The mechanism of acceptor reactions of leuconostoc mesenteroides B-512F dextransucrase

Reactions of dextransucrase and sucrose in the presence of sugars (acceptors) of low molecular weight have been observed to give a dextran of low molecular weight and a series of oligosaccharides. The acceptor reaction of dextransucrase was examined in the absence and presence of sucrose by using d-[¹⁴C]glucose, d-[¹⁴C]fructose, and ¹⁴C-reducing-end labeled maltose as acceptors. A purified dextransucrase was pre-incubated with sucrose, and the resulting d-fructose and unreacted sucrose were removed from the enzyme by chromatography on columns of Bio-Gel P-6. The enzyme, which migrated at the void volume, was collected and referred to as “charged enzyme”. The charged enzyme was incubated with ¹⁴C-acceptor in the absence of sucrose. Each of the three acceptors gave two fractions of labeled products, a high molecular weight product, identified as dextran, and a product of low molecular weight that was an oligosaccharide. It was found that all three of the acceptors were incorporated into the products at the reducing end. Similar results were obtained when the reactions were performed in the presence of sucrose, but higher yields of labeled products were obtained and a series of homologous oligosaccharides was produced when d-glucose or maltose was the acceptor. We propose that the acceptor reaction proceeds by nucleophilic displacement of glucosyl and dextranosyl groups from a covalent enzyme-complex by a specific, acceptor hydroxyl group, and that this reaction effects a glycosidic linkage between the d-glucosyl and dextranosyl groups and the acceptor. We conclude that the acceptor reactions serve to terminate polymerization of dextran by displacing the growing dextran chain from the active site of the enzyme; the acceptors, thus, do not initiate dextran polymerization by acting as primers.     

Structural characterization of enzymatically synthesized dextran and oligosaccharides from Leuconostoc mesenteroides NRRL B-1426 dextransucrase

Leuconostoc mesenteroides NRRL B-1426 dextransucrase synthesized a high molecular mass dextran (>2 × 10⁶ Da) with ～85.5% α-(1→6) linear and ～14.5% α-(1→3) branched linkages. This high molecular mass dextran containing branched α-(1→3) linkages can be readily hydrolyzed for the production of enzyme-resistant isomalto-oligosaccharides. The acceptor specificity of dextransucrase for the transglycosylation reaction was studied using sixteen different acceptors. Among the sixteen acceptors used, isomaltose was found to be the best, having 89% efficiency followed by gentiobiose (64%), glucose (30%), cellobiose (25%), lactose (22.5%), melibiose (17%), and trehalose (2.3%) with reference to maltose, a known best acceptor. The β-linked disaccharide, gentiobiose, showed significant efficiency for oligosaccharide production that can be used as a potential prebiotic.     

Mechanism of chain initiation by dextransucrase: Absence of terminal ‘sucrose’ linkage in newly synthesized dextran chains

Dextran was synthesized using dextransucrase from Streptococus sanguis 10558 and (F)-[¹⁴C]sucrose as substrate to test the possibility that sucrose may be the initial acceptor for glucose. If sucrose is the initial acceptor, then dextran chains should have [¹⁴C] fructose in a terminal ‘sucrose’ linkage which can be cleaved under mild conditions. Although incorporation of [¹⁴C]fructose into dextran was observed, the label was not released by mild hydrolysis, indicating that sucrose is not the initiator for dextran synthesis. Incorporation of [¹⁴C]fructose into dextran might represent its ability to act as an acceptor, as suggested by the isolation of leucrose as a by-product in the reaction.     

Characterization of a novel dextransucrase from Weissella confusa isolated from sourdough

