Biosynthetic studies on N-acetylmannosaminuronic acid containing teichuronic acid in Bacillus megaterium

The particulate enzymes obtained from four strains of Bacillus megaterium AHU 1240, AHU 1373, AHU 1375, and T catalyzed the synthesis of a polysaccharide and glycolipids from UDP-N-acetylmannosaminuronic acid, UDP-N-acetylglucosamine, and UDP-glucose. Chemical studies involving Smith degradation, acid hydrolysis, and N-acetylation revealed that the polysaccharide product has a backbone made up of trisaccharide repeating units comprising glucose, N-acetylmannosaminuronic acid, and N-acetylglucosamine and that the main oligosaccharide moieties of the glycolipids were identical with N-acetylmannosaminuronosyl-N-acetylglucosamine and glucosyl-N-acetylmannosaminuronosyl-N-acetylglucosamine. Incubation of the disaccharide-linked lipid with each particulate enzyme in the presence of UDP-glucose produced the trisaccharide-linked lipid and a polysaccharide. It is therefore suggested that in this polysaccharide-synthesizing system the repeating unit is formed on a carrier lipid from appropriate nucleotide derivatives first and the polymerization of the units then occurs to synthesize the backbone while the growing chain remains in pyrophosphate linkage to the carrier lipid presumed to be undecaprenol.     

Synthesis and enzymatic susceptibility of a series of novel GM2 analogs

A series of GM2 analogs in which GM2 epitope was coupled to a variety of glycosyl lipids were designed and synthesized to investigate the mechanism of enzymatic hydrolysis of GM2 ganglioside. The coupling of N-Troc-protected sialic acid and p-methoxyphenyl galactoside acceptor gave the crystalline disaccharide, which was further coupled with galactosamine donor to give the desired GM2 epitope trisaccharide. After conversion into the corresponding glycosyl donor, the trisaccharide was coupled with galactose, glucose and artificial ceramide (B30) to give the final compounds. The result on hydrolysis of GM2 analogs indicates that GM2 activator protein requires one spacer sugar between GM2 epitope and the lipid moiety to assist the hydrolysis of the terminal GalNAc residue. Synthetic studies on sialoglycoconjugates, Part 140. For part 139, see Ref [1].     

Synthesis of polyprenol-monophosphate-β-galactose by Acetobacter xylinum

Summary A particulate enzyme preparation fromAcetobacter xylinum synthesizes ficaprenol-monophosphate--galactose from ficaprenol monophosphate (FMP) and UDP-galactose in the presence of detergent. The product has the same properties as those previously reported for the compound formed with the endogenous acceptor. Dolichol-monophosphate (DolMP) is also a good galactose acceptor but the product obtained has different properties. Lipid extracts fromAcetobacter contain galactose acceptor capacity which is lost by mild acid treatment. FMP behaves in a similar manner but DolMP is resistant to this treatment. It is concluded that the endogeneous acceptor is an allylic phosphate ester of a polyprenol.Abbreviations FMP ficaprenol monophosphate - DolMP dolichol monophosphate Dedicated to ProfessorLuis F. Leloir on the occasion of his 70^th birthday.     

Exopolysaccharide Biosynthesis in Lactococcus lactis NIZO B40: Functional Analysis of the Glycosyltransferase Genes Involved in Synthesis of the Polysaccharide Backbone

We used homologous and heterologous expression of the glycosyltransferase genes of the Lactococcus lactis NIZO B40 eps gene cluster to determine the activity and substrate specificities of the encoded enzymes and established the order of assembly of the trisaccharide backbone of the exopolysaccharide repeating unit. EpsD links glucose-1-phosphate from UDP-glucose to a lipid carrier, EpsE and EpsF link glucose from UDP-glucose to lipid-linked glucose, and EpsG links galactose from UDP-galactose to lipid-linked cellobiose. Furthermore, EpsJ appeared to be involved in EPS biosynthesis as a galactosyl phosphotransferase or an enzyme which releases the backbone oligosaccharide from the lipid carrier.     

