Substrate Selectivity in Aspergillus niger KU-8 Acid Phosphatase II Using Phosphoryl Oligosaccharides

The intracellular acid phosphatase II (ACPase II) produced by Aspergillus niger KU-8 preferentially dephosphorylates C-6 phosphate groups rather than C-3 phosphate groups of phosphoryl oligosaccharides. In this study, the kinetic parameters of ACPase II were measured. 3²-phosphoryl maltotriose and 6²-phosphoryl maltotriose, which differ only in the binding position of the phosphate group, were prepared and used as the substrates. The K _m for both substrates were similar. However, the k _cat value for the 6²-phosphoryl maltotriose was about three-fold of that for the 3²-phosphoryl maltotriose.     

Trichoderma harzianum Produces a New Thermally Stable Acid Phosphatase,with Potential for Biotechnological Application

Acid phosphatases (ACPases) are produced by a variety of fungi and have gained attention due their biotechnological potential in industrial, diagnosis and bioremediation processes. These enzymes play a specific role in scavenging, mobilization and acquisition of phosphate, enhancing soil fertility and plant growth. In this study, a new ACPase from Trichoderma harzianum, named ACPase II, was purified and characterized as a glycoprotein belonging to the acid phosphatase family. ACPase II presents an optimum pH and temperature of 3.8 and 65°C, respectively, and is stable at 55°C for 120 min, retaining 60% of its activity. The enzyme did not require metal divalent ions, but was inhibited by inorganic phosphate and tungstate. Affinity for several phosphate substrates was observed, including phytate, which is the major component of phosphorus in plant foods. The inhibition of ACPase II by tungstate and phosphate at different pH values is consistent with the inability of the substrate to occupy its active site due to electrostatic contacts that promote conformational changes, as indicated by fluorescence spectroscopy. A higher affinity for tungstate rather than phosphate at pH 4.0was observed, in accordance with its highest inhibitory effect. Results indicate considerable biotechnological potential of the ACPase II in soil environments.     

The effect of glucose, insulin and noradrenaline on lipolysis and on the concentrations of adenosine 3′:5′-cyclic monophosphate and adenosine 5′-triphosphate in adipose tissue

1. The deoxyfluoro-d-glucopyranose 6-phosphates were prepared from the corresponding deoxyfluoro-d-glucoses and ATP by using hexokinase. 2. 3-Deoxy-3-fluoro- and 4-deoxy-4-fluoro-d-glucose 6-phosphate were substrates for glucose phosphate isomerase, and in addition the products of this reaction, 3-deoxy-3-fluoro- and 4-deoxy-4-fluoro-d-fructose 6-phosphate respectively, were good substrates for phosphofructokinase. 3. Some C-2-substituted derivatives of d-glucose 6-phosphate were found to be competitive inhibitors of glucose phosphate isomerase. 4. The possible role of the hydroxyl groups in the binding of d-glucose 6-phopshate to glucose phosphate isomerase is discussed.     

Transglycosylic reactions of deoxysugars. Biosynthesis of deoxy analogues of starch.

The biosynthesis of starch was investigated in the reaction catalyzed by plant alpha(1 leads to 4)-glucan phosphorylase using alpha-D-glucopyranosyl phosphate and its deoxy analogues as substrates. It was found that the hydroxyl groups at the positions C-2, C-3, C-4 and C-6 in the glucose moiety of the molecule of alpha-D-glucopyranosyl phosphate are not essential for its substrate properties in the transglycosylic reaction. The affinity of plant (alpha(1 leads to 4)-glucan phosphorylase and the rate of hexose incorporation into alpha(1 leads to 4)-glucan decreases in the following sequence: alpha-D-glucopyranosyl phos-phosphate, 2-deoxy-, 6-deoxy, 4-deoxy, and 3-deoxy-alpha-D-glucopyranosyl phosphate. The deoxyglucosyl analogues of alpha-D-glucosylpyranosyl phosphate act as competitive inhibitors on the elongation reaction of the alpha(1 leads to 4) chains of starch. It was found that more than one residue of 2-deoxy-D-glucose or 6-deoxy-D-glucose can be incorporated into the nonreducing terminus of alpha(1 leads to 4)-glucan chains of starch.     

