Characterization of α2,3- and α2,6-sialyltransferases from Helicobacter acinonychis

Genome sequence data were used to clone and express two sialyltransferase enzymes of the GT-42 family from Helicobacter acinonychis ATCC 51104, a gastric disease isolate from Cheetahs. The deposited genome sequence for these genes contains a large number of tandem repeat sequences in each of them: HAC1267 (RQKELE)(15) and HAC1268 (EEKLLEFKNI)(13). We obtained two clones with different numbers of repeat sequences for the HAC1267 gene homolog and a single clone for the HAC1268 gene homolog. Both genes could be expressed in Escherichia coli and sialyltransferase activity was measured using synthetic acceptor substrates containing a variety of terminal sugars. Both enzymes were shown to have a preference for N-acetyllactosamine, and they each made a product with a different linkage to the terminal galactose. HAC1267 is a mono-functional α2,3-sialyltransferase, whereas HAC1268 is a mono-functional α2,6-sialyltransferase and is the first member of GT-42 to show α2,6-sialyltransferase activity.     

Specific dissociation of αB subunits from α-crystallin

Exposure of bovine α-crystallin to 0.1 M glycine at pH 7 decreases the average molar mass of the protein from 700 to 420 kDa. When the pH is lowered to 2.5, in the same buffer, the αB chains specifically dissociate from the aggregates, leaving a particle of 290 kDa containing only αA chains. The decrease in the molar mass corresponds to the mass of the αB chains in the original aggregate. The pH-dependent dissociation is fully reversible. Similar changes were observed with rat and kangaroo α-crystallins but the dogfish protein was not affected. Sedimentation velocity analyses and fluorescence spectroscopy yielded a pK, for the dissociation, of 3.7 for α-crystallin and 4.0 for a homopolymer constructed from purified αB₂ polypeptides. An αA₂ homopolymer was virtually unaffected by the lowering of pH. The products from the dissociation were isolated and their properties studied by sedimentation analysis and acrylamide quenching of tryptophan fluorescence. The αB chains were found to be completely denatured, whereas the structure of the αA chains, in the 290 kDa, particle, were only slightly altered. Comparisons of the sequences of the various proteins examined suggested that decreased ionization of aspartic acid 127 in the αB chain was responsible for the specific dissociation of this polypeptide.     

Hydrolysis of α-1,6-Glucosidic Linkages by an α-Amylase from Thermoactinomyces vulgaris R-47

We studied DNA breakage by phenyl compounds present in foodstuffs in vitro using γDNA and in cultured mammalian cells using RFL and HeLa cells. Strong in vitro activity was detected in o- and p-dihydroxyphenols, but the m-isomer had no activity. The same results were obtained with aminophenols and phenylenediamines. In flavonoids, the 3-OH group seemed to be active in the DNA breakage, in addition to the odiphenol group. Cellular DNA breakage by the compounds was different from their in vitro activity and varied with the cell lines. RFL cells were preferable to HeLa cells for screening for DNA breaking substances, because of their greater sensitivity.     

Extracellular α-amylase from Streptomyces rimosus

Summary A purification procedure for an extracellular -amylase from Streptomyces rimosus, oxytetracycline-producing strain, is described. The enzyme obtained was shown to be an acidic (pI 4.75) monomer with a relative molecular mass (M_r) of 43 000, containing three cysteines involved in the catalytic activity of the enzyme. Its amino-terminal part has 57–67% homology with amylases from other Streptomyces species. S. rimosus -amylase is sensitive to higher temperatures, and partially stabilized by Ca²⁺ ions. It hydrolyses starch (optimum at pH 5.0–6.0) in an endohydrolase manner giving rise to maltotriose, maltotetraose and higher oligosaccharides. Starch granules, except those from rice, were not significantly affected by the isolated -amylase.     

Purification of α-galactosidase from coconut

α-Galactosidase from coconut endosperm was purified to homogeneity with a 490-fold increase in specific activity. The yield was 70%, and the specific activity was 24.5 units/mg protein. The purification procedure included extraction, acidification, ammonium sulphate fractionation and hydrophobic chromatography. The hydrophobic gel (Sepharose-4B-capranilide) had a capacity of 0.63 mg of α-galactosidase per ml of gel. Purified α-galactosidase was a glycoprotein with a carbohydrate content of 12%. The molar extinction coefficient was 8.7 x 10⁴/M/cm.     

