pancreatic alpha-amylase (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1) has been examined by nuclear magnetic resonance (NMR) at subzero temperatures by using [1-13C]maltotetraose as substrate. Spectral summation and difference techniques revealed a broad resonance peak, whose chemical shift, relative signal intensity and time-course appearance corresponded to a beta-carboxyl-acetal ester covalent enzyme-glycosyl intermediate. This evidence supports a double-displacement covalent mechanism for porcine pancreatic alpha-amylase-catalyzed hydrolysis of glycosidic linkages, based on the presence of catalytic aspartic acid residues within the active site of this enzyme.
Animals↗
Synthesis of 4,6-dideoxysucrose, and inhibition studies of Leuconostoc and Streptococcus D-glucansucrases with deoxy and chloro derivatives of sucrose modified at carbon atoms 3, 4, and 6.
Starting from sucrose, 2,3,1',3',4',6'-hexa-O-benzoyl-6-deoxy-6-iodosucrose (1) was synthesized. Reaction of 1 with sulfuryl chloride in pyridine gave 2,3,1',3',4',6'-hexa-O-benzoyl-4-chloro-4,6-dideoxy-6-iodogalactosucr ose (2). Compound 2 was treated with tributyltin hydride in toluene in the presence of a radical initiator, alpha, alpha-azobis(isobutanonitrile) (AIBN), to remove iodine and chlorine groups and give hexa-O-benzoyl-4,6-dideoxysucrose. Benzoyl groups were removed by sodium methoxide in methanol to give 4,6-dideoxysucrose. Sucrose was modified at carbon atom 3, carbon atom 4, or carbon atoms 4 and 6, and these analogs were tested as inhibitors of the D-glucansucrases (D-glucosyltransferases) of Streptococcus mutans 6715 and Leuconostoc mesenteroides B-512F. Sucrose analogs used in this study are 4-deoxysucrose and 4-chloro-4-deoxygalactosucrose with S. mutans 6715 D-glucansucrases (GTF-S and GTF-I), and 3-deoxysucrose, 4-deoxysucrose, 4-chloro-4-deoxygalactosucrose, 6-deoxysucrose, and 4,6-dideoxysucrose with L. mesenteroides B-512F D-glucansucrase. The data indicate that 3-deoxysucrose, 4-deoxysucrose, and 4-chloro-4-deoxygalactosucrose are weak noncompetitive inhibitors for B-512F dextransucrase, with Ki values of 530, 201, and 202mM respectively. For the same enzyme, 6-deoxysucrose was a strong competitive inhibitor, with Ki of 1.60mM, and 4,6-dideoxysucrose was a good competitive inhibitor, with Ki of 20.3mM. 4-Deoxysucrose was a weak noncompetitive inhibitor for both GTF-I and GTF-S, with Ki values of 672 and 608mM, respectively. 4-Chloro-4-deoxygalactosucrose was also a weak noncompetitive inhibitor for GTF-I and GTF-S with Ki values of 391 and 308mM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Glucosyltransferases↗
Essential histidine residues in dextransucrase: chemical modification by diethyl pyrocarbonate and dye photo-oxidation.
Treatment of Leuconostoc mesenteroides B-512F dextransucrase with diethyl pyrocarbonate (DEP) at pH 6.0 and 25 degrees or photo-oxidation in the presence of Rose Bengal or Methylene Blue at pH 6.0 and 25 degrees, caused a rapid decrease of enzyme activity. Both types of inactivation followed pseudo-first-order kinetics. Enzyme partially inactivated by DEP could be completely reactivated by treatment with 100 mM hydroxylamine at pH 7 and 4 degrees. The presence of dextran partially protected the enzyme from inactivation. At pH 7 or below, DEP is relatively specific for the modification of histidine. DEP-modified enzyme showed an increased absorbance at 240 nm, indicating the presence of (ethoxyformyl)ated histidine residues. DEP modification of the sulfhydryl group of cysteine and of the phenolic group of tyrosine was ruled out by showing that native and DEP-modified enzyme had the same number of sulfhydryl and phenolic groups. DEP modification of the epsilon-amino group of lysine was ruled out by reaction at pH 6 and reactivation with hydroxylamine, which has no effect on DEP-modified epsilon-amino groups. The photo-oxidized enzyme showed a characteristic increase in absorbance at 250 nm, also indicating that histidine had been oxidized, and no decrease in the absorbance at 280 nm, indicating that tyrosine and tryptophan were not oxidized. A statistical, kinetic analysis of the data on inactivation by DEP showed that two histidine residues are essential for the enzyme activity. Previously, it was proposed that two nucleophiles at the active site attack bound sucrose, to give two covalent D-glucosyl-enzyme intermediates. We now propose that in addition, two imidazolium groups of histidine at the active site donate protons to the leaving, D-fructosyl moieties. The resulting imidazole groups then facilitate the formation of the alpha-(1----6)-glycosidic linkage by abstracting protons from the C-6-OH groups, and become reprotonated for the next series of reactions.