Determination of substituents distribution in carboxymethylpullulans by NMR spectroscopy

The distribution of carboxymethyl substituents in the alpha-(1 --> 6)-linked maltotriosyl repeating units of a carboxymethylpullulan (CMP) series was investigated by high resolution NMR spectroscopy on very short oligomers (DPn = 1.2-1.5) obtained by acid hydrolysis. A series of 2D NMR experiments on parent pullulan, hydrolysed pullulan and CMP was used to assign the proton and carbon chemical shifts of CMP acid hydrolysates. The degree of substitution (DS) and the relative distribution of -CH2COONa groups at OH-2, OH-3, OH-4 and OH-6 of glucose residues (DSi) were determined from 1H NMR measurements. From a set of CMP samples, widely different in degree of substitution, it was observed that the substitution at C-2 is predominant and decreases according to the order C-2 > C-3 > C-6 > C-4. Taking into account the availability of each OH group in the parent pullulan, an order of relative reactivity of hydroxyl groups is defined according to the relation: Ri = DSi/ni, where ni is the number of free OH groups in a maltotriose unit (MTU) for a given site C-i, the reactivity order was found to be OH-2 > OH-4 > OH-6 > OH-3.     

New insights on the specificity of heparin and heparan sulfate lyases from Flavobacterium heparinum revealed by the use of synthetic derivatives of K5 polysaccharide from E. coli. and 2-O-desulfated heparin

The capsular polysaccharide from E. Coli, strain K5 composed of ...-->4)beta-D-GlcA(1-->4)alpha-D-GlcNAc(1-->4)beta-D-GlcA (1-->..., chemically modified K5 polysaccharides, bearing sulfates at C-2 and C-6 of the hexosamine moiety and at the C-2 of the glucuronic acid residues as well as 2-O desulfated heparin were used as substrates to study the specificity of heparitinases I and II and heparinase from Flavobacterium heparinum. The natural K5 polysaccharide was susceptible only to heparitinase I forming deltaU-GlcNAc. N-deacetylated, N-sulfated K5 became susceptible to both heparitinases I and II producing deltaU-GlcNS. The K5 polysaccharides containing sulfate at the C-2 and C-6 positions of the hexosamine moiety and C-2 position of the glucuronic acid residues were susceptible only to heparitinase II producing deltaU-GlcNS,6S and deltaU,2S-GlcNS,6S respectively. These combined results led to the conclusion that the sulfate at C-6 position of the glucosamine is impeditive for the action of heparitinase I and that heparitinase II requires at least a C-2 or a C-6 sulfate in the glucosamine residues of the substrate for its activity. Iduronic acid-2-O-desulfated heparin was susceptible only to heparitinase II producing deltaU-GlcNS,6S. All the modified K5 polysaccharides as well as the desulfated heparin were not substrates for heparinase. This led to the conclusion that heparitinase II acts upon linkages containing non-sulfated iduronic acid residues and that heparinase requires C-2 sulfated iduronic acid residues for its activity.     

Synthesis of very short chain lysophosphatidyloligodeoxynucleotides

Very short chain 5'-O-lysophosphatidyloligonucleotides [5'-O-(1-O-palmitoyl-sn-glycero-3-phosphoryl)oligodeoxynucleotides, (5'-LyPOdNs)] were synthesized following a two-step chemoenzymatic synthesis. 5'-O-(sn-Glycero-3-phosphoryl)oligodeoxynucleotides (5'-GPOdNs) were first prepared by simply using a phosphoramidite of [(4S)-2,2-dimethyl-1,3-dioxolan-4-yl]methanol (1) in a further coupling step after the solid-phase elongation of each desired oligodeoxynucleotide. Next, the regioselective palmitoylation at the C-1 hydroxyl of the glycerol moiety of 5'-GPOdNs was achieved by a lipase-catalyzed transacylation with trifluoroethyl palmitate in organic solvent. Despite of the molecular bulkiness of 5'-GPOdNs, 2-, 3-, and 4-mer 5'-LyPOdNs were prepared by this procedure. Although in very low yield, 5- and 6-mer 5'-LyPOdNs were also obtained by this way.     