Cytosolic Carboxypeptidase 5 Removes α- and γ-Linked Glutamates from Tubulin

Cytosolic carboxypeptidase 5 (CCP5) is a member of a subfamily of enzymes that cleave C-terminal and/or side chain amino acids from tubulin. CCP5 was proposed to selectively cleave the branch point of glutamylated tubulin, based on studies involving overexpression of CCP5 in cell lines and detection of tubulin forms with antisera. In the present study, we examined the activity of purified CCP5 toward synthetic peptides as well as soluble α- and β-tubulin and paclitaxel-stabilized microtubules using a combination of antisera and mass spectrometry to detect the products. Mouse CCP5 removes multiple glutamate residues and the branch point glutamate from the side chains of porcine brain α- and β-tubulin. In addition, CCP5 excised C-terminal glutamates from detyrosinated α-tubulin. The enzyme also removed multiple glutamate residues from side chains and C termini of paclitaxel-stabilized microtubules. CCP5 both shortens and removes side chain glutamates from synthetic peptides corresponding to the C-terminal region of β3-tubulin, whereas cytosolic carboxypeptidase 1 shortens the side chain without cleaving the peptides'' γ-linked residues. The rate of cleavage of α linkages by CCP5 is considerably slower than that of removal of a single γ-linked glutamate residue. Collectively, our data show that CCP5 functions as a dual-functional deglutamylase cleaving both α- and γ-linked glutamate from tubulin.     

Evidence for α-1,6 and α-1,4-glucosidic bond cleavage in highly branched glycogen by amylopullulanase from Thermoanaerobacter ethanolicus

Summary Activity of amylopullulanase from Thermoanaerobacter ethanolicus 39E on -1,6 and -1,4-glucosidic linkages in highly branched mammalian glycogen was analyzed by paper chromatography and ¹³C nuclear magnetic resonance (NMR) spectroscopy. Paper chromatography analysis showed that the glycogen hydrolysate consisted of glucose, maltose, maltotriose and maltotetraose. NMR spectroscopy confirmed that no hydrolysate products of -1,6 linkage were present resulting from treatment with the amylopullulanase. Therefore, the amylopullulanase efficiently hydrolyzed glycogen both at -1,6- and at -1,4-glucosidic linkages into oligosaccharides.     

Soluble adenosine diphosphate glucose–α-1,4-glucan α-4-glucosyltransferases from spinach leaves

Multiple forms of ADP-glucose-alpha-1,4-glucan alpha-4-glucosyltransferase were obtained from spinach leaves by gradient elution from a DEAE-cellulose column. In the presence of high concentrations of some salts and bovine serum albumin, unprimed activity was found in one (transglucosylase III) of the four fractions eluted from the column. In addition to having unprimed activity, transglucosylase III had a lower K(m) for ADP-glucose, a much higher K(m) for oyster glycogen, greater heat sensitivity and lower affinity for maltose, maltotriose and amylopectin beta-limit dextrin than fractions I, II and IV. In addition, the kinetics at low concentrations of amylose, amylopectin and rabbit liver glycogen were non-linear for transglucosylase III. The properties of transglucosylases I, II and IV were generally similar to each other. Rates of the unprimed reaction at physiological concentrations of ADP-glucose were greater than those found for the primed reaction of fraction III. The product formed by the unprimed reaction was a glucan containing principally alpha-1,4 linkages with some alpha-1,6 linkages. The primer, maltose, at a concentration of 0.5m inhibited the synthesis of the unprimed product.     