Weissella confusa and Weissella cibaria isolated from wheat sourdoughs produce, from sucrose, linear dextrans due to a single soluble dextransucrase. In this study, the first complete gene sequence encoding dextransucrase from a W. confusa strain (LBAE C39-2) along with the one from a W. cibaria strain (LBAE K39) were reported. Corresponding gene cloning was achieved using specific primers designed on the basis of the draft genome sequence of these species. Deduced amino acid sequence of W. confusa and W. cibaria dextransucrase revealed common structural features of the glycoside hydrolase family 70. Notably, the regions located in the vicinity of the catalytic triad (D, E, D) are highly conserved. However, comparison analysis also revealed that Weissella dextransucrases form a distinct phylogenetic group within glucansucrases of other lactic acid bacteria. We then cloned the W. confusa C39-2 dextransucrase gene and successfully expressed the mature corresponding enzyme in Escherichia coli. The purified recombinant enzyme rDSRC39-2 catalyzed dextran synthesis from sucrose with a K _m of 8.6 mM and a V _max of 20 μmol/mg/min. According to ¹H and ¹³C NMR analysis, the polymer is a linear class 1 dextran with 97.2 % α-(1→6) linkages and 2.8 % α-(1→3) branch linkages, similar to the one produced by W. confusa C39-2 strain. The enzyme exhibited optimum catalytic activity for temperatures ranging from 35 to 40 °C and a pH of 5.4 in 20 mM sodium acetate buffer. This novel dextransucrase is responsible for production of dextran with predominant α-(1→6) linkages that could find applications as food hydrocolloids.     

Dextransucrase and the mechanism for dextran biosynthesis

Remaud-Simeon and co-workers [Moulis, C.; Joucla, G.; Harrison, D.; Fabre, E.; Potocki-Veronese, G.; Monsan, P.; Remaud-Simeon, M. J. Biol. Chem., 2006, 281, 31254-31267] have recently proposed that a truncated Escherichia coli recombinant B-512F dextransucrase uses sucrose and the hydrolysis product of sucrose, d-glucose, as initiator primers for the nonreducing-end synthesis of dextran. Using ¹⁴C-labeled d-glucose in a dextransucrase-sucrose digest, it was found that <0.02% of the d-glucose appears in a dextran of M_n 84,420, showing that d-glucose is not an initiator primer, and when the dextran was treated with 0.01 M HCl at 80 °C for 90 min and a separate sample with invertase at 50 °C for 24 h, no d-fructose was formed, indicating that sucrose is not present at the reducing-end of dextran, showing that sucrose also was not an initiator primer. It is further shown that both d-glucose and dextran are covalently attached to B-512FMC dextransucrase at the active site during polymerization. A pulse reaction with [¹⁴C]-sucrose and a chase reaction with nonlabeled sucrose, followed by dextran isolation, reduction, and acid hydrolysis, gave ¹⁴C-glucitol in the pulsed dextran, which was significantly decreased in the chased dextran, showing that the d-glucose moieties of sucrose are added to the reducing-ends of the covalently linked growing dextran chains. The molecular size of dextran is shown to be inversely proportional to the concentration of the enzyme, indicating a highly processive mechanism in which d-glucose is rapidly added to the reducing-ends of the growing chains, which are extruded from the active site of dextransucrase. It is also shown how the three conserved amino acids (Asp551, Glu589, and Asp 622) at the active sites of glucansucrases participate in the polymerization of dextran and related glucans from a single active site by the addition of the d-glucose moiety of sucrose to the reducing-ends of the covalently linked glucan chains in a two catalytic-site, insertion mechanism.     

Purification of membrane-bound dopamine beta-monooxygenase from chromaffin granules: relation to soluble dopamine beta-monooxygenase

Previous studies have indicated that α-d-1-fluoroglucose is a glycosyl donor for glucosyl transferases (5, 6) including dextransucrases formed by Leuconostoc and Streptococcus mutans. The present report confirms these observations with dextransucrase isolated from S. sanguis and conclusively establishes the details of this reaction as well as proving that mechanism of fluoroglucose transfer is comparable to that glucosyl transfer from sucrose. A new procedure for monitoring the reaction is reported, and is based on the measurement of proton formation using the pH indicator, bromcresol purple. Production of F^? was found to be stoichiometric with proton production. Rate studies with the substrate indicate that α-1-fluoroglucose undergoes spontaneous hydrolysis, which is greatly increased in the presence of nucleophilic buffers. When [¹⁴C]maltose and α-1-fluoroglucose or [¹⁴C]α-1-fluoroglucose and maltose were incubated with dextransucrase, a series of oligosaccharide products was observed. The results indicate that the glucosyl moiety of α-1-fluoroglucose transferred to the acceptor. The nature of formation of the products are consistent with a series of precursor-product reactions. Product analysis of the saccharides by borohydride reduction analysis demonstrated that the glucosyl unit was added to the nonreducing end of maltose. When either [¹⁴C]fructose or [¹⁴C]-α-1-fluoroglucose were incubated with enzyme, a reaction was observed which was analogous to the isotopic-exchange reaction catalyzed by the enzyme in the presence of [¹⁴C]fructose and sucrose.     

