scholarly journals An Orthologue of Bacteroides fragilis NanH Is the Principal Sialidase in Tannerella forsythia

2009 ◽  
Vol 191 (11) ◽  
pp. 3623-3628 ◽  
Author(s):  
Hayley Thompson ◽  
Karen A. Homer ◽  
Susmitha Rao ◽  
Veronica Booth ◽  
Arthur H. F. Hosie
Keyword(s):  
Sialic Acid ◽  
Biochemical Characterization ◽  
Bacteroides Fragilis ◽  
Periodontal Pathogen ◽  
Putative Virulence Factor ◽  
Tannerella Forsythia ◽  
Sialidase Activity ◽  
Acetylneuraminic Acid ◽  
Ph Range

ABSTRACT Sialidase activity is a putative virulence factor of the anaerobic periodontal pathogen Tannerella forsythia, but it is uncertain which genes encode this activity. Characterization of a putative sialidase, SiaHI, by others, indicated that this protein alone may not be responsible for all of the sialidase activity. We describe a second sialidase in T. forsythia (TF0035), an orthologue of Bacteroides fragilis NanH, and its expression in Escherichia coli. Sialidase activity of the expressed NanH was confirmed by using 2′-(4-methylumbelliferyl)-α-d-N-acetylneuraminic acid as a substrate. Biochemical characterization of the recombinant T. forsythia NanH indicated that it was active over a broad pH range, with optimum activity at pH 5.5. This enzyme has high affinity for 2′-(4-methylumbelliferyl)-α-d-N-acetylneuraminic acid (Km of 32.9 ± 10.3 μM) and rapidly releases 4-methylumbelliferone (V max of 170.8 ± 11.8 nmol of 4-methylumbelliferone min−1 mg of protein−1). E. coli lysates containing recombinant T. forsythia NanH cleave sialic acid from a range of substrates, with a preference for α2-3 glycosidic linkages. The genes adjacent to nanH encode proteins apparently involved in the metabolism of sialic acid, indicating that the NanH sialidase is likely to be involved in nutrient acquisition.

Biochemical characterization of a glucoamylase from Saccharomycopsis fibuligera R64

Biologia ◽  
2011 ◽  
Vol 66 (1) ◽  
Author(s):  
Dessy Natalia ◽  
Keni Vidilaseris ◽  
Pasjan Satrimafitrah ◽  
Wangsa Ismaya ◽  
Purkan ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Amino Acid ◽  
Amino Acid Sequence Identity ◽  
Biochemical Characterization ◽  
Saccharomycopsis Fibuligera ◽  
Sequence Identity ◽  
Ic50 Value ◽  
High Amino Acid ◽  
Ph Range

AbstractGlucoamylase from the yeast Saccharomycopsis fibuligera R64 (GLL1) has successfully been purified and characterized. The molecular mass of the enzyme was 56,583 Da as determined by mass spectrometry. The purified enzyme demonstrated optimum activity in the pH range of 5.6–6.4 and at 50°C. The activity of the enzyme was inhibited by acarbose with the IC50 value of 5 μM. GLL1 shares high amino acid sequence identity with GLU1 and GLA1, which are Saccharomycopsis fibuligera glucoamylases from the strains HUT7212 and KZ, respectively. The properties of GLL1, however, resemble that of GLU1. The elucidation of the primary structure of GLL1 contributes to the explanation of this finding.

scholarly journals Kinetic studies on the acid hydrolysis of the methyl ketoside of unsubstituted and O-acetylated N-acetylneuraminic acid

1973 ◽  
Vol 133 (4) ◽  
pp. 623-628 ◽  
Author(s):  
A. Neuberger ◽  
Wendy A. Ratcliffe
Keyword(s):  
Sialic Acid ◽  
Acid Hydrolysis ◽  
Model Compound ◽  
Kinetic Studies ◽  
Order Rate Constant ◽  
Neuraminic Acid ◽  
Acetylneuraminic Acid ◽  
Rate Of Hydrolysis ◽  
Ph Range ◽  
Hydrolysis Of