A dolichol-linked trisaccharide from central nervous tissue: structure and biosynthesis.

Calf brain membranes have previously been shown to enzymatically transfer N-acetyl[¹⁴C]glucosamine from UDP-N-acetyl[¹⁴C]glucosamine into N-acetyl[¹⁴C]glucosami-nylpyrophosphoryldolichol, N,N′-diacetyl[¹⁴C]chitobiosylpyrophosphoryldolichol and a minor labeled product with the chemical and chromatographic properties of a [¹⁴C]trisaccharide lipid (Waechter, C. J., and Harford, J. B. (1977) Arch. Biochem. Biophys.181, 185–198). This paper demonstrates that incubating calf brain membranes containing endogenous, prelabeled N-acetyl[¹⁴C]glucosaminyl lipids with unlabeled GDP-mannose enhances the formation of the [¹⁴C]trisaccharide lipid. The intact [¹⁴C]trisaccharide lipid behaves like a dolichol-bound trisaccharide, in which the glycosyl group is linked via a pyrophosphate bridge, when chromatographed on SG-81 paper or DEAE-cellulose. Mild acid treatment releases a water-soluble product that comigrates with authentic β-Man-(1→4)-β-GlcNAc(1→4)-GlcNAc. The free [¹⁴C]trisaccharide is converted to N,N′-diacetyl[¹⁴C]chitobiose by incubation with a highly purified β-mannosidase. These findings indicate that the trisaccharide lipid formed by calf brain membranes is β-mannosyl-N,N′-diacetylchito-biosylpyrophosphoryldolichol. The two glycosyltransferases responsible for the enzymatic conversion of the N-acetylglucosaminyl lipid to the trisaccharide lipid have been studied using exogenous, purified [¹⁴C]glycolipid substrates. Calf brain membranes enzymatically transfer N-acetylglucosamine from UDP-N-acetylglucosamine to exogenous N-acetyl[¹⁴C] glucosaminylpyrophosphoryldolichol to form [¹⁴C]disaccharide lipid. The biosynthesis of [¹⁴C]disaccharide lipid is stimulated by unlabeled UDP-N-acetylglucosamine under conditions that inhibit N-acetylglucosaminylpyrophosphoryldolichol synthesis. Unlike the formation of N-acetylglucosaminylpyrophosphoryldolichol the enzymatic addition of the second N-acetylglucosamine residue is not inhibited by tunicamycin. Exogenous purified [¹⁴C] disaccharide lipid is enzymatically mannosylated by calf brain membranes to form the [¹⁴C] trisaccharide lipid. The formation of the [¹⁴C]trisaccharide lipid from exogenous [¹⁴C] disaccharide lipid is stimulated by unlabeled GDP-mannose and Mg²⁺, and inhibited by EDTA. Exogenous dolichyl monophosphate is also inhibitory. These results strongly suggest that the calf brain mannosyltransferase involved in the synthesis of the trisaccharide lipid requires a divalent cation and utilizes GDP-mannose, not mannosylphosphoryldolichol, as the direct mannosyl donor.     

Dolichyl monophosphate and its sugar derivatives in plants.