Alternansucrase acceptor products

The regioselectivity of alternansucrase (EC 2.4.1.140) differs from dextransucrase (EC 2.4.1.5) in ways that can be useful for the synthesis of novel oligosaccharide structures. For example, it has been recently shown that the major oligosaccharides produced when maltose is the acceptor include one trisaccharide structure, two tetrasaccharides, one pentasaccharide, two hexasaccharides, one heptasaccharide, and at least two octasaccharides, containing no adjacent α-(1→3) linkages and no more than two consecutive α-(1→6) linkages. This may shed some light on how the enzyme works to produce the alternating structure. Another characteristic of alternansucrase that distinguishes it from dextransucrase is its greater ability to use leucrose as an acceptor. Leucrose, produced by glucosyl transfer to fructose released from the initial sucrose substrate, represents a very poor substrate for Leuconostoc mesenteroides NRRL B-512F dextransucrase. Alternansucrase, however, continues to transfer glucosyl units to leucrose, resulting in some unusual glucosyl-fructose oligosaccharides.     

Oligomannoside biosynthesis in Mycobacterium smegmatis

A cell-free particulate enzyme system of Mycobacterium smegmatis ATCC 607 was shown to catalyze the incorporation of labeled mannose from GDP-[¹⁴C]mannose into endogenous acceptors to form a series of labeled neutral oligomannosides. These oligomannosides were devoid of amino sugar. The major oligomannoside product was characterized to be a trimannoside, O-α-d-mannopyranosyl-(1 → 2)-O-α-d-mannopyranosyl(1 → 2)-d-mannose and represented 46% of the total labeled oligomannoside product. The higher oligomannosides were shown to have either/or both α(1 → 2) and α(1 → 6) glycosidic linkages. A series of unlabeled endogenous oligosaccharides was isolated from the 105,000g supernatant fractions of the cell-free extracts of M. smegmatis and found to be chromatographically similar to the labeled oligomannosides synthesized by the cell-free system. The nature of the endogenous acceptor was not determined.     

An enzyme from Streptococcus mutans forms branches on dextran in the absence of sucrose

An enzyme in glucosyltransferase preparations from Streptococcus mutans catalyzed the transfer of [14C]glucopyranoside from purified isomaltosaccharides, each containing [14C]glucopyranoside at its non-reducing terminus, to acceptor dextran, in the absence of sucrose. Half of the radioactivity present in the resulting [14C]dextrans was resistant to hydrolysis by amylo-1,6-glucosidase. Treatment of the [14C]dextrans with endodextranase resulted in extensive hydrolysis and produced [14C]-labeled limit oligosaccharides containing branch sites. Acetolysis of the [14C]-labeled limit oligosaccharides yielded [14C]nigerose, thus indicating the formation of branch sites on dextran in the absence of sucrose. The enzyme catalyzing this reaction has not been identified but appears to be independent of the major extracellular glucosyltransferases of S. mutans.     

Dextran acceptor reaction of Streptococcus sobrinus glucosyltransferase GTF-I as revealed by using uniformly 13C-labeled sucrose.

A sucrose glucosyltransferase GTF-I from cariogenic Streptococcus sobrinus transferred the uniformly 13C-labeled glucosyl residue ([U-(13)C]Glc) from [U-(13)C]sucrose to exogenous dextran T500 at the non-reducing-end, mostly by alpha-(1-->6) linkages and partially by alpha-(1-->3) linkages, as revealed by the 13C-(13)C NMR coupling pattern. With increasing amounts of [U-(13)C]sucrose, transfer of [U-(13)C]Glc to the alpha-(1-->3)-linked chain became predominant without increase in the number of chains. The transfer of [U-(13)C]Glc to an isomaltopentaose acceptor occurred similarly to its transfer to T500. alpha-(1-->3)-branches in the [U-(13)C]dextran, specifically synthesized from [U-(13)C]sucrose by a Streptococcus bovis dextransucrase, were not formed by GTF-I, as judged by the observation that a newly-formed alpha-1,3,6-branched [U-(13)C]Glc was not detected, which could have been formed by transferring the unlabeled Glc from sucrose to the internal alpha-(1-->6)-linked [U-(13)C]Glc at C-3. The 13C-(13)C one-bond coupling constants (1J) were also recorded for the C-1--C-6 bond of the internal alpha-(1-->6)-linked [U-(13)C]Glc and of the non-reducing-end [U-(13)C]Glc.     