The hydrolysis of the model compound 2-O-methyl-4,7,8,9-tetra-O-acetyl-N-acetyl-α-d-neuraminic acid and neuraminidase (Vibrio cholerae) closely resembled that of the O-acetylated sialic acid residues of rabbit Tamm–Horsfall glycoprotein. This confirmed that O-acetylation was responsible for the unusually slow rate of acid hydrolysis of O-acetylated sialic acid residues observed in rabbit Tamm–Horsfall glycoprotein and their resistance to hydrolysis by neuraminidase. The first-order rate constant of hydrolysis of 2-methyl-N-acetyl-α-d-neuraminic acid by 0.05m-H2SO4 was 56-fold greater than that of 2-O-methyl-4,7,8,9-tetra-O-acetyl-N-acetyl -α-d-neuraminic acid. Kinetic studies have shown that in the pH range 1.00–3.30, the observed rate of hydrolysis of 2-methyl-N-acetyl-α-d-neuraminic acid can be attributed to acid-catalysed hydrolysis of the negatively charged CO2− form of the methyl ketoside.

scholarly journals Characterization of the bacteriocin-producing strain Lactobacillus paracasei subsp. paracasei BGUB9

2010 ◽  
Vol 62 (4) ◽  
pp. 889-899 ◽  
Author(s):  
Maja Tolinacki ◽  
M. Kojic ◽  
Jelena Lozo ◽  
Amarela Terzic-Vidojevic ◽  
L. Topisirovic ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
Biochemical Characterization ◽  
Proteolytic Enzymes ◽  
Lactobacillus Paracasei ◽  
Plasmid Curing ◽  
Closely Related Species ◽  
Antimicrobial Spectrum ◽  
Activity Test ◽  
Ph Range

The strain Lactobacillus paracasei subsp. paracasei BGUB9 that was isolated from traditionally homemade hard cheese produces bacteriocin designated as BacUB9, with an approximate molecular mass of 4 kDa. Biochemical characterization and the antimicrobial activity test of BacUB9 were performed. The onset of BacUB9 biosynthesis was observed at the end of an exponential phase of growth. Bacteriocin UB9 retained the antimicrobial activity within the pH range from 1 to 10 and after treatment at 100oC for 30 min. The bacteriocin is susceptible to the activity of proteolytic enzymes. Bacteriocin BacUB9 has a very narrow antimicrobial spectrum, limited to several strains that belong to closely related species. The effect of BGUB9 on the growth of the strain Lactobacillus paracasei subsp. paracasei BGHN14 in a mixed culture was monitored. The mode of action of BacUB9 on the strain BGHN14 was identified as bacteriostatic. Plasmid curing results indicated that a plasmid, designated as pUB9, seemed to be responsible for both bacteriocin BacUB9 production and host immunity.

scholarly journals Biochemical Characterization of a New β-Agarase from Cellulophaga Algicola

2019 ◽  
Vol 20 (9) ◽  
pp. 2143 ◽  
Author(s):  
Han ◽  
Zhang ◽  
Yang
Keyword(s):  
Structural Model ◽  
Biochemical Characterization ◽  
Maximum Activity ◽  
Optimal Temperature ◽  
Salt Tolerant ◽  
Wide Ph Range ◽  
Specific Activities ◽  
Ph Range ◽  
Layer Chromatography

Cellulophaga algicola DSM 14237, isolated from the Eastern Antarctic coastal zone, was found to be able to hydrolyze several types of polysaccharide materials. In this study, a predicted β-agarase (CaAga1) from C. algicola was heterologously expressed in Escherichia coli. The purified recombinant CaAga1 showed specific activities of 29.39, 20.20, 14.12, and 8.99 U/mg toward agarose, pure agar, and crude agars from Gracilaria lemaneiformis and Porphyra haitanensis, respectively. CaAga1 exhibited an optimal temperature and pH of 40 oC and 7, respectively. CaAga1 was stable over a wide pH range from 4 to 11. The recombinant enzyme showed an unusual thermostability, that is, it was stable at temperature below or equal to 40oC and around 70 oC, but was thermolabile at about 50 oC. With the agarose as the substrate, the Km and Vmax values for CaAga1 were 1.19 mg/mL and 36.21 U/mg, respectively. The reducing reagent (dithiothreitol) enhanced the activity of CaAga1 by more than one fold. In addition, CaAga1 was salt-tolerant given that it retained approximately 70% of the maximum activity in the presence of 2 M NaCl. The thin layer chromatography results indicated that CaAga1 is an endo-type β-agarase and efficiently hydrolyzed agarose into neoagarotetraose (NA4) and neoagarohexaose (NA6). A structural model of CaAga1 in complex with neoagarooctaose (NA8) was built by homology modeling and explained the hydrolysis pattern of CaAga1.

