The enzymic synthesis of ribose-1,5-bisphosphate: studies of its role in metabolism

Ribose-1,5-bisphosphate is synthesized in a reaction that uses ribose-1(or 5)-P as the phosphoryl acceptor and the acyl-P of 3-phosphoglyceryl phosphate as the donor. Glucose-1,6-bisphosphate is synthesized in a similar reaction. The relative activity with the two substrates remains unchanged over almost 300-fold purification of the enzyme, indicating that glucose-1,6-bisphosphate synthase catalyzes both reactions. The relative V/Km values for alternative phosphoryl acceptors are ribose-1-P (1); glucose-1-P (0.30); mannose-1-P and ribose-5-P (0.11); glucose-6-P (0.10); 2-deoxyglucose-6-P (0.03); and 2-deoxyribose-5-P (0.02). Fructose-1- and 6-phosphates are not substrates. The synthesis of both ribose-1,5-bisphosphate and glucose-1,6-bisphosphate is inhibited by physiologically significant levels of fructose-1,6-bisphosphate, glycerate-2,3-bisphosphate, glycerate-3-phosphate, citrate, and inorganic phosphate. Ribose-1,5-bisphosphate is a strong activator of brain phosphofructokinase.     

Denitrification at extremely high pH values by the alkaliphilic, obligately chemolithoautotrophic, sulfur-oxidizing bacterium Thioalkalivibrio denitrificans strain ALJD

Thioalkalivibrio denitrificans is the first example of an alkaliphilic, obligately autotrophic, sulfur-oxidizing bacterium able to grow anaerobically by denitrification. It was isolated from a Kenyan soda lake with thiosulfate as electron donor and N2O as electron acceptor at pH 10. The bacterium can use nitrite and N2O, but not nitrate, as electron acceptors during anaerobic growth on reduced sulfur compounds. Nitrate is only utilized as nitrogen source. In batch culture at pH 10, rapid growth was observed on N2O as electron acceptor and thiosulfate as electron donor. Growth on nitrite was only possible after prolonged adaptation of the culture to increasing nitrite concentrations. In aerobic thiosulfate-limited chemostats, Thioalkalivibrio denitrificans strain ALJD was able to grow between pH values of 7.5 and 10.5 with an optimum at pH 9.0. Growth of the organism in continuous culture on N2O was more stable and faster than in aerobic cultures. The pH limit for growth on N2O was 10.6. In nitrite-limited chemostat culture, growth was possible on thiosulfate at pH 10. Despite the observed inhibition of N2O reduction by sulfide, the bacterium was able to grow in sulfide-limited continuous culture with N2O as electron acceptor at pH 10. The highest anaerobic growth rate with N2O in continuous culture at pH 10 was observed with polysulfide (S8(2-)) as electron donor. Polysulfide was also the best substrate for oxygen-respiring cells. Washed cells at pH 10 oxidized polysulfide to sulfate via elemental sulfur in the presence of N2O or O2. In the absence of the electron acceptors, elemental sulfur was slowly reduced which resulted in regeneration of polysulfide. Cells of strain ALJD grown under anoxic conditions contained a soluble cd1-like cytochrome and a cytochrome-aa3-like component in the membranes.     

Ability of adenosine-2'(3')-deoxy-3'(2')-triphosphates and related analogues to replace ATP as phosphate donor for all human and Drosphila melanogaster deoxyribonucleoside kinases

Six non-conventional adenosine-2'- and 3'-triphosphate analogues of ATP were tested as potential phosphate donors for all four human, and D. melanogaster, deoxyribonucleoside kinases. With dCK (only dAdo as acceptor), TK1, TK2 and dNK only 3'-deoxyadenosine-2'-triphosphate was an effective donor (5-60% that for ATP). With dCK (dCyd as acceptor) and dGK (dGuo as acceptor), sharing 45% sequence identity, donor activities ranged from 13 to 119% that for ATP. Products were 5'-phosphates. In some instances, kinetics are dependent on the nature of the acceptor, and donor and acceptors properties are mutually interdependent. Results are highly relevant to studies on the modes of interaction with the enzymes, and to interpretations of reported crystal structures of dCK and dNK with bound ligands.     