Characterization of the extracellular,water-insoluble α-D-glucans of oral streptococci by methylation analysis,and by enzymic synthesis and degradation

Methylation analysis of water-insoluble α-D-glucans synthesized from sucrose by culture filtrates from several strains of Streptococcus spp. has proved that all of the glucans were highly branched and that the chains contained (1→6)- and (1→3)-linked D-glucose residues not involved in branch points. Hydrolysis of the glucans with a specific endo-(1→3)-α-D-glucanase demonstrated that the majority of the (1→3)-linked glucose residues were arranged in sequences. D-Glucose was the major product of the hydrolysis, and a small proportion of nigerose was also released. The use of a specific endo-(1→6)-α-D-glucanase similarly indicated that the glucans also contained sequences of (1→6)-linked α-D-glucose residues, and that those chains were branched. Two D-glucosyltransferases (GTF-S and GTF-I), which reacted with sucrose to synthesize a soluble glucan and a water-insoluble glucan, respectively, were separated from culture filtrates of S. mutans OMZ176. The soluble glucan was characterized as a branched (1→6)-α-D-glucan, whereas the insoluble one was a relatively linear (1→3)-α-D-glucan. The hypothesis is advanced that the glucosyltransferases can transfer glucan sequences by means of acceptor reactions similar to those proposed by Robyt for dextransucrase, leading to the synthesis of a highly branched glucan containing both types of chain. The resulting structure is consistent with the evidence obtained from methylation analysis and enzymic degradations, and explains the synergy displayed when the two D-glucosyltransferases interact with sucrose. Variations in one basic structure can account for the characteristics of water-insoluble glucans from S. sanguis and S. salivarius, and for the strain-dependent diversity of S. mutans glucans.     

A technique for the isolation of yeast alcohol dehydrogenase mutants with altered substrate specificity.

α-Amylases have been found to convert starch and glycogen, in part, to products other than hemiacetal-bearing entities (maltose, maltodextrins, etc.)—hitherto, the only products obtained from natural α-glucans by α-amylolysis. Glycosides of maltosaccharides were synthesized by purified α-amylases acting on starch or bacterial glycogen in the presence of p-nitrophenyl α- or β-d-glucoside. From a digest with crystallized B. subtilis var. amyloliquefaciens α-amylase, containing 4 mg/ml of [¹⁴C]glycogen and 40 mmp-NP β-d-glucoside, three pairs of correspondingly labeled glycosides and sugars were recovered: p-NP α-d-[¹⁴C]glucopyranosyl (1 → 4) β-d-glucopyranoside, and [¹⁴C]glucose; p-NP α-[¹⁴C]maltosyl (1 → 4) β-d-glucopyranoside, and [¹⁴C]maltose; p-NP α-[¹⁴C]maltotriosyl (1 → 4) β-d-glucopyranoside, and [¹⁴C]maltotriose. The three glycosides accounted for 11.4% of the [¹⁴C]glycogen donor substrate; the three comparable sugars, for 30.4%; higher maltodextrins, for 58.2%. Calculations based on the molar yields of all reaction products show that [¹⁴C]glycosyl moieties were transferred from donor to p-NP β-d-glucoside with a frequency of 0.234 relative to all transfers to water. This is a very high value considering the minute molar ratio (0.0007) of β-d-glucoside-to-water concentration. Less striking but similar findings were obtained with cryst. hog pancreatic and Aspergillus oryzae α-amylases. The results extend earlier findings (Hehre et al., Advan. Chem. Ser. (1973) 117, 309) in showing that α-amylases have a substantial capacity to utilize the C₄-carbinols of certain d-glucosyl compounds as acceptor sites.     