A glucose acceptor was isolated from soya beans by extraction with chloroform/methanol (2:1, v/v), followed by DEAE-cellulose column chromatography of the extract. This acceptor could not be distinguished from liver dolichyl monophosphate by t.l.c. It could replace dolichyl monophosphate as a mannose acceptor with a liver enzyme and its glucosylated derivative could replace dolichyl monophosphate glucose as a glucose donor in the same system. These results, together with those already reported [Pont Lezica, Brett, Romero Martinez & Dankert (1975) Biochem, Biophys. Res. Commun. 66, 980-987], indicate that the acceptor from soya bean is a dolichyl monophosphate. Gel filtration of its glucosylated derivative on Sephadex G-75 in the presence of sodium deoxycholate indicated that the acceptor contained 17 or 18 isoprene units. An enzyme preparation from pea seedlings was shown to use endogenous acceptors to form lipid phosphate sugars containing mannose and N-acetylglucosamine from GDP-mannose and UDP-N-acetylglucosamine. Chromatographic and degradative techniques indicated that the compounds formed were lipid monophosphate mannose, lipid pyrophosphate N-acetylglucosamine, lipid pyrophosphate chitobiose and a series of lipid pyrophosphate oligosaccharides containing both mannose and N-acetylglucosamine. None of these compounds was degraded by catalytic hydrogenation, and so the lipid moiety in each case was probably an alpha-saturated polyprenol. The endogenous acceptors for mannose and N-acetylglucosamine in peas may therefore be dolichyl monophosphate, as has been found in mammalian systems.     

Role of a sugar-lipid intermediate in colanic acid synthesis by Escherichia coli.

Membrane fractions from a lon strain of Escherichia coli but not a wild-type strain catalyze the incorporation of fucose from guanosine 5'-diphosphate-fucose into a lipid and into polymeric material. Both incorporation reactions specifically require only uridine 5'-diphosphate (UDP)-glucose. The sugar lipid was shown to be an intermediate in the synthesis of the polymer which was related to colanic acid. The sugar lipid had the structure (fucose3, glucose2)-glucose P-P-lipid. Its behavior on column and thin-layer chromatography, the rates of its hydrolysis in acid and base, and the response of its synthesis to inhibitors are all identical to the other sugar-lipid intermediates which have been shown to contain sugars attached to the C55-polyisoprenol, undecaprenol, by a pyrophosphate linkage. The membrane fractions from both the lon strain and the wild-type strain also catalyzed the incorporation of either glucose from UDP-glucose or galactose from UDP-galactose into a lipid fraction which was shown to contain the free sugar attached by a monophosphate linkage to an undecaprenol-like lipid. This lipid was isolated and its nuclear magnetic resonance spectra was identical to undecaprenol. The membrane fractions from both strains also incorporated glucose from UDP-glucose into glycogen and into a polymer that behaved like Escherichia coli lipopolysaccharide. Conditions were found where the incorporation of glucose could be directed specifically into each compound by adding the appropriate inhibitors.     

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.     

A mutant β-glucosidase increases the rate of the cellulose enzymatic hydrolysis

The enzymatic hydrolysis of cellulose and lignocellulosic materials is marked by a rate decrease along the reaction time. Cellobiohydrolase slow dissociation from the substrate and its inhibition by the cellobiose produced are relevant factors associated to the rate decrease. In that sense, addition of β-glucosidases to the enzyme cocktails employed in cellulose enzymatic hydrolysis not only produces glucose as final product but also reduces the cellobiohydrolase inhibition by cellobiose. The digestive β-glucosidase GH1 from the fall armyworm Spodoptera frugiperda, hereafter called Sfβgly, containing the mutation L428V showed an increased k_cat for cellobiose hydrolysis. In comparison to assays conducted with the wild-type Sfβgly and cellobiohydrolase TrCel7A, the presence of the mutant L428V increased in 5 fold the initial rate of crystalline cellulose hydrolysis and reduced to one quarter the time needed to TrCel7A produce the maximum glucose yield. As our results show that mutant L428V complement the action of TrCel7A, the introduction of the equivalent replacement in β-glucosidases is a promising strategy to reduce costs in the enzymatic hydrolysis of lignocellulosic materials.     