Carbohydrates↗
Neisseria perflava amylosucrase: characterization of its product polysaccharide and a study of its inhibition by sucrose derivatives.
Neisseria perflava amylosucrase forms from sucrose a polysaccharide very similar to glycogen, except that a larger proportion of its D-glucosyl residues are in short branches. Iodine staining of samples taken during polysaccharide formation indicate that the initial product is less branched than that formed at longer times. This glycogen-like polysaccharide has an estimated molecular mass range of 1 MD to 20 MD. Sucrose derivatives modified at C-3 (3-deoxysucrose and alpha-D-allopyranosyl beta-D-fructofuranoside), C-6 (6-deoxysucrose and 6-deoxy-6-fluorosucrose), and both C-4 and C-6 (4,6-dideoxysucrose) were tested as inhibitors of amylosucrase. Derivatives modified at C-6 were potent competitive inhibitors, with Ki values of 6.2 +/- 0.3 mM (6-deoxysucrose) and 0.50 +/- 0.06 mM (6-deoxy-6-fluorosucrose). The KM value of sucrose is 26.5 +/- 4.6 mM. Sucrose derivatives modified at C-3 were not significantly inhibitory over the concentration range tested. 4,6-Dideoxysucrose gave an unusual, non-competitive inhibition, in that, increasing its concentration did not produce a commensurate increase in the level of inhibition, which instead appeared to approach a limit. None of these sucrose derivatives was a substrate for amylosucrase, nor were they glycosyl donors to maltotriose.
Glucosyltransferases↗
Synthesis of 6-deoxy-6-fluorosucrose, and its inhibition of Leuconostoc and Streptococcus D-glucansucrases.
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Glycoside Hydrolases↗
Inhibition- and acceptor-reaction studies of Streptococcus mutans 6715 glucosyltransferases with 3-deoxysucrose, 3-deoxy-3-fluorosucrose, and alpha-D-allopyranosyl beta-D-fructofuranoside.
Three new sucrose analogs modified at C-3 have been studied as inhibitors and substrates for the glucosyltransferases (glucansucrases) of Streptococcus mutans 6715. Although none of the analogs were found to be substrates for polymer synthesis with either the soluble-polysaccharide producing enzyme, GTF-S, or the insoluble-polysaccharide producing enzyme, GTF-I, 3-deoxysucrose and 3-deoxy-3-fluorosucrose were able to donate glycosyl residues for acceptor reactions with both enzymes. Modification at C-3 considerably decreased the binding at the active site of both enzymes, since all of the analogs had inhibition constants at least one order of magnitude greater than the Km value for sucrose.
Chromatography, Thin Layer↗
Detection of dextransucrase and levansucrase on polyacrylamide gels by the periodic acid-Schiff stain: staining artifacts and their prevention.
One use of the periodic acid-Schiff (PAS) stain is to detect dextransucrase and levansucrase activities on polyacrylamide gels by staining their polysaccharide products, dextran and levan. When gels with heavy dextran or levan bands were PAS stained, proteins other than dextransucrase and levansucrase also were stained, and a high background developed during storage. The staining of proteins other than dextransucrase and levansucrase is caused by the diffusion of the periodate-oxidized carbohydrate before and after staining. This diffusion could be greatly slowed, and the staining artifact decreased, by following the PAS stain by a crosslinking treatment of the carbohydrate-dye complex. Protein staining artifacts could be prevented by using chymotrypsin to remove the protein from the gel at the stage after polysaccharide synthesis but before the PAS stain.
Chymotrypsin↗