Glycoprotein biosynthesis: studies on thyroid mannosyltransferases. II. Characterization of a polyisoprenyl mannosyl phosphate and evaluation of its intermediary role in the glycosylation of exogenous acceptors

The transfer of mannose from GDP-mannonse to exogenous glycopeptides and simple glycosides has been shown to be carried out by calf thyroid particles (Adamany, A. M., and Spiro, R. G. (1975) J. Biol. Chem. 250, 2830-2841). The present investigation indicates that this mannosylation process is accomplished through two sequential enzymatic reactions. The first involves the transfer of mannose from the sugar nucleotide to an endogenous acceptor to form a compound which has the properties of dolichyl mannosyl phosphate, while in the properties of dolichyl mannosyl phosphate, while in the second reaction this mannolipid serves as the glycosyl donor to exogenous acceptors. The particle-bound enzyme which catalyzed the first reaction utilized GDP-mannose (Km = 0.29 microM) as the most effective mannosyl donor, required a divalent cation, preferably manganese or calcium, and acted optimally at pH 6.3. Mannolipid synthesis was reversed by addition of GDP and a ready exchange of the mannose moiety was observed between [14C]mannolipid and unlabeled GDP-mannose. Exogenously supplied dolichyl phosphate, and to a lesser extent ficaprenyl phosphate, served as acceptors for the transfer reaction. The 14C-labeled endogenous lipid had the same chromatographic behavior as synthetic dolichyl mannosyl phosphate and enzymatically mannosylated dolichyl phosphate. The mannose component in the endogenous lipid was not susceptible to reduction with sodium borohydride and was released by mild acid hydrolysis. Alkaline treatment of the mannolipid released a phosphorylated mannose with properties consistent with that of mannose 2-phosphate. The formation of this compound which can arise from a cyclic 1,2-phosphate indicated, on the basis of steric considerations, that the mannose is present in beta linkage to the phosphate of the lipid. An intermediate role of the mannolipid in the glycosylation of exogenous acceptors was suggested by the observation that addition of dolichyl phosphate to thyroid particles resulted in a marked enhancement of mannose transfer from GDP-mannose to methyl-alpha-D-mannopyranoside acceptor while the presence of the glycoside caused a decrease in the mannolipid level. The glycosyl donor function of the polyisoprenyl mannosyl phosphate in the second reaction of the mannosylation sequence could be directly demonstrated by the transfer of [14C]mannose from purified endogenous mannolipid to either methyl-alpha-D-mannoside or dinitrophenyl unit A glycopeptides by thyroid enzyme in the presence of Triton X-100. The mannosylation of the glycoside was not inhibited by EDTA whereas the transfer of mannose to glycopeptide was cation-dependent. While dolichyl [14C]mannosyl phosphate, prepared from exogenous dolichyl phosphate, served as a donor of mannose to exogenous acceptor, this function could not be fulfilled by ficaprenyl [14C]mannosyl phosphate. The two-step reaction sequence carried out by thyroid enzymes which leads to the formation of an alpha-D-manno-pyranosyl-D-mannose linkage in exogenous acceptors by transfer of mannose from GDP-mannose through a beta-linked intermediate appears to involve a double inversion of anomeric configuration of this sugar.     