Enzymatic synthesis of polyprenol monophosphate mannose in insects

Summary The microsomal fraction of insects was found to contain an enzyme which transfers mannose from guanosine diphosphate mannose to an endogenous or exogenous insect lipid and to other acceptors such as dolichol monophosphate or ficaprenol monophosphate. This activity depended on the presence of Triton X-100 and magnesium ions, the optimal concentration of the latter being 10mM. The optimal temperature of the reaction was 25 °C and the maximal activity was obtained at pH 7.9. The mannolipid formed behaved as a monophosphodiester when chromatographed on DEAE-cellulose. Weak acid treatment of the product liberated mannose. Its behaviour both on thin layer and Sephadex G-150 chromatography would indicate the presence of a number of isoprenyl units similar to the dolichol and different from the ficaprenol derivative. Stability to phenol treatment indicated that the lipid fraction of the mannolipid is an±-saturated polyprenol phosphate similar to dolichol monophosphate.Abbreviations DoIMP dolichol monophosphate - FMP ficaprenol monophosphate - IGAL insect glycosyl acceptor lipid Dedicated to ProfessorLuis F. Leloir on the occasion of his 70th birthday.     

A new ADP (UDP)--glucose glucosyltransferase activity in disrupted starch grains.

Disrupted potato starch granules obtained in the presence of 8 M urea were shown to increase [¹⁴C] glucose incorporation from labeled ADP-glucose or UDP-glucose into starch, as compared to intact grains. Labeled glucose or maltose units were found to be incorporated through a linkage that produced cyclic phosphate esters upon mild alkaline treatment and was sensitive to hydrolysis at pH 2.0. Both properties of this linkage strongly resembled those of the pyrophosphate bond of ADP-glucose or UDP-glucose.     

Kinetics of dextran-independent α-(1→3)-glucan synthesis by Streptococcus sobrinus glucosyltransferase I

Glucosyltransferase (GTF)-I from cariogenic Streptococcus sobrinus elongates the α-(1→3)-linked glucose polymer branches on the primer dextran bound to the C-terminal glucan-binding domain. We investigated the GTF-I-catalyzed glucan synthesis reaction in the absence of the primer dextran. The time course of saccharide production during dextran-independent glucan synthesis from sucrose was analyzed. Fructose and glucose were first produced by the sucrose hydrolysis. Leucrose was subsequently produced, followed by insoluble glucan [α-(1→3)-linked glucose polymers] after a lag phase. High levels of intermediate nigerooligosaccharide series accumulation were characteristically not observed during the lag phase. The results from the enzymatic activity of the acceptor reaction for the nigerooligosaccharide with a degree of polymerization of 2-6 and methyl α-D-glucopyranoside as a glucose analog indicate that the activity increased with an increase in the degree of polymerization. The production of insoluble glucan was numerically simulated using the fourth-order Runge-Kutta method with the kinetic parameters estimated from the enzyme assay. The simulated time course provided a profile similar to that of experimental data. These results define the relationship between the kinetic properties of GTF-I and the time course of saccharide production. These results are discussed with respect to a mechanism that underlies efficient glucan synthesis.     

Inhibition of beta-1,4-Glucan Biosynthesis by Deoxyglucose : The Effect on the Glucosylation of Lipid Intermediates

GDP- and UDP-deoxyglucose inhibit the incorporation of glucose from UDP-glucose into dolichyl phosphate glucose and dolichyl pyrophosphate oligosaccharides. GDP-deoxyglucose inhibits by competing with the physiological nucleotide sugars for dolichyl phosphate, and dolichyl phosphate deoxyglucose is formed. This inhibition is reversed by excess of dolichyl phosphate. UDP-deoxyglucose does not give rise to a lipid-linked derivative, and inhibition by this analog is not reversed by dolichyl phosphate. The UDP- and GDP-derivatives of deoxyglucose inhibit the incorporation of glucose into glucose-containing glycoproteins. This effect seems to be the result of the inhibition of lipid intermediates glucosylation and is comparable to the effect produced by coumarin. Cellulose synthetase activity is not affected by UDP- or GDP-deoxyglucose. On the other hand, deoxyglucose inhibits the formation of β-1,4-glucans in vivo.     