scholarly journals Characterization of a sialate-O-acetylesterase (NanS) from the oral pathogen Tannerella forsythia that enhances sialic acid release by NanH, its cognate sialidase

2015 ◽  
Vol 472 (2) ◽  
pp. 157-167 ◽  
Author(s):  
Chatchawal Phansopa ◽  
Radoslaw P. Kozak ◽  
Li Phing Liew ◽  
Andrew M. Frey ◽  
Thomas Farmilo ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Periodontal Disease ◽  
High Activity ◽  
Tannerella Forsythia ◽  
Oral Pathogen ◽  
Oral Pathogens ◽  
Acid Release

We characterize a novel bacterial sialate-O-acetylesterase potentially important for the nutrition of oral pathogens causing periodontal disease by enhancing their ability to harvest sialic acid sugar. Its high activity and stability indicate that it can also be used in glycan pharmacoanalytics.

scholarly journals Sialic acid acquisition in bacteria–one substrate, many transporters

2016 ◽  
Vol 44 (3) ◽  
pp. 760-765 ◽  
Author(s):  
Gavin H. Thomas
Keyword(s):  
Sialic Acid ◽  
Immune Evasion ◽  
Sialic Acids ◽  
Major Facilitator Superfamily ◽  
Acid Uptake ◽  
Gram Negative Bacteria ◽  
Acetylneuraminic Acid ◽  
Wide Range ◽  
Major Facilitator

The sialic acids are a family of 9-carbon sugar acids found predominantly on the cell-surface glycans of humans and other animals within the Deuterostomes and are also used in the biology of a wide range of bacteria that often live in association with these animals. For many bacteria sialic acids are simply a convenient source of food, whereas for some pathogens they are also used in immune evasion strategies. Many bacteria that use sialic acids derive them from the environment and so are dependent on sialic acid uptake. In this mini-review I will describe the discovery and characterization of bacterial sialic acids transporters, revealing that they have evolved multiple times across multiple diverse families of transporters, including the ATP-binding cassette (ABC), tripartite ATP-independent periplasmic (TRAP), major facilitator superfamily (MFS) and sodium solute symporter (SSS) transporter families. In addition there is evidence for protein-mediated transport of sialic acids across the outer membrane of Gram negative bacteria, which can be coupled to periplasmic processing of different sialic acids to the most common form, β-D-N-acetylneuraminic acid (Neu5Ac) that is most frequently taken up into the cell.

Inhibition of Sialic Acid Loss Greatly Improves Survival of Refrigerated Platelets

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1133-1133
Author(s):  
Gerard Jansen ◽  
Emma C Josefsson ◽  
Qiyong Peter Liu ◽  
Viktoria Rumjantseva ◽  
Herve Falet ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Sodium Salt ◽  
Platelet Transfusion ◽  
Blood Components ◽  
Sialidase Activity ◽  
Acetylneuraminic Acid ◽  
Sialidase Inhibitor ◽  
Rapid Clearance ◽  
Receptor Shedding ◽  
Von Willebrand