Ability of Adenosine-2′(3′)-deoxy-3′(2′)-triphosphates and Related Analogues to Replace ATP as Phosphate Donor for all Human and Drosphila melanogaster Deoxyribonucleoside Kinases

Abstract

Six non-conventional adenosine-2′- and 3′-triphosphate analogues of ATP were tested as potential phosphate donors for all four human, and D. melanogaster, deoxyribonucleoside kinases. With dCK (only dAdo as acceptor), TK1, TK2 and dNK only 3′-deoxyadenosine-2′-triphosphate was an effective donor (5–60% that for ATP). With dCK (dCyd as acceptor) and dGK (dGuo as acceptor), sharing 45% sequence identity, donor activities ranged from 13 to 119% that for ATP. Products were 5′-phosphates. In some instances, kinetics are dependent on the nature of the acceptor, and donor and acceptors properties are mutually interdependent. Results are highly relevant to studies on the modes of interaction with the enzymes, and to interpretations of reported crystal structures of dCK and dNK with bound ligands.     

Enzymatic glycosidation of sugar oxazolines having a carboxylate group catalyzed by chitinase

Enzymatic glycosidation using sugar oxazolines 1-3 having a carboxylate group as glycosyl donors and compounds 4-6 as glycosyl acceptors was performed by employing a chitinase from Bacillus sp. as catalyst. All the glycosidations proceeded with full control in stereochemistry at the anomeric carbon of the donor and regio-selectivity of the acceptor. The N,N'-diacetyl-6'-O-carboxymethylchitobiose oxazoline derivative 1 was effectively glycosidated, under catalysis by the enzyme, with methyl N,N'-diacetyl-beta-chitobioside (4), pent-4-enyl N-acetyl-beta-D-glucosaminide (5), and methyl N-acetyl-beta-D-glucosaminide (6), affording in good yields the corresponding oligosaccharide derivatives having 6-O-carboxymethyl group at the nonreducing GlcNAc residue. The N,N'-diacetyl-6-O-carboxymethylchitobiose oxazoline derivative 2 was subjected to catalysis by the enzyme catalysis; however, no glycosidated products were produced through the reactions with 4, 5, and 6. Glycosidation reactions of the beta-d-glucosyluronic-(1-->4)-N-acetyl-D-glucosamine oxazoline derivative 3 proceeded with each of the glycosyl acceptors, giving rise to the corresponding oligosaccharide derivative having a GlcA residue at their nonreducing termini in good yields.     

Synthesis of unnatural sugar nucleotides and their evaluation as donor substrates in glycosyltransferase-catalyzed reactions

New unnatural sugar nucleotides, UDP-Fuc and CDP-Fuc were synthesized from fucose-beta-1-phosphate and nucleotide monophosphates activated as morpholidates. Furthermore, a nucleotide analogue was prepared by phosphorylation of 1-(beta-D-ribofuranosyl)cyanuric acid, itself obtained as a protected derivative by condensation of the persilylated derivative of cyanuric acid with 1-O-acetyl-2,3,5-tri-O-benzoyl-beta-D-ribofuranose in 74% yield. This phosphate activated according to the same procedure was condensed with fucose-beta-1-phosphate, affording a new sugar nucleotide conjugate (NDP-Fuc) which was evaluated together with UDP-Fuc, CDP-Fuc and ADP-Fuc, as fucose donors in alpha-(1-->4/3)-fucosyltransferase (FucT-III) catalyzed reaction. Fucose transfer could be observed with each of the donors and kinetic parameters were determined using a fluorescent acceptor substrate. Efficiency of the four analogues towards FucT-III was in the following order: UDP-Fuc=ADP-Fuc>NDP-Fuc>CDP-Fuc. According to the same strategy ADP-GlcNAc was prepared from AMP-morpholidate and N-acetylglucosamine-alpha-1-phosphate; tested as a glucosaminyl donor towards Neisseria meningitidis N-acetylglucosaminyl transferase (LgtA), ADP-GlcNAc was recognized with 0.1% efficiency as compared with UDP-GlcNAc, the natural donor substrate.     

Palmitoyl diol S- and O-phosphoroamidates of some sterols.