Evolutionary History of Eukaryotic α-Glucosidases from the α-Amylase Family

Although some α-glucosidases from the α-amylase family (glycoside hydrolase family GH13) have been studied extensively, their exact number, organization on the chromosome, and orthology/paralogy relationship were unknown. This was true even for important disease vectors where gut α-glucosidase is known to be receptor for the Bin toxin used to control the population of some mosquito species. In some cases orthologs from related species were studied intensively, while potentially important paralogs were omitted. We have, therefore, used a bioinformatics approach to identify all family GH13 α-glucosidases from the selected species from Metazoa (including three mosquito species: Aedes aegypti, Anopheles gambiae, and Culex quinquefasciatus) as well as from Fungi in an effort to characterize their arrangement on the chromosome and evolutionary relationships among orthologs and among paralogs. We also searched for pseudogenes and genes coding for enzymatically inactive proteins with a possible new function. We have found GH13 α-glucosidases mostly in Arthropoda and Fungi where they form gene families, as a result of multiple lineage-specific gene duplications. In mosquito species we have identified 14 α-glucosidase (Aglu) genes of which only five have been biochemically characterized so far, two are putative pseudogenes and the rest remains uncharacterized. We also revealed quite a complex evolutionary history of the eukaryotic α-glucosidases probably involving multiple losses of genes or horizontal gene transfer from bacteria.     

Genetics of α-amylases from rye endosperm

Summary Fifteen inbred lines of rye, F₁ and F₂ progenies from crosses between lines were studied using polyacrylamide gel electrophoresis. Conventional genetic analysis of -amylase zymograms showed that the 19 bands detected in the endosperm of germinating caryopses were controlled by three linked structural loci and one independent modifying locus, which influenced the electrophoretic mobility of isozymes. Two codominant alleles were found at the -Amy1, -Amy2 structural loci and the M--Amy modifying locus while the -Amy3 locus had three alleles. Double-banded expression of the -amylase alleles was probably due to the simultaneous presence of modified and unmodified forms of isozymes on the zymogram.This work was supported by Polish Academy of Sciences under project MR-II/7 and was also a part of the author's PhD Thesis     

Residue-specific α-helix propensities from molecular simulation

Formation of α-helices is a fundamental process in protein folding and assembly. By studying helix formation in molecular simulations of a series of alanine-based peptides, we obtain the temperature-dependent α-helix propensities of all 20 naturally occurring residues with two recent additive force fields, Amber ff03w and Amber ff99SB¹. Encouragingly, we find that the overall helix propensity of many residues is captured well by both energy functions, with Amber ff99SB¹ being more accurate. Nonetheless, there are some residues that deviate considerably from experiment, which can be attributed to two aspects of the energy function: i), variations of the charge model used to determine the atomic partial charges, with residues whose backbone charges differ most from alanine tending to have the largest error; ii), side-chain torsion potentials, as illustrated by the effect of modifications to the torsion angles of I, L, D, N. We find that constrained refitting of residue charges for charged residues in Amber ff99SB¹ significantly improves their helix propensity. The resulting parameters should more faithfully reproduce helix propensities in simulations of protein folding and disordered proteins.     

Enzymatic production of α-dehydrobiotin from biotin

The enzymatic production of α-dehydrobiotin (α-DHB), an antibiotic, from biotinyl-CoA using acyl-CoA oxidase and from biotin using a coupling system of biotinyl-CoA synthetase and acyl-CoA oxidase was developed. Acyl-CoA oxidase was found to show activity for biotinyl-CoA. K_m and V_max values of acyl-CoA oxidase for biotinyl-CoA were 75 μM and 3.92 μmol min⁻¹ mg⁻¹, respectively. Optimum reaction conditions for the α-DHB production from biotin were examined. The maximum production of α-DHB (4.29 μmol ml⁻¹) was obtained, when the reaction was carried out at 30°C for 36 h in a mixture consisting of 100 mM potassium phosphate buffer (pH 8.0), 20 mM biotin, 20 mM ATP, 60 mM CoA, 20 mM MgCl₂, 2 units of biotinyl-CoA synthetase, 90 units of acyl-CoA oxidase and 25 units of catalase in a total volume of 0.6 ml under aerobic conditions. The product was purified from 14 ml of the reaction mixture and 10 mg of crystals with white needle form were obtained. From NMR, mass spectra and other physical analyses, this compound was identified as (+)-trans-α-DHB.     