Abstract Abstract 1133 Platelets have the shortest shelf life of all major blood components and are the most difficult to store, a fact that complicates platelet transfusion practices. Platelet refrigeration could slow bacterial growth and possibly retard the loss of platelet function following storage. However, in contrast to other blood components, platelets do not tolerate refrigeration and are rapidly cleared from the circulation. We demonstrated that two distinct pathways recognizing GPIba remove refrigerated platelets in recipient's livers: 1) αMβ2 integrins (Mac-1) on hepatic resident macrophages (Kupffer cells) selectively recognize irreversibly clustered b-N-acetylglucosamine (β-GlcNAc)–terminated glycans on GPIbα, and 2) hepatic Asialoglycoprotein (Asg) receptors (Ashwell Morell receptors) recognize desialylated GPIba. We here investigated the mechanism of sialic acid loss during refrigeration. We show, that when refrigerated platelets are rewarmed, they secrete active sialidases, including the lysosomal sialidase Neu1 that remove sialic acid from platelet receptors, specifically from GPIbα. Platelets also express Neu3 on their surfaces, however Neu3 expression appears to be unaffected by platelet refrigeration. Importantly, the recovery and circulation of refrigerated platelets is greatly improved by storage in the presence of the competitive sialidase inhibitor N-Acetylneuraminic Acid, 2,3-Dehydro-2-deoxy-Sodium Salt (DANA). Desialylated von Willebrand receptor (vWfR) complex is also a target for metalloproteinases (MMPs), as GPIbα and GPV are cleaved from the surface of refrigerated platelets. Receptor shedding is inhibited by the metalloproteinase inhibitor GM6001 and does not occur in ADAM17ΔZn/ΔZn platelets expressing inactive ADAM17. Critically, desialylation in the absence of metalloproteinase-mediated receptor shedding is sufficient to induce the rapid clearance of platelets from circulation. Desialylation of platelet vWfR therefore triggers platelet clearance, and primes GPIbα and GPV for metalloproteinase-dependent cleavage. We conclude that desialylation of platelets is caused by increased surface sialidase activity following refrigeration and desialylation of glycoproteins, specifically of GPIbα, promotes receptor cleavage by MMPs. Disclosures: Liu: Velicomedical, Inc: Employment.

scholarly journals A Novel Sialic Acid Utilization and Uptake System in the Periodontal Pathogen Tannerella forsythia

2010 ◽  
Vol 192 (9) ◽  
pp. 2285-2293 ◽  
Author(s):  
Sumita Roy ◽  
C. W. Ian Douglas ◽  
Graham P. Stafford
Keyword(s):  
Escherichia Coli ◽  
Sialic Acid ◽  
Outer Membrane ◽  
Cloning And Expression ◽  
Periodontal Pathogen ◽  
Acid Uptake ◽  
Uptake System ◽  
Tannerella Forsythia ◽  
Inner Membrane ◽  
Biofilm Growth

ABSTRACT Tannerella forsythia is a key contributor to periodontitis, but little is known of its virulence mechanisms. In this study we have investigated the role of sialic acid in biofilm growth of this periodontal pathogen. Our data show that biofilm growth of T. forsythia is stimulated by sialic acid, glycolyl sialic acid, and sialyllactose, all three of which are common sugar moieties on a range of important host glycoproteins. We have also established that growth on sialyllactose is dependent on the sialidase of T. forsythia since the sialidase inhibitor oseltamivir suppresses growth on sialyllactose. The genome of T. forsythia contains a sialic acid utilization locus, which also encodes a putative inner membrane sialic acid permease (NanT), and we have shown this is functional when it is expressed in Escherichia coli. This genomic locus also contains a putatively novel TonB-dependent outer membrane sialic acid transport system (TF0033-TF0034). In complementation studies using an Escherichia coli strain devoid of its outer membrane sialic acid transporters, the cloning and expression of the TF0033-TF0034 genes enabled an E. coli nanR nanC ompR strain to utilize sialic acid as the sole carbon and energy source. We have thus identified a novel sialic acid uptake system that couples an inner membrane permease with a TonB-dependent outer membrane transporter, and we propose to rename these novel sialic acid uptake genes nanO and nanU, respectively. Taken together, these data indicate that sialic acid is a key growth factor for this little-characterized oral pathogen and may be key to its physiology in vivo.

scholarly journals Identification and Biochemical Characterization of the Novel α2,3-Sialyltransferase WbwA from Pathogenic Escherichia coli Serotype O104

2015 ◽  
Vol 197 (24) ◽  
pp. 3760-3768 ◽  
Author(s):  
Diana Czuchry ◽  
Paul Desormeaux ◽  
Melissa Stuart ◽  
Donald L. Jarvis ◽  
Khushi L. Matta ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Sialic Acid ◽  
Biochemical Characterization ◽  
Enzyme Reaction ◽  
T Antigen ◽  
Glycan Structure ◽  
Content Type ◽  
E Coli ◽  
Pathogenic Escherichia Coli