Purification and substrate specificity of honeybee, Apis mellifera L., alpha-glucosidase III

Alpha-glucosidase III, which was different in substrate specificity from honeybee alpha-glucosidases I and II, was purified as an electrophoretically homogeneous protein from honeybees, by salting-out chromatography, DEAE-cellulose, DEAE-Sepharose CL-6B, Bio-Gel P-150, and CM-Toyopearl 650M column chromatographies. The enzyme preparation was confirmed to be a monomeric protein and a glycoprotein containing about 7.4% of carbohydrate. The molecular weight was estimated to approximately 68,000, and the optimum pH was 5.5. The substrate specificity of alpha-glucosidase III was kinetically investigated. The enzyme did not show unusual kinetics, such as the allosteric behaviors observed in alpha-glucosidases I and II, which are monomeric proteins. The enzyme was characterized by the ability to rapidly hydrolyze sucrose, phenyl alpha-glucoside, maltose, and maltotriose, and by extremely high Km for substrates, compared with those of alpha-glucosidases I and II. Especially, maltotriose was hydrolyzed over 3 times as rapidly as maltose. However, maltooligosaccharides of four or more in the degree of polymerization were slowly degraded. The relative rates of the k0 values for maltose, sucrose, p-nitrophenyl alpha-glucoside and maltotriose were estimated to be 100, 527, 281 and 364, and the Km values for these substrates, 11, 30, 13, and 10 mM, respectively. The subsite affinities (Ai's) in the active site were tentatively evaluated from the rate parameters for maltooligosaccharides. In this enzyme, it was peculiar that the Ai value at subsite 3 was larger than that of subsite 1.     

Structure of the lipoteichoic acids from Bifidobacterium bifidum spp. pennsylvanicum

The lipoteichoic acids from Bifidobacterium bifidum spp. pennsylvanicum were extracted from cytoplasmic membranes or from disintegrated bacteria with aqueous phenol and purified by gel chromatography. The lipoteichoic acid preparations contained phosphate, glycerol, galactose, glucose and fatty acids in a molar ratio of 1.0:1.0:1.3:1.2:0.3. Chemical analysis and NMR studies of the native preparations and of products from various acid and alkaline hydrolysis procedures gave evidence for the structure of two lipoteichoic acids. The lipid anchor appeared to be 3-O-(6'-(sn-glycero-1-phosphoryl)diacyl-beta-D-galactofuranosyl)-sn-1, 2-diacylglycerol. The polar part showed two structural features not previously described for lipoteichoic acids. A 1,2-(instead of the usual 1,3-) phosphodiester-linked sn-glycerol phosphate chain is only used substituted at the terminal glycerol unit with a linear polysaccharide, containing either beta(1----5)-linked D-galactofuranosyl groups or beta(1----6)-linked D-glucopyranosyl groups.     

Structural studies of lipid A from Pseudomonas aeruginosa PAO1: occurrence of 4-amino-4-deoxyarabinose.

Lipid A derived from Pseudomonas aeruginosa PAO1 contains a biphosphorylated 1-6-linked glucosamine disaccharide backbone. The reducing glucosamine has an unsubstituted glycosidically linked phosphate at C-1. The nonreducing glucosamine has an ester-bound phosphate at C-4' which is nonstoichiometrically substituted with 4-amino-4-deoxyarabinose. Induction of 4-amino-4-deoxyarabinose was dependent on cultural conditions. No pyrophosphate groups were detected. Acyloxyacyl diesters are formed by esterification of the amide-bound 3-hydroxydodecanoic acid with dodecanoic acid and 2-hydroxydodecanoic acids in an approximate molar ratio of 2:1. Dodecanoic and 3-hydroxydecanoic acids are esterified to positions C-3 and C-3' in the sugar backbone. All hydroxyl groups of the glucosamine disaccharide except C-4 and C-6' are substituted. Lipopolysaccharide chemical analyses measured glucose, rhamnose, heptose, galactosamine, alanine, phosphate, and glucosamine. The proposed lipid A structure differs from previous models. There are significant differences in acyloxyacyl diesters, and the proposed model includes an aminopentose substituent.     