Synthesis of α-carboxyphosphinopeptides derived from norleucine

In the present study, we describe in detail the synthesis of a relatively rare class of phosphorus compounds, α-carboxyphosphinopeptides. We prepared several norleucine-derived α-carboxyphosphinic pseudopeptides of the general formula Nle-Ψ[PO(OH)]-Gly. These compounds could have important applications as transition state-mimicking inhibitors for methionine or leucine aminopeptidases or other enzymes. For the preparation of the key α-carboxyphosphinate protected precursors, we investigated, compared and improved two different synthetic methods described in literature: the Arbuzov reaction of a silylated N-protected phosphinic acid with a bromoacetate ester and the nucleophilic addition of a mixed O-methyl S-phenyl N-protected phosphonic acid or a methyl N-protected phosphonochloridate with tert-butyl lithioacetate. We also prepared two N-Fmoc protected synthons, Fmoc-Nle-Ψ[PO(OH)]-Gly-COOH and Fmoc-Nle-Ψ[PO(OAd)]-Gly-COOH, and demonstrated that these precursors are suitable building blocks for the solid-phase synthesis of α-carboxyphosphinopeptides.     

Chemical modification of α-galactosidase from coconut

α-Galactosidase from coconut kernel was inhibited by chemical modification of its tyrosine, tryptophan and carboxyl groups. Treatment with N-bromosuccinamide and tetranitromethane indicated that modification of one tryptophan and one tyrosine residue inhibited enzyme activity by 55 and 84%, respectively. Modification of carboxyl groups by carbodiimide indicated that inhibition was due to modification of two carboxyl groups. In the presence of the competitive inhibitor D-galactose, α-galactosidase was protected from inhibition by N-bromosuccinamide, tetranitromethane and carbodiimide. These results indicate that a tryptophan, tyrosine and two carboxyl groups are at or near the active site of α-galactosidase.     

Cloning and functional characterization of an α-1,3-fucosyltransferase from Bacteroides fragilis

Bacteroides fragilis is a clinically important anaerobic pathogen present in the human gastrointestinal tract and is involved in a high number of anaerobic peritoneal infections. The complete genome sequence of B. fragilis NCTC 9343 revealed the presence of several putative fucosyltransferase gene homologues known as alpha-1,3-fucosyltransferases (α-1,3-FucTs). However, their expression and functional activities have not been studied. Here, we report the molecular cloning, functional expression, and characterization of the alpha-1,3-fucosyltransferase 3 (α-1,3-FucT3) enzyme from B. fragilis NCTC 9343. The polymerase chain reaction (PCR)-based approach was used to clone the 331 amino acid long (MW, ～39 kDa) PCR product encoding fucosyltransferase enzyme. The enzyme had low identity of 30–40% with other known α-1,3-FucTs from Azospirillum sp, Rickettsia bellii, and different strains of Helicobacter pylori. An in vitro enzyme reaction analysis showed the ability of the enzyme to transfer the fucose moiety from guanosine-5′-diphosphate β-l-fucose to the N-acetyllactosamine to produce Lewis X. The reaction product, Lewis X was confirmed by thin layer chromatography, liquid chromatography-mass spectroscopy, and ¹H-nuclear magnetic resonance analyses.     

Subunit Composition of the Zinc Proteins α- and β-Lipovitellin from Chicken

Chicken α- and β-lipovitellin are derived from parent vitellogenin proteins and contain four subunits (125, 80, 40, and 30 kDa) and two subunits (125 and 30 kDa), respectively. Metal analyses demonstrate both are zinc proteins containing 2.1 ± 0.2 mol of zinc/275 kDa per α-lipovitellin and 1.4 ± 0.2 mol of zinc/155 kDa per β-lipovitellin, respectively. The subunits of β-lipovitellin, Lv 1 (MW 125 kDa) and Lv 2 (MW 30 kDa), are separated by gel exclusion chromatography in the presence of zwittergent 3–16. Zinc elutes with Lv 1, suggesting that this subunit binds zinc in the absence of Lv 2. The subunits of α- and β-lipovitellin were separated by SDS-PAGE, digested with trypsin, and mapped by reverse-phase HPLC. The peptide maps of the 125-kDa subunits from α- and β-lipovitellin are essentially identical. Similar results are obtained for the 30-kDa subunits of both lipovitellins. The sequences of five and four peptides of the 125-kDa subunit of α- and β-Lv, respectively, and two peptides of the 30-kDa subunit of α- and β-lipovitellin were determined and match those predicted from the gene for vitellogenin II, Vtg II. Comparison of the amino acid composition of the 125- and 30-kDa subunits of α- and β-lipovitellin support the conclusion that they originate from the same gene. The sequences of peptides from the 80- and 40-kDa subunits of α-lipovitellin have not been found in the NCBI nonredundant data bank. The 27-amino acid N-terminal sequence of the 40-kDa protein is 56% similar to the last third of the Lv 1-coding region of the Vtg II gene, suggesting it may come from an analogous region of the Vtg I gene. We propose a scheme for the precursor—product relationship of Vtg I.     