The hexamethylphosphorus triamide activated by the addition of iodine at the optimum molar ratio 1.05:0.05 was used as a phosphorylating reagent to synthesize 1-palmitoyloxyethyl-2-O-, 1-palmitoyloxypropyl-3-O- and 1-palmitoyloxybutyl-4-O-(N,N-dimethylamido)thiophosphate and -phosphate derivatives of beta-sitosterol, cholesterol and stigmasterol in a one-pot procedure with overall yields of 80-87%. 1-Palmitoyloxypropyl-3-O-(cholesteryl-3-O)-(N,N-dimethylamido++ +) phosphite was used as a model synthon for the preparation of transamidated morpholido-thiophosphate and -phosphate analogues with final yields of 82-86%.     

Comparison of Energy and Growth Yields for Desulfitobacterium dehalogenans during Utilization of Chlorophenol and Various Traditional Electron Acceptors

Desulfitobacterium dehalogenans grew with formate as the electron donor and 3-chloro-4-hydroxyphenylacetate (3-Cl-4-OHPA) as the electron acceptor, yielding Y(X/formate), Y(X/2e), and Y(X/ATP) ranging from 3.2 to 11.3 g of biomass (dry weight)/mol, thus indicating that energy was conserved through reductive dechlorination. Pyruvate was utilized as the electron donor and acceptor, yielding stoichiometric amounts of acetate and lactate, respectively, and a Y(X/reduced acceptor) of 13.0 g of biomass (dry weight)/mol. The supplementation of pyruvate-containing medium with additional electron acceptors, such as 3-Cl-4-OHPA, nitrate, fumarate, or sulfite, caused pyruvate to be replaced as the electron acceptor and nearly doubled the Y(X/ATP) (Y(X/acetate formed)). A comparison of the yields for 3-Cl-4-OHPA with those for other traditional electron acceptors indicates that the dehalogenation reaction led to the formation of similar amounts of energy equivalents. The various electron acceptors were used concomitantly with 3-Cl-4-OHPA in nonacclimated cultures, but the utilization rates and amounts utilized differed.     

Transglycosylation by Streptococcus mutans GS-5 glucosyltransferase-D: acceptor specificity and engineering of reaction conditions

The acceptor specificity of Streptococcus mutans GS-5 glucosyltransferase-D (GTF-D) was studied, particular the specificity toward non-saccharide compounds. Dihydroxy aromatic compounds like catechol, 4-methylcatechol, and 3-methoxycatechol were glycosylated by GTF-D with a high efficiency. Transglycosylation yields were 65%, 50%, and 75%, respectively, using 40 mM acceptor and 200 mM sucrose as glucosyl donor. 3-Methoxylcatchol was also glycosylated, though at a significantly lower rate. A number of other aromatic compounds such as phenol, 2-hydroxybenzaldehyde, 1,3-dihydroxybenzene, and 1, 2-phenylethanediol were not glycosylated by GTF-D. Consequently GTF-D aromatic acceptors appear to require two adjacent aromatic hydroxyl groups. In order to facilitate the transglycosylation of less water-soluble acceptors the use of various water miscible organic solvents (cosolvents) was studied. The flavonoid catechin was used as a model acceptor. Bis-2-methoxyethyl ether (MEE) was selected as a useful cosolvent. In the presence of 15% (v/v) MEE the specific catechin transglucosylation activity was increased 4-fold due to a 12-fold increase in catechin solubility. MEE (10-30% v/v) could also be used to allow the transglycosylation of catechol, 4-methylcatechol, and 3-methoxycatechol at concentrations (200 mM) otherwise inhibiting GTF-D transglycosylation activity.     

3-Ribosyl-6-methyluracil as a phosphate acceptor in the synthesis of the internucleotide bond catalyzed by pancreatic ribonuclease.