Heterogeneity of lipid A: structural determination by 13C and 31P NMR of lipid A fractions from lipopolysaccharide of Escherichia coli 0111

Purified lipid A from Escherichia coli 0111 was fractionated by thin-layer chromatography, and seven major bands were studied by 13C and 31P NMR. All lipid A fractions except one had fatty acids, 3-hydroxytetradecanoic acid, 3-(acyloxy)tetradecanoic acid, and phosphate groups bonded to the diglucosamine backbone. The remaining fraction was shown to be phosphatidylethanolamine. The number of substituents found showed that in all fractions all sites available for C-acylation (C-3, C-4, and C-3') and N-acylation (C-2 and C-2') carried acylic substituents. The number, ranging from four to six, and type of ester-bound carboxylic acid residues as well as the number of phosphate groups differed among the fractions. The three fastest moving bands all had three unsubstituted hydroxy fatty acids and one phosphate group (C-4'), while the slower moving bands had four hydroxy fatty acids and two phosphate groups. Unsubstituted 3-hydroxytetradecanoic acid residues were amide-bound to the disaccharide in all but one of the fractions. In summary, the heterogeneity of E. coli 0111 lipid A is found to be a consequence of a variation of the number and composition of carboxylic acid residues and of varying phosphate content.     

Ribosomal proteins P0, P1, and P2 are phosphorylated by casein kinase II at their conserved carboxyl termini

A potential casein kinase II (CK II) recognition site is located within the conserved carboxyl (COOH) terminus of the ribosomal P (phospho) proteins P0, P1, and P2. To determine whether the COOH termini of the P proteins are physiological substrates for CK II, we studied the phosphorylation of the P proteins in vitro and in intact cells. The results show that the addition of exogenous purified CK II and ATP to intact ribosomes in vitro resulted in the relatively selective phosphorylation of all three P proteins. A synthetic peptide corresponding to the COOH-terminal 22 amino acids of P2 (C-22) was also phosphorylated by CK II with a Km of 13.4 microM. An endogenous ribosome-associated, CK II-like enzyme also phosphorylated the P proteins relatively selectively in the presence of 10 mM Mg2+ and ATP. The endogenous kinase was inhibited by heparin, utilized either ATP or GTP as a phosphate donor, and phosphorylated casein. A CK II-specific peptide (Arg-Arg-Arg-Glu-Glu-Glu-Thr-Glu-Glu-Glu) and the C-22 peptide inhibited the phosphorylation of the P proteins by the endogenous kinase, providing further evidence for its CK II-like properties and for localization of the CK II phosphorylation site to the COOH termini of the P proteins. Tryptic phosphopeptide maps of P1 and P2 phosphorylated by exogenous CK II and the endogenous ribosome-bound kinase were virtually identical. These phosphopeptides comigrated with the tryptic digest of C-22 and with the tryptic phosphopeptides derived from P1 and P2 isolated from intact cells metabolically labeled with [32P]orthophosphate in vivo. These studies demonstrate that exogenous CK II and a ribosome-bound, CK II-like enzyme phosphorylate the ribosomal P proteins in vitro and localize the target site for phosphorylation to the COOH terminus. The incorporation of phosphate into the same target site in intact cells indicates that the P proteins are in vivo substrates of CK II.     

Chemical structure of the lipid A component of Plesiomonas shigelloides and its taxonomical significance

Abstract The chemical structure of the lipid A moiety of the lipopolysaccharide of the type strain of Plesiomonas shigelloides was elucidated. It consists of a β-(1 → 6)-linked glucosamine disaccharide carrying phosphate groups at C-1 of the reducing and at C-4' of the non-reducing glucosamine. It contains a total of 6 residues of fatty acids, 2 amide-linked and 4 ester-linked. The amino groups of the backbone disaccharide are N -acylated by substituted 3-hydroxyacyl residues: at the reducing glucosamine by 3-O-(14:0)14:0; and at the non-reducing glucosamine by 3-O-(12:0)14:0.
Two residues of 3-hydroxytetradecanoic acid are linked to C-3 and C-3' of the glucosamine residues; the hydroxy groups of these ester-linked 3-hydroxytetradecanoic acids are unsubstituted. In free lipid A, the hydroxyl groups at C-4 and C-6' are unsubstituted, indicating that the 2-keto-3-deoxyoctonic acid (KDO) is linked to C-6' of the non-reducing glucosamine, as was shown with enterobacterial lipid A. The taxonomical significance of these structural details is discussed.     