Expression and characterization of Pichia etchellsii β-glucosidase in Escherichia coli

The β-glucosidase enzyme is important as the terminal enzyme involved in hydrolysis of cellobiose and short-chain cellodextrins generated during enzymatic cellulose degradation. Under controlled reaction conditions the enzyme also displays cello-oligosaccharide synthesizing ability (based on either the thermodynamic or kinetic approach). We present here the purification of the enzyme β-glucosidase (BGL) of Pichia etchellsii from recombinant pBG55 Escherichia coli clone. The kinetic parameters, substrate specificity and oligosaccharide synthesizing ability of the purified enzyme are also reported. The purified 200-kDa protein (tetramer of 50 kDa) was identified as a broad-substrate-specificity enzyme exhibiting increased temperature and glucose tolerance compared to the native yeast enzyme. Temperature directed substrate specificity for aryl β,1–4 linkage, and β(1–2), β(1–4), β(1–6) and β(2-1) linkages in various natural disaccharides was observed. Glycosylation of the enzyme was found to be unimportant for enzyme activity. With both cellobiose and glucose, oligosaccharide synthesis was detected. The implications of this information with regard to cellulose hydrolysis and oligosaccharide synthesis are discussed.     

CST-II's recognition domain for acceptor substrates in α-(2→8)-sialylations

CST-II is a bacterial sialyltransferase known for its ability to perform α-(2→8)-sialylations using GM(3) related trisaccharide substrates. Previously, we probed the enzyme's substrate specificity and developed an efficient synthesis for α-(2→8)-oligosialosides, and we suggested that CST-II could have a very small substrate recognition domain. Here we report our full studies on CST-II's recognition feature for acceptor substrates. The current study further demonstrates the versatility of CST-II in preparing complex oligosaccharides that contain α-(2→8)-oligosialyl moieties.     

The synthesis of p-(2-acetamido-2-deoxy-alpha-D-glucopyranosyl) P2-ficaprenyl pyrophosphate

2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-α,β-D-glucopyranosylammonium phosphate was prepared by the action of crystalline phosphoric acid on 2-acetamido-1,3,4,6-tetra-O-acetyl-β-D-glucopyranose. The α-D anomer (3) was the main product, and was isolated pure by preparative thin-layer chromatography or by removal of the β-D anomer (6) by selective acid hydrolysis. Ficaprenyl phosphate was prepared from ficaprenol, obtained as an isomeric mixture (mainly C₅₅) from an extract of Ficus elastica. Compound 3 was converted into the free acid and then into the tributyl-ammonium salt, which was treated with P¹-diphenyl P²-ficaprenyl pyrophosphate to give the acetylated pyrophosphate diester 8, characterized by analytical, spectral, and hydrogenolytic studies. Deacetylation of 8 gave the synthetic “lipid intermediate”, P¹-(2-acetamido-2-deoxy-D-glucopyranosyl) P²-ficaprenyl pyrophosphate (9), the properties of which were compared with those of natural substances considered to be active in the biosynthesis of teichoic acids.     

A novel biochemical route for fuels and chemicals production from cellulosic biomass

The conventional biochemical platform featuring enzymatic hydrolysis involves five key steps: pretreatment, cellulase production, enzymatic hydrolysis, fermentation, and product recovery. Sugars are produced as reactive intermediates for subsequent fermentation to fuels and chemicals. Herein, an alternative biochemical route is proposed. Pretreatment, enzymatic hydrolysis and cellulase production is consolidated into one single step, referred to as consolidated aerobic processing, and sugar aldonates are produced as the reactive intermediates for biofuels production by fermentation. In this study, we demonstrate the viability of consolidation of the enzymatic hydrolysis and cellulase production steps in the new route using Neurospora crassa as the model microorganism and the conversion of cellulose to ethanol as the model system. We intended to prove the two hypotheses: 1) cellulose can be directed to produce cellobionate by reducing β-glucosidase production and by enhancing cellobiose dehydrogenase production; and 2) both of the two hydrolysis products of cellobionate--glucose and gluconate--can be used as carbon sources for ethanol and other chemical production. Our results showed that knocking out multiple copies of β-glucosidase genes led to cellobionate production from cellulose, without jeopardizing the cellulose hydrolysis rate. Simulating cellobiose dehydrogenase over-expression by addition of exogenous cellobiose dehydrogenase led to more cellobionate production. Both of the two hydrolysis products of cellobionate: glucose and gluconate can be used by Escherichia coli KO 11 for efficient ethanol production. They were utilized simultaneously in glucose and gluconate co-fermentation. Gluconate was used even faster than glucose. The results support the viability of the two hypotheses that lay the foundation for the proposed new route.     