The synthesis of uridylyl-(3'-5')-3-ribosyl-6-methyluracil (UprmU) catalyzed by pancreatic ribonuclease (EC 3.4.1.22) has been performed using uridine 2', 3'-cyclic phosphate (U greater than p) as phosphate donor and 3-ribosyl-6-methyluracil (rmU) as phosphate acceptor. The rate of synthesis of UprmU is much higher than that of uridylyl-(3'-5')-uridine (UpU) in a control experiment under the same conditions with uridine as acceptor. The yields of UpU and UprmU were 20 and 17% respectively. The competitive hydrolysis of the initial U greater than p also proceeds faster when rmU is used as the acceptor. The relationship between the conformation of this nucleoside and its acceptor activity in the enzymatic synthesis of the internucleotide bond is discussed.     

Interaction of phosphate analogues with glyceraldehyde-3-phosphate dehydrogenase.

The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase catalyzes the oxidative phosphorylation of D-glyceraldehyde 3-phosphate. A variety of phosphonates have been shown to substitute for phosphate in this reaction [Gardner, J. H., & Byers, L. D., (1977) J. Biol. Chem. 252, 5925--5927]. The dependence of the logarithm of the equilibrium constant for the reaction on the pKa2 value of the phosphonate is characterized by a Br?nsted coefficient, betaeq, of approximately 1. This represents the sensitivity of the transfer of the phosphoglyceroyl group between the active-site sulfhydryl residue (in the acyl-enzyme intermediate) and the acyl acceptor on the basicity of the acyl acceptor. Molybdate (MoO42-) can also serve as an acyl acceptor in the glyceraldehyde-3-phosphate dehydrogenase catalyzed reaction. The second-order rate constant for the reaction with molybdate is only approximately 12 times lower than the reaction with phosphate even though the pKa2 of molybdate is 3.1 units lower than the pKa2 of phosphate. The immediate product of the molybdate reaction is the acyl molybdate, 1-molybdo-3-phosphoglycerate. The acyl molybdate, like the acyl arsenate (the immediate product of the reaction when arsenate is the acyl acceptor), is kinetically unstable. At pH 7.3 (25 degrees C), the half-life for hydrolysis of the acyl molybdate, or the acyl arsenate, is less than 2.5 s. Thus, hydrolysis of 1-molybdo- and 1-arseno-3-phosphoglycerate is at least 2000 times faster than hydrolysis of 1,3-diphosphoglycerate under the same conditions. Glyceraldehyde-3-phosphate dehydrogenase has a fairly broad specificity for acyl acceptors. Most tetrahedral oxy anions tested are substrates for the enzyme (except SO4(2-) and SeO4(2-)). Tetrahedral monoanions such as ReO4- and GeO(OH)3- are not substrates but do bind to the enzyme. These results suggest the requirement of at least one anionic site on the acyl acceptor required for binding and another anionic group on the acyl receptor required for nucleophilic attack on the acyl enzyme.     

Study of the acyl transfer activity of a recombinant amidase overproduced in an Escherichia coli strain. Application for short-chain hydroxamic acid and acid hydrazide synthesis

The study of acyl transfer activity of a wide spectrum amidase from Rhodococcus sp. R312, overproduced in an Escherichia coli strain, revealed that the ‘bi-bi-ping-pong’ type reaction was efficient with only four very-short chain (C₂–C₃) aliphatic amides as substrates. The optimum working pH was 7.0 for all neutral amides. Very short-chain aliphatic carboxylic acids were 10–1000-fold less efficient and the corresponding optimum working pH values depended on the acid used. Very polar molecules, such as water, hydroxylamine and hydrazine, were good acyl acceptors. An [acyl donor]/[acyl acceptor] ratio lower than 0.3-0.5 had to be maintained to avoid enzyme inhibition by excess acyl donor. The different acyl-enzyme complexes generally exhibited high affinity for hydroxylamine or hydrazine (except the propionyl-enzyme complex), so that the residual hydrolysis activities were almost totally inhibited at appropriate acyl acceptor concentrations. Molar conversion yields were higher with hydrazine as acyl acceptor (e.g., 97% with acetamide as acyl donor) because of the higher V_max values, but in all cases, interesting quantities of short-chain hydroxamic acids (2.9-6.5 g l⁻¹) and acid hydrazides (6.4–7.8 g l⁻¹) could be quickly obtained (10–60 min) with small amounts of enzyme (0.04-0.20 g l⁻¹).     