Kinetics of the hydrolysis of di- and trisaccharides with Aspergillus niger glucoamylases I and II

Near-homogeneous forms of glucoamylases I and II, previously purified from an industrial Aspergillus niger preparation, were used to hydrolyze a number of di- and trisaccharides linked by alpha-D-glucosidic bonds. Maximum rates and Michaelis constants were obtained at various temperatures and pH values with glucoamylase I for the disaccharides beta,alpha-trehalose, kojibiose, nigerose, maltose, and isomaltose and the trisaccharides panose and iso-maltotriose, and with glucoamylase II for maltose, maltotriose, and isomaltotriose. Maximum rates were highest and energies of activation were lowest for maltose, maltotriose, and panose, the only three substrates containing alpha-D-(1, 4)-glucosidic bonds. Michaelis constants were lowest and standard heats of binding were most negative for maltose and maltotriose. The variation of maximum rates and Michaelis constants with varying pH values suggested that two carboxyl groups were involved in substrate binding.     

Purification and Substrate Specificity of Honeybee,Apis mellifera L., α-Glucosidase III

α-Glucosidase III, which was different in substrate specificity from honeybee α-glucosidases I and II, was purified as an electrophoretically homogeneous protein from honeybees, by salting-out chromatography, DEAE-cellulose, DEAE-Sepharose CL-6B, Bio-Gel P-150, and CM-Toyopearl 650M column chromatographies. The enzyme preparation was confirmed to be a monomeric protein and a glycoprotein containing about 7.4% of carbohydrate. The molecular weight was estimated to approximately 68,000, and the optimum pH was 5.5. The substrate specificity of α-glucosidase III was kinetically investigated. The enzyme did not show unusual kinetics, such as the allosteric behaviors observed in α-glucosidases I and II, which are monomeric proteins. The enzyme was characterized by the ability to rapidly hydrolyze sucrose, phenyl α-glucoside, maltose, and maltotriose, and by extremely high K_m for substrates, compared with those of α-glucosidases I and II. Especially, maltotriose was hydrolyzed over 3 times as rapidly as maltose. However, maltooligosaccharides of four or more in the degree of polymerization were slowly degraded. The relative rates of the k₀ values for maltose, sucrose, p-nitrophenyl α-glucoside and maltotriose were estimated to be 100, 527, 281 and 364, and the K_m values for these substrates, 11, 30, 13, and 10 mM, respectively. The subsite affinities (A_i’s) in the active site were tentatively evaluated from the rate parameters for maltooligosaccharides. In this enzyme, it was peculiar that the A_i value at subsite 3 was larger than that of subsite 1.     

Microbial transformations of flavanone and 6-hydroxyflavanone by Aspergillus niger strains

Flavanone (1) and 6-hydroxyflavanone (2) were subjected to transformation by means of Aspergillus niger strains (one wild and three UV mutants). For both substrates the biotransformation resulted in reduction of the carbonyl group (products 5 and 7) and dehydrogenation at C-2 and C-3 (3 and 8). Additionally, for flavanone (1) reduction of C-4 together with hydroxylation at C-7 (6) and dehydrogenation at C-2, C-3 along with hydroxylation at C-3 (4) were observed.     

Isotope effects in 3-dehydroquinate synthase and dehydratase. Mechanistic implications.