ABSTRACTThe sialyl-T antigen sialylα2-3Galβ1-3GalNAc is a common O-glycan structure in human glycoproteins and is synthesized by sialyltransferase ST3Gal1. The enterohemorrhagicEscherichia coliserotype O104 has the rare ability to synthesize a sialyl-T antigen mimic. We showed here that thewbwAgene of theE. coliO104 antigen synthesis gene cluster encodes an α2,3-sialyltransferase WbwA that transfers sialic acid from CMP-sialic acid to Galβ1-3GalNAcα-diphosphate-lipid acceptor. Nuclear magnetic resonance (NMR) analysis of purified WbwA enzyme reaction product indicated that the sialyl-T antigen sialylα2-3Galβ1-3GalNAcα-diphosphate-lipid was synthesized. We showed that the conserved His-Pro (HP) motif and Glu/Asp residues of two EDG motifs in WbwA are important for the activity. The characterization studies showed that WbwA fromE. coliO104 is a monofunctional α2,3-sialyltransferase and is distinct from human ST3Gal1 as well as all other known sialyltransferases due to its unique acceptor specificity. This work contributes to knowledge of the biosynthesis of bacterial virulence factors.IMPORTANCEThis is the first characterization of a sialyltransferase involved in the synthesis of an O antigen inE. coli. The enzyme contributes to the mimicry of human sialyl-T antigen and has unique substrate specificity but very little sequence identity to other sialyltransferases. Thus, the bacterial sialyltransferase is related to the human counterpart only by the similarity of biochemical activity.

scholarly journals Uptake of N-acetylneuraminic acid by Escherichia coli K-235. Biochemical characterization of the transport system

1987 ◽  
Vol 246 (2) ◽  
pp. 287-294 ◽  
Author(s):  
L B Rodríguez-Aparicio ◽  
A Reglero ◽  
J M Luengo
Keyword(s):  
Escherichia Coli ◽  
Sialic Acid ◽  
Transport System ◽  
Biochemical Characterization ◽  
Osmotic Shock ◽  
Metabolic Inhibitors ◽  
Acid Uptake ◽  
Uptake System ◽  
Acid Transport ◽  
Acetylneuraminic Acid

Kinetic measurement of the uptake of N-acetyl[4,5,6,7,8,9-14C]neuraminic acid by Escherichia coli K-235 was carried out in vivo at 37 degrees C in 0.1 M-Tris/maleate buffer, pH 7.0. Under these conditions uptake was linear for at least 30 min and the Km calculated for sialic acid was 30 microM. The transport system was osmotic-shock-sensitive and was strongly inhibited by uncouplers of oxidative phosphorylation [2,4-dinitrophenol (100%); NaN3 (66%]) and by the metabolic inhibitors KCN (84%) and sodium arsenate (76%). The thiol-containing compounds mercaptoethanol, glutathione, cysteine, dithiothreitol and cysteine had no significant effect on the sialic acid-transport rate, whereas the thiol-modifying reagents N-ethylmaleimide, iodoacetate and p-chloromercuribenzoate almost completely blocked (greater than 94%) the uptake of this N-acetyl-sugar. N-Acetylglucosamine inhibited non-competitively the transport of N-acetylneuraminic acid, whereas other carbohydrates (hexoses, pentoses, hexitols, hexuronic acids, disaccharides, trisaccharides) and N-acetyl-sugars or amino acid derivatives (N-acetylmannosamine, N-acetylcysteine, N-acetylproline and N-acetylglutamic acid) did not have any effect. Surprisingly, L-methionine and its non-sulphur analogue L-norleucine partially blocked the transport of this sugar (50%), whereas D-methionine, D-norleucine, several L-methionine derivatives (L-methionine methyl ester, L-methionine ethyl ester, L-methionine sulphoxide) and other amino acids did not affect sialic acid uptake. The N-acetylneuraminic acid-transport system is induced by sialic acid and is strictly regulated by the carbon source used for E. coli growth, arabinose, lactose, glucose, fructose and glucosamine being the carbohydrates that cause the greatest repressions in this system. Addition of cyclic AMP to the culture broth reversed the glucose effect, indicating that the N-acetylneuraminic acid-uptake system is under catabolic regulation. Protein synthesis is not needed for sialic acid transport.

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