The stereochemical course of the reaction mechanism of trehalose phosphorylase from Schizophyllum commune

Phosphorolysis of α,α-trehalose catalyzed by trehalose phosphorylase from the basidiomycete Schizophyllum commune proceeds via net retention of anomeric configuration and yields α- -glucose 1-phosphate and α- -glucose as the products. In reverse reaction, only the α-anomers of -glucose 1-phosphate and -glucose are utilized as glucosyl donor and acceptor, respectively, and give exclusively the α,α-product. Trehalose phosphorylase converts α- -glucose 1-fluoride and phosphate into α- -glucose 1-phosphate, a reaction requiring the stereospecific protonation of the glucosyl fluoride by a Brønsted acid. The results are discussed with regard to a plausible reaction mechanism of fungal trehalose phosphorylase.     

Biosynthesis of wall teichoic acids in Staphylococcus aureus H, Micrococcus varians and Bacillus subtilis W23. Involvement of lipid intermediates containing the disaccharide N-acetylmannosaminyl N-acetylglucosamine

The precursors for linkage unit (LU) synthesis in Staphylococcus aureus H were UDP-GlcNAc, UDP-N-acetylmannosamine (ManNAc) and CDP-glycerol and synthesis was stimulated by ATP. Moraprenol-PP-GlcNAc-ManNAc-(glycerol phosphate)1-3 was formed from chemically synthesised moraprenol-PP-GlcNAc, UDP-ManNAc and CDP-glycerol in the presence of Triton X-100. LU intermediates formed under both conditions served as acceptors for ribitol phosphate residues, from CDP-ribitol, which comprise the main chain. The initial transfer of GlcNAc-1-phosphate from UDP-GlcNAc was very sensitive to tunicamycin whereas the subsequent transfer of ManNAc from UDP-ManNAc was not. Poly(GlcNAc-1-phosphate) and LU synthesis in Micrococcus varians, with endogenous lipid acceptor, UDP-GlcNAc and CDP-glycerol, was stimulated by UDP-ManNAc. Synthesis of LU on exogenous moraprenol-PP-GlcNAc, with Triton X-100, was dependent on UDP-ManNAc and CDP-glycerol and the intermediates formed served as substrates for polymer synthesis. Membranes from Bacillus subtilis W23 had much lower levels of LU synthesis, but UDP-ManNAc was again required for optimal synthesis in the presence of UDP-GlcNAc and CDP-glycerol. Conditions for LU synthesis on exogenous moraprenol-PP-GlcNAc were not found in this organism. LU synthesis on endogenous acceptor in the absence of UDP-ManNAc was explained by contamination of membranes with UDP-GlcNAc 2-epimerase. Under appropriate conditions, low levels of this enzyme were sufficient to convert UDP-GlcNAc into a mixture of UDP-Glc-NAc and UDP-ManNAc and account for LU synthesis. The results indicate the formation of prenol-PP-GlcNAc-ManNAc-(glycerol phosphate)1-3 which is involved in the synthesis of wall teichoic acids in S. aureus H, M. varians and B. subtilis W23 and their attachment to peptidoglycan.     