Role of mannosyl phosphoryl polyisoprenol in biosynthesis of mammary glycoproteins

When a membrane preparation from the lactating bovine mammary gland is incubated with GDP-[¹⁴C] mannose, mannose is incorporated into a [¹⁴C] mannolipid, a [Man-¹⁴C] oligosaccharide-lipid, and metabolically stable endogenous acceptor(s). The rate of mannosyl incorporation is the fastest into [¹⁴C] mannolipid, intermediate in [Man-¹⁴C] oligosaccharide-lipid, and least into [Man-¹⁴C] endogenous acceptor(s). The [¹⁴C] mannolipid has been partially purified and characterized. Mild acid hydrolysis of this compound gives [¹⁴C] mannose, whereas alkaline hydrolysis yielded [¹⁴C] mannose phosphate as the labeled product. The t_½ of hydrolysis of the mannolipid under the acidic and basic conditions are comparable to values obtained for mannosyl phosphoryl dolichol in other systems. The mannolipid is chromatographically indistinguishable from calf brain mannosyl phosphoryl polyisoprenol and chemically synthesized β-mannosyl phosphoryl dolichol. Exogenous dolichol phosphate stimulates the synthesis of mannolipid in mammary particulate preparations 8.5-fold. Synthesis of mannolipid is freely reversible; in the presence of GDP, the transfer of mannosyl moiety from endogenously labeled mannolipid to GDP-mannose is obtained. All of these results indicate that the structure of mannolipid is mannosyl phosphoryl polyisoprenol. Even though the precise chain length of the polyisoprenol portion has not been established, it is tentatively suggested to be dolichol. Partially purified [¹⁴C] mannolipid can directly serve as a mannosyl donor in the synthesis of [Man-¹⁴C] oligosaccharide-lipid and [Man-¹⁴C] endogenous acceptor(s). Pulse and chase kinetics utilizing GDP-mannose to chase the mannosyl transfer from GDP-[¹⁴C] mannose in the mammary membrane incubations caused an immediate and rapid turnover of [¹⁴C] mannose from [¹⁴C] mannolipid while the incorporation of label in [Man-¹⁴C] oligosaccharide-lipid and radioactive endogenous acceptor(s) continued for a short period before coming to a halt. Both gel filtration and electrophoresis indicate that the endogenous acceptor(s) are a mixture of 2 or more glycoproteins since incubation with proteases releases all of the radioactivity into water soluble low-molecular-weight components, perhaps glycopeptides. All of the above evidence is consistent with the following precursor-product relationship: GDP-mannose ? mannosyl phosphoryl polyisoprenol → mannosyl-oligosaccharide-lipid → mannosyl-proteins. The exact structure of the oligosaccharide-lipid and the endogenous glycoproteins is unknown.     

Comparative study of the efficacies of nine assay methods for the dextransucrase synthesis of dextran

A comparative study of nine assay methods for dextransucrase and related enzymes has been made. A relatively widespread method for the reaction of dextransucrase with sucrose is the measurement of the reducing value of d-fructose by alkaline 3,5-dinitrosalicylate (DNS) and thereby the amount of d-glucose incorporated into dextran. Another method is the reaction with ¹⁴C-sucrose with the addition of an aliquot to Whatman 3MM paper squares that are washed three times with methanol to remove ¹⁴C-d-fructose and unreacted ¹⁴C-sucrose, followed by counting of ¹⁴C-dextran on the paper by liquid scintillation counting (LSC). It is shown that both methods give erroneous results. The DNS reducing value method gives extremely high values due to over-oxidation of both d-fructose and dextran, and the ¹⁴C-paper square method gives significantly low values due to the removal of some of the ¹⁴C-dextran from the paper by methanol washes. In the present study, we have examined nine methods and find two that give values that are identical and are an accurate measurement of the dextransucrase reaction. They are (1) a ¹⁴C-sucrose/dextransucrase digest in which dextran is precipitated three times with three volumes of ethanol, dissolved in water, and added to paper and counted in a toluene cocktail by LSC; and (2) precipitation of dextran three times with three volumes of ethanol from a sucrose/dextransucrase digest, dried, and weighed. Four reducing value methods were examined to measure the amount of d-fructose. Three of the four (two DNS methods, one with both dextran and d-fructose and the other with only d-fructose, and the ferricyanide/arsenomolybdate method with d-fructose) gave extremely high values due to over-oxidation of d-fructose, d-glucose, leucrose, and dextran.     