A novel two-step synthesis of α-linked mannobioses based on an acid-assisted reverse hydrolysis reaction

Instead of an enzyme-assisted reverse hydrolysis reaction for the synthesis of manno-oligosaccharides, we propose here a versatile new approach. By Fischer type glycosylation, a D-mannose solution of extremely high concentration (approximately 83% (w/w)) was incubated at 60°C for 65 h in 0.5 M HCl. After dilution and neutralization, the small amount of formed β-linked oligosaccharides was hydrolyzed by β-mannosidase. The yields of α-D-Manp-(1→2)-D-Manp (7.9%), α-D-Manp-(1→3)-D-Manp (7.9%), and α-D-Manp-(1→6)-D-Manp (29.1%) isolated by an activated carbon column chromatography were almost identical to those of the enzymatic reaction, but the yield of α-D-Manp-(1→3)-D-Manp increased enormously by the present method.     

Biosynthesis of dolichyl phosphate: characterization and site of synthesis in algae

This is the first report not only on the presence of polyprenyl phosphates and their site of synthesis in algae, but also on the formation of their sugar derivatives in this system.

A glucose acceptor lipid was isolated from the nonphotosynthetic alga Prototheca zopfii. The lipid was acidic and resistant to mild acid and alkaline treatments. The glucosylated lipid was labile to mild acid hydrolysis and resistant to phenol treatment and catalytic hydrogenation, as dolichyl phosphate glucose is. These results are consistent with the properties of an α-saturated polyprenyl phosphate.

The polyprenylic nature of the lipid was confirmed by biosynthesis from radioactive mevalonate. The [¹⁴C]lipid had the same chromatographic properties as dolichyl phosphate in DEAE-cellulose and Sephadex LH-20. Strong alkaline treatment and enzymic hydrolysis liberated free alcohols with chain lengths ranging from C₉₀ to C₁₀₅, C₉₅ and C₁₀₀ being the most abundant molecular forms. The glucose acceptor activity of the biosynthesized polyprenyl phosphate was confirmed.

The ability of different subcellular fractions to synthesize dolichyl phosphate was studied. Mitochondria and the Golgi apparatus were the sites of dolichyl phosphate synthesis from mevalonate.

     

Enzymatic hydrolysis of cellulose to glucose lung immobilized β-glucosidase

The production of sugars by enzymatic hydrolysis of cellulose is a multistep process which includes conversion of the intermediate cellobiose to glucose by β-glucosidase. Aside from its role as an intermediate, cellobiose inhibits the endoglucanase components of typical cellulase enzyme systems. Because these enzyme systems often contain insufficient concentrations of β-glucosidase to prevent accumulation of inhibitory cellobiose, this research investigated the use of supplemental immobilized β-glucosidase to increase yield of glucose. Immobilized β-glucosidase from Aspergillus phoenicis was produced by sorption at controlled-pore alumina with about 90% activity retention. The product lost only about 10% of the original activity during an on-stream reaction period of 500 hr with cellobiose as substrate; maximum activity occurred near pH 3.5 and the apparent activation energy was about 11 kcal/mol. The immobilized β-glucosidase was used together with Trichoderma reesei cellulase to hydrolyze cellulosic materials, such as Solka Floc, corn stove and exploded wood. Increased yields of glucose and greater conversions of cellobiose of glucose were observed when the reaction systems contained supplemental immobilized β-glucosidase.