Recombinant sucrose phosphorylase from Leuconostoc mesenteroides: characterization, kinetic studies of transglucosylation, and application of immobilised enzyme for production of alpha-D-glucose 1-phosphate

Sucrose phosphorylase catalyzes the reversible conversion of sucrose (alpha-D-glucopyranosyl-1,2-beta-D-fructofuranoside) and phosphate into D-fructose and alpha-D-glucose 1-phosphate. We report on the molecular cloning and expression of the structural gene encoding sucrose phosphorylase from Leuconostoc mesenteroides (LmSPase) in Escherichia coli DH10B. The recombinant enzyme, containing an 11 amino acid-long N-terminal metal affinity fusion peptide, was overproduced 60-fold in comparison with the natural enzyme. It was purified to apparent homogeneity using copper-loaded Chelating Sepharose and obtained in 20% yield with a specific activity of 190 Umg(-1). LmSPase was covalently attached onto Eupergit C with a binding efficiency of 50% and used for the continuous production of alpha-D-glucose 1-phosphate from sucrose and phosphate (600 mM each) in a packed-bed immobilised enzyme reactor (30 degrees C, pH 7.0). The reactor was operated at a stable conversion of 91% (550 mM product) and productivity of approximately 11 gl(-1)h(-1) for up to 600 h. A kinetic study of transglucosylation by soluble LmSPase was performed using alpha-d-glucose 1-phosphate as the donor substrate and various alcohols as acceptors. D- and L-arabitol were found to be good glucosyl acceptors.     

Enzymatic synthesis of aliphatic beta-lactosides as mimic units of glycosphingolipids by use of Trichoderma reesei cellulase

Aliphatic beta-lactosides were directly synthesized by beta-lactosyl transfer reaction from p-nitrophenyl beta-lactoside (Lac beta-pNP) to various 1-alkanols (n = 2-12), utilizing commercially available cellulase preparation of Trichoderma reesei C1. With ethanol acceptor, the enzyme induced ethyl beta-lactoside (1) in 18% yield based on the donor added in aqueous buffer system. When 1-octanol and dodecanol were acceptors, octyl beta-lactoside (2) and dodecyl beta-lactoside (3) were also obtained as transfer products, respectively. In both cases, the addition of sodium cholate as detergent to the reaction system ensured a sufficient solubility of these acceptors and resulted in a remarkable increase of the desired compounds (5-13% yields based on the donor added). Furthermore, the enzyme catalyzed the N-acetyllactosaminyl transfer reaction from p-nitrophenyl beta-N-acetyllactosaminide (LacNAc beta-pNP) not only to 1-alkanol, but also to the OH-4 position of Man and Glc to produce the trisaccharides, Gal beta1-4GlcNAc beta1-4Man (4) and Gal beta1-4GlcNAc beta1-4Glc (5), respectively. The enzyme activities transferring lactosyl and N-acetyllactosaminyl groups were not separated by chromatographies using DEAE-Sepharose Fast Flow and Sephadex 75 pg columns, indicating that the two reactions were catalyzed by a single enzyme. It was specified that a single enzyme works both transglycosylations, based on the substrate competition assay on hydrolysis.     

Modeling bacterial UDP-HexNAc: polyprenol-P HexNAc-1-P transferases

Protein N-glycosylation in eukaryotes and peptidoglycan biosynthesis in bacteria are both initiated by the transfer of a D-N-acetylhexosamine 1-phosphate to a membrane-bound polyprenol phosphate. These reactions are catalyzed by a family of transmembrane proteins known as the UDP-D-N-acetylhexosamine: polyprenol phosphate D-N-acetylhexosamine 1-phosphate transferases. The sole eukaryotic member of this family, the d-N-acetylglucosamine 1-phosphate transferase (GPT), is specific for UDP-GlcNAc as the donor substrate and uses dolichol phosphate as the membrane-bound acceptor. The bacterial translocases, MraY, WecA, and WbpL, utilize undecaprenol phosphate as the acceptor substrate, but differ in their specificity for the UDP-sugar donor substrate. The structural basis of this sugar nucleotide specificity is uncertain. However, potential carbohydrate recognition (CR) domains have been identified within the C-terminal cytoplasmic loops of MraY, WecA, and WbpL that are highly conserved in family members with the same UDP-N-acetylhexosamine specificity. This review focuses on the catalytic mechanism and substrate specificity of these bacterial UDP-D-N-acetylhexosamine: polyprenol phosphate D-N-acetylhexosamine 1-P transferases and may provide insights for the development of selective inhibitors of cell wall biosynthesis.     