Structural analysis and properties of dextran produced by Leuconostoc mesenteroides NRRL B-640

A water-soluble dextran was produced by purified dextransucrase from Leuconostoc mesenteroides NRRL B-640. The dextran was purified by alcohol precipitation. The structure of dextran was determined by FT-IR, ¹H NMR, ¹³C NMR and 2-dimensional NMR spectroscopic techniques. NMR techniques (1D ¹H, ¹³C and 2D HMQC) were used to fully assign the ¹H and ¹³C spectra. All the spectral data showed that the dextran contains d-glucose residues in a linear chain with consecutive α(1 → 6) linkages. No branching was observed in the dextran structure. The viscosity of dextran solution decreased with the increase in shear rate exhibiting a typical non-Newtonian pseudoplastic behavior. The surface morphology of dried and powdered dextran studied using Scanning electron microscopy revealed the cubical porous structure.     

The dextran acceptor reaction of dextransucrase from Streptococcus mutans K1-R

Soluble dextransucrase activity(ies) was eluted with a solution of clinical dextran from the insoluble dextran-cell complex produced by Streptococcus mutans K1-R grown in the presence of sucrose. Studies of the dextran acceptor-reaction of the soluble enzyme-preparation indicate that it is highly specific for dextran of high molecular weight. Increased dextran synthesis in the presence of dextran acceptor and the apparent inhibition of this stimulation by higher concentrations of dextran result from product modification rather than a direct effect on the level of enzyme activity. The results demonstrate that the potentially water-insoluble structure synthesized by dextransucrase on exogenous, soluble dextran acts as a more-efficient acceptor than the soluble dextran. The role of the acceptor reaction in the biosynthesis of complex dextrans is discussed.     

Activation and inhibition of dextransucrase by calcium

Initial rate kinetics of dextran synthesis by dextransucrase (sucrose:1,6-alpha-D-glucan-6-alpha-D-glucosyltransferase, EC 2.4.1.5) from Leuconostoc mesenteroides NRRL B-512F showed that below 1 mM, Ca2+ activated the enzyme by increasing Vmax and decreasing the Km for sucrose. Above 1 mM, Ca2+ was a weak competitive inhibitor (Ki = 59 mM). Although it was an activator at low concentration, Ca2+ was not required for dextran synthesis, either of main chain or branch linkages. Neither was it required for sucrose hydrolysis, acceptor reactions, or enzyme renaturation after SDS-polyacrylamide gel electrophoresis. A model for dextran synthesis is proposed in which dextransucrase has two Ca2+ sites, one activating and one inhibitory. Ca2+ at the inhibitory site prevents the binding of sucrose.     

Lipid-mediated glycosylation of a white matter membrane glycoprotein with an apparent molecular weight of 24,000.

White matter membrane preparations from pig brain catalyze the transfer of [¹⁴C]mannose from exogenous [¹⁴C]mannosylphosphoryldolichol into an endogenous oligosaccharide lipid. Under the same incubation conditions label is also incorporated into endogenous membrane glycoproteins. The enzymatic labeling of both classes of endogenous acceptors is stimulated by the addition of Ca²⁺. Several enzymatic properties of the mannosyltransferase activity responsible for the transfer of mannose from mannosylphosphoryldolichol into the oligosaccharide lipid intermediate have been examined. The [Man-¹⁴C] oligosaccharide lipid synthesized by this in vitro system has the solubility, hydrolytic and chromatographic characteristics of a pyrophosphate-linked oligosaccharide derivative of dolichol. The free [Man-¹⁴C]oligosaccharide liberated from the carrier lipid by mild acid treatment is estimated to contain 8 glycose units. All of the [¹⁴C]mannosyl units in the [Man-¹⁴C]oligosaccharide derived from exogenous [¹⁴C]mannosylphosphoryldolichol are released as free [¹⁴C]mannose by an α-mannosi-dase. No [¹⁴C]mannose is released during incubation with a β-mannosidase. The presence of an N,N′-diacetylchitobiose unit at the reducing end of the lipid-bound [Man-¹⁴C]oligosaccharide is indicated by its susceptibility to digestion by endo-β-N-acetylglucosaminidase H. Pronase digestion of the enzymatically labeled [Man-¹⁴C]glycoprotein yields a single [Man-¹⁴C]gly-copeptide fraction on Bio-Gel P-6 that appears to be slightly larger than the free [Man-¹⁴C]oligosac-charide released from the carrier lipid by mild acid hydrolysis. The [Man-¹⁴C]glycopeptide is cleaved by endo-β-N-acetylglucosaminidase H, and the neutral [Man-¹⁴C]oligosaccharide product appears to be identical to the product formed when the lipid-bound [Man-¹⁴C]oligosaccharide is degraded by the endoglycosidase. The glycopeptide linkage in the [Man-¹⁴C]glycoprotein is stable to mild alkali treatment. These results are consistent with the dolichol-linked [Man-¹⁴C]oligosaccharide, mannosy-lated via exogenous [¹⁴C]mannosylphosphoryldoiichol, being subsequently transferred en bloc from dolichyl pyrophosphate to asparagine residues in endogenous membrane polypeptide acceptors. SDS-polyacrylamide gel electrophoresis of the [Man-¹⁴C]glycoprotein, labeled when white matter membranes are incubated with [¹⁴C]mannosylphosphoryldolichol. revealed a major labeled polypeptide with an apparent mol wt of 24,000. A minor labeled membrane glycoprotein is also seen, having an apparent mol wt of 105,000.     