Serum α-6-l-fucosyltransferase is released from platelets during clotting of blood

Activity of serum α-6-l-fucosyltransferase is strictly correlated with the original concentration of blood platelets. The plasma enzyme activity is confined to intact platelets and appears in serum during the course of coagulation of blood. Activity of serum α-3-l-fucosyltransferase does not depend on platelets.     

Properties of an α-1,3-Glucanase from Streptomyces sp. KI-8

Srome properties were examined of purified α-l,3-glucanase isolated from the culture supernatant of the soil microorganism Streptomyces KI-8.

The optimum pH and temperature were pH 5.4 and 60°C, respectively. The α-1,3-glucanase was stable up to 50°C on heating for 10 min. This enzyme hydrolyzed the substrate α-l,3-glucan into glucose and nigerose by an endo-type of action. Nigerotriose, nigerotetraose and nigeropentaose were hydrolyzed into glucose and nigerose, whereas nigerose was not attacked. The degree of hydrolysis of pseudonigeran, Lentinus α-1,3-glucan, mutan IG-1 (less soluble fraction) and IG-2 (more soluble fraction) by the α-1,3-glucanase were 28.5%, 14.3%, 8.8% and 10.0%, respectively. Km values (mg/ml) for pseudonigeran, Lentinus α-l,3-glucan, mutan IG-1 and IG-2 were 1.12, 1.98, 8.00 and 5.00. The enzyme solubilized 50 to 80% of mutan by concerted action with dextranase.     

Production of hydrogen from α-1,4- and β-1,4-linked saccharides by marine hyperthermophilic Archaea

Nineteen hyperthermophilic heterotrophs from deep-sea hydrothermal vents, plus the control organism Pyrococcus furiosus, were examined for their ability to grow and produce H₂ on maltose, cellobiose, and peptides and for the presence of the genes encoding proteins that hydrolyze starch and cellulose. All of the strains grew on these disaccharides and peptides and converted maltose and peptides to H₂ even when elemental sulfur was present as a terminal electron acceptor. Half of the strains had at least one gene for an extracellular starch hydrolase, but only P. furiosus had a gene for an extracellular β-1,4-endoglucanase. P. furiosus was serially adapted for growth on CF11 cellulose and H₂ production, which is the first reported instance of hyperthermophilic growth on cellulose, with a doubling time of 64 min. Cell-specific H₂ production rates were 29 fmol, 37 fmol, and 54 fmol of H₂ produced cell⁻¹ doubling⁻¹ on α-1,4-linked sugars, β-1,4-linked sugars, and peptides, respectively. The highest total community H₂ production rate came from growth on starch (2.6 mM H₂ produced h⁻¹). Hyperthermophilic heterotrophs may serve as an important alternate source of H₂ for hydrogenotrophic microorganisms in low-H₂ hydrothermal environments, and some are candidates for H₂ bioenergy production in bioreactors.     

Inhibitors of α-glucosidase, α-amylase and lipase from Chrysanthemum morifolium

A new endoperoxysesquiterpene lactone, 10α-hydroxy-1α,4α-endoperoxy-guaia-2-en-12,6α-olide (1), together with a flavanone, eriodictyol (2), and two flavone glycosides, acacetin-7-O-β-d-glucopyranoside (3) and acacetin-7-O-α-l-rhamopyranoside (4), were isolated from the methanol extract of Chrysanthemum morifolium flowers by a bioassay-guided fractionation. Compound 1 showed strong inhibitory effects against α-glucosidase and lipase activities, with IC₅₀ values of 229.3 and 161.0 μM, respectively. The flavone glycosides 3 and 4 inhibited both α-glucosidase and α-amylase, while flavanone 2 was only effective against α-amylase.