Some Properties of Potato Tuber UDPGd-fructose-2-glucosyltransferase (E.C. 2.4.1.14) and UDPGd-fructose-6-phosphate-2-glucosyltransferase (E.C. 2.4.1.13)

Sucrose and sucrose 6-phosphate synthetase were isolated from potato tubers, partially purified and their properties studied. The sucrose synthetase showed optimum activity at 45° and was inhibited competitively by ADP and some phenolic glucosides. The Ki′s for these inhibitors were determined. Mg²⁺ was found to activate this enzyme. Activity toward UDP-glucose or ADP-glucose formation was measured. The optimum conditions for sucrose and UDP-glucose formation were found to differ. The specificity for the glucosyl donor and acceptor were determined.

The optimum conditions for sucrose 6-phosphate synthetase activity were studied. This enzyme was not inhibited by either ADP or phenolic glucosides; UDP-glucose was the only glucosyl donor for sucrose 6-phosphate formation.

     

Efficient chemoenzymatic oligosaccharide synthesis by reverse phosphorolysis using cellobiose phosphorylase and cellodextrin phosphorylase from Clostridium thermocellum

Inverting cellobiose phosphorylase (CtCBP) and cellodextrin phosphorylase (CtCDP) from Clostridium thermocellum ATCC27405 of glycoside hydrolase family 94 catalysed reverse phosphorolysis to produce cellobiose and cellodextrins in 57% and 48% yield from α-d-glucose 1-phosphate as donor with glucose and cellobiose as acceptor, respectively. Use of α-d-glucosyl 1-fluoride as donor increased product yields to 98% for CtCBP and 68% for CtCDP. CtCBP showed broad acceptor specificity forming β-glucosyl disaccharides with β-(1→4)- regioselectivity from five monosaccharides as well as branched β-glucosyl trisaccharides with β-(1→4)-regioselectivity from three (1→6)-linked disaccharides. CtCDP showed strict β-(1→4)-regioselectivity and catalysed linear chain extension of the three β-linked glucosyl disaccharides, cellobiose, sophorose, and laminaribiose, whereas 12 tested monosaccharides were not acceptors. Structure analysis by NMR and ESI-MS confirmed two β-glucosyl oligosaccharide product series to represent novel compounds, i.e. β-d-glucopyranosyl-[(1→4)-β-d-glucopyranosyl]_n-(1→2)-d-glucopyranose, and β-d-glucopyranosyl-[(1→4)-β-d-glucopyranosyl]_n-(1→3)-d-glucopyranose (n = 1–7). Multiple sequence alignment together with a modelled CtCBP structure, obtained using the crystal structure of Cellvibrio gilvus CBP in complex with glucose as a template, indicated differences in the subsite +1 region that elicit the distinct acceptor specificities of CtCBP and CtCDP. Thus Glu636 of CtCBP recognized the C1 hydroxyl of β-glucose at subsite +1, while in CtCDP the presence of Ala800 conferred more space, which allowed accommodation of C1 substituted disaccharide acceptors at the corresponding subsites +1 and +2. Furthermore, CtCBP has a short Glu496-Thr500 loop that permitted the C6 hydroxyl of glucose at subsite +1 to be exposed to solvent, whereas the corresponding longer loop Thr637–Lys648 in CtCDP blocks binding of C6-linked disaccharides as acceptors at subsite +1. High yields in chemoenzymatic synthesis, a novel regioselectivity, and novel oligosaccharides including products of CtCDP catalysed oligosaccharide oligomerisation using α-d-glucosyl 1-fluoride, all together contribute to the formation of an excellent basis for rational engineering of CBP and CDP to produce desired oligosaccharides.