4,6-α-Glucanotransferase activity occurs more widespread in Lactobacillus strains and constitutes a separate GH70 subfamily

Family 70 glycoside hydrolase glucansucrase enzymes exclusively occur in lactic acid bacteria and synthesize a wide range of α-d-glucan (abbreviated as α-glucan) oligo- and polysaccharides. Of the 47 characterized GH70 enzymes, 46 use sucrose as glucose donor. A single GH70 enzyme was recently found to be inactive with sucrose and to utilize maltooligosaccharides [(1→4)-α-d-glucooligosaccharides] as glucose donor substrates for α-glucan synthesis, acting as a 4,6-α-glucanotransferase (4,6-αGT) enzyme. Here, we report the characterization of two further GH70 4,6-αGT enzymes, i.e., from Lactobacillus reuteri strains DSM 20016 and ML1, which use maltooligosaccharides as glucose donor. Both enzymes cleave α1→4 glycosidic linkages and add the released glucose moieties one by one to the non-reducing end of growing linear α-glucan chains via α1→6 glycosidic linkages (α1→4 to α1→6 transfer activity). In this way, they convert pure maltooligosaccharide substrates into linear α-glucan product mixtures with about 50% α1→6 glycosidic bonds (isomalto/maltooligosaccharides). These new α-glucan products may provide an exciting type of carbohydrate for the food industry. The results show that 4,6-αGTs occur more widespread in family GH70 and can be considered as a GH70 subfamily. Sequence analysis allowed identification of amino acid residues in acceptor substrate binding subsites +1 and +2, differing between GH70 GTF and 4,6-αGT enzymes.     

Cleavage of oligosaccharides by rat kidney sialidase Influence of substrate structure

The specificity of the sialidase activity present in rat kidney cortex (12 000 × g pellet) was studied with various tritiated oligosaccharidic substrates: (i) αNeuAc2 → 3βGall → 4Glc-itol[³H], αNeuAc2 → 6βGall → 4Glc-itol[³H] and αNeuAc2 → 8αNeuAc2 → 3βGall → 4Glc-itol[³H] from bovine colostrum; (ii) α-NeuAc2 → 6βGall → 4βGlcNAc-itol[³H], αNeuAc2 → 3βGal1 → 4βGlcNAcl → 2αManl → 3βMan1 → 4GlcNAc-itol[³H]. αNeuAc2 → 6βGall → 4βGlcNAcl → 2αManl α 3(βGall → 4GlcNAcl → 2αManl → 6)βManl → 4GlcNAc-itol [³H]et αNeuAc2 → 6βGall → 4βGlcNAcl → 2αManl-3(αNeuAc2 → 6βGall → 4βGlcNAcl → 2αManl → 6)βManl 4GlNAc-itol[³H] isolated from the urine of a patient with mucolipidosis I. The enzyme cleaves α2 → 3 and α2 → 8 linkages at a greater rate than the α2 → 6 bonds. Its activity decreases with the length of the oligosaccharidic chain. Substitution of a glucose moiety by Nacetylglucosamine results in diminished activity. The specificity of rat kidney sialidase differs from that reported for other mammalian of viral sialidases.