The mechanism of 3-dehydroquinate synthase was explored by incubating partially purified enzyme with mixtures of [1-14C]3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP) and one of the specifically tritiated substrates [4-3H]DAHP, [5-3H]DAHP, [6-3H]DAHP, (7RS)-[7-3H]DAHP, (7R)-[7-3H]DAHP, or (7S)-[7-3H]DAHP. Kinetic and secondary 3H isotope effects were calculated from 3H:14C ratios obtained in unreacted DAHP, 3-dehydroquinate, and 3-dehydroshikimate. 3H was not incorporated from the medium into 3-dehydroquinate, indicating that a carbanion (or methyl group) at C-7 is not formed. A kinetic isotope effect kH/k3H of 1.7 was observed at C-5, and afforded support for a mechanism involving oxidation of C-5 with NAD. A similar kinetic isotope effect was found at C-6 owing to removal of a proton in elimination of phosphate, which is reasonably assumed to be the next step in 3-dehydroquinate synthase. Hydrogen at C-7 of DAHP was not lost in the cyclization step of the reaction, indicating that the enol formed in phosphate elimination participated directly in an aldolase-type reaction with the carbonyl at C-2. In the dehydration of 3-dehydroquinate to 3-dehydroshikimate the (7R) proton from (7RS)- or (7R)-[7-3H]DAHP is lost, indicating that the 7R proton occupies the 2R position in dehydroquinate. Hence the cyclization step occurs with inversion of configuration at C-7. A kinetic isotope effect kH/k3H = 2.3 was observed in the conversion of (2R)-[2-3H]dehydroquinate to dehydroshikimate. Hence loss of a proton from the enzyme-dehydroquinate imine contributed to rate limitation in the reaction.     

Localization of acid phosphatase activity in the apoplast of pea (Pisum sativum L.) root nodules grown under phosphorus deficiency

ACPase activity was localized in the apoplast of pea root nodules under phosphorus deficiency. Pea plants (Pisum sativum L. cv. Sze ciotygodniowy) where inoculated with Rhizobium leguminosarum bv. viciae 248 and were cultured on nitrogen-free medium with phosphate (−N/+P) or phosphate-deficient (−N/−P) one. In comparison with control nodules, P-deficient nodules showed the increase of ACPase activity in plant cell walls and the infection threads. The increase in bacterial ACPase activity under P-deficiency may reflect higher demand for inorganic phosphorus that is necessary for bacteria multiplication within the infection threads. The increase of ACPase activity in nodule apoplast under P stress may enlarge the availability of phosphate for plant and bacteria.     

The fructose 6-phosphate site of phosphofructokinase. Epimeric specificity.

The epimeric specificity of the catalytic site of rabbit muscle phosphofructokinase was investigated by testing three ketose phosphates as alternate substrates. These (and their epimeric carbons) included: D-psicose-6-P (C-3), D-tagatose-6-P (C-4), and L-sorbose-6-P (C-5). The Michaelis constants (and relative maximal velocities) were: 3.0 mM (45%), 0.054 mM (104%), and 11 mM (15%), respectively. Under the same conditions, D-fructose-6-P had a Km of 0.043 mM and an arbitrary Vmax of 100%. The low affinity of the enzyme for D-psicose-6-P indicates that the L configuration at C-3 is required for effective binding, a specificity similar to several other fructose-metabolizing enzymes. The D configuration at C-5 is also important for tight binding and the proper orientation of the phosphate group of the substrate. The kinetic constants of D-tagatose-6-P were identical with those of D-fructose-6-P, within experimental error. Thus, the configuration at C-4 is not essential for activity; an indication that D-tagatose may be utilized in mammalian tissues. A novel method for the synthesis of D-psicose-6-P and an improved procedure for the synthesis of D-tagatose-6-P are described. All products and intermediates were characterized unequivocally by chemical and physical methods.     

Phosphonomethyl analogues of hexose phosphates.

The analogue of fructose 1,6-bisphosphate in which the phosphate group, -O-PO3H2, on C-6 is replaced by the phosphonomethyl group, -CH2-PO3H2, was made enzymically from the corresponding analogue of 3-phosphoglycerate. It was a substrate for aldolase, which was used to form it, but not for fructose 1,6-bisphosphatase. It was hydrolysed chemically to yield the corresponding analogue of fructose 6-phosphate [i.e. 6-deoxy-6-(phosphonomethyl)-D-fructose, or, more strictly, 6,7-dideoxy-7-phosphono-D-arabino-2-heptulose]. This proved to be a substrate for the sequential actions of glucose 6-phosphate isomerase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. Thus seven out of the nine enzymes of the glycolytic and pentose phosphate pathways so far tested catalyse the reactions of the phosphonomethyl isosteres of their substrates.