scholarly journals Mechanistic and structural studies into the biosynthesis of the bacterial sugar pseudaminic acid (Pse5Ac7Ac)

2020 ◽  
Vol 18 (5) ◽  
pp. 799-809 ◽  
Author(s):  
Harriet S. Chidwick ◽  
Martin A. Fascione
Keyword(s):  
Structural Studies ◽  
Human Pathogens ◽  
Structural Characterisation ◽  
Nonulosonic Acid ◽  
Pseudaminic Acid

The nonulosonic acid sugar pseudaminic acid, Pse5Ac7Ac, is present on the surface of a number of human pathogens, herein we review the mechanistic and structural characterisation of the enzymes responsible for its biosynthesis in bacteria.

scholarly journals Novel serine/threonine-O-glycosylation with N-acetylneuraminic acid and 3-deoxy-D-manno-octulosonic acid by bacterial flagellin glycosyltransferases

Glycobiology ◽  
2020 ◽  
Author(s):  
Aasawari Khairnar ◽  
Sonali Sunsunwal ◽  
Ponnusamy Babu ◽  
T N C Ramya
Keyword(s):  
Escherichia Coli ◽  
Clostridium Botulinum ◽  
Associated Factors ◽  
Nucleotide Sugar ◽  
Acetylneuraminic Acid ◽  
Geobacillus Kaustophilus ◽  
Nonulosonic Acid ◽  
Glycosyl Donor ◽  
Pseudaminic Acid ◽  
Substrate Promiscuity

Abstract Some bacterial flagellins are O-glycosylated on surface-exposed serine/threonine residues with nonulosonic acids such as pseudaminic acid, legionaminic acid and their derivatives by flagellin nonulosonic acid glycosyltransferases, also called motility-associated factors (Maf). We report here two new glycosidic linkages previously unknown in any organism, serine/threonine-O-linked N-acetylneuraminic acid (Ser/Thr-O-Neu5Ac) and serine/threonine-O-linked 3-deoxy-D-manno-octulosonic acid or keto-deoxyoctulosonate (Ser/Thr-O-KDO), both catalyzed by Geobacillus kaustophilus Maf and Clostridium botulinum Maf. We identified these novel glycosidic linkages in recombinant G. kaustophilus and C. botulinum flagellins that were coexpressed with their cognate recombinant Maf protein in Escherichia coli strains producing the appropriate nucleotide sugar glycosyl donor. Our finding that both G. kaustophilus Maf (putative flagellin sialyltransferase) and C. botulinum Maf (putative flagellin legionaminic acid transferase) catalyzed Neu5Ac and KDO transfer on to flagellin indicates that Maf glycosyltransferases display donor substrate promiscuity. Maf glycosyltransferases have the potential to radically expand the scope of neoglycopeptide synthesis and posttranslational protein engineering.

scholarly journals Cloning, purification and preliminary crystallographic analysis of theHelicobacter pyloripseudaminic acid biosynthesisN-acetyltransferase PseH

2014 ◽  
Vol 70 (9) ◽  
pp. 1276-1279 ◽  
Author(s):  
Yu C. Liu ◽  
Abu I. Ud-Din ◽  
Anna Roujeinikova
Keyword(s):  
Crystallographic Analysis ◽  
Diffusion Method ◽  
X Ray Diffraction ◽  
Data Set ◽  
Cell Parameters ◽  
Nonulosonic Acid ◽  
Pseudaminic Acid ◽  
The Common ◽  
H Pylori ◽  
Development Of Gastric Cancer

Helicobacter pyloriinfection is the common cause of gastritis and duodenal and stomach ulcers, which have been linked to a higher risk of the development of gastric cancer. The motility that facilitates persistent infection requires functional flagella that are heavily glycosylated with 5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-L-manno-nonulosonic acid (pseudaminic acid). Pseudaminic acid biosynthesis protein H (PseH) catalyzes the third step in its biosynthetic pathway, producing UDP-2,4-diacetamido-2,4,6-trideoxy-β-L-altropyranose. Crystals ofH. pyloriPseH have been grown by the hanging-drop vapour-diffusion method using diammonium tartrate as a precipitating agent. The crystals belonged to space groupI222 orI212121, with unit-cell parametersa= 107.8,b= 145.4,c= 166.3 Å. A complete X-ray diffraction data set has been collected to 2.5 Å resolution using cryocooling conditions and synchrotron radiation.

X-Ray Structural Studies of Small-Bite Ligands on Large Cations – Lanthanide(III) Ions and Dimethylphosphate

2020 ◽  
Vol 73 (6) ◽  
pp. 539 ◽  
Author(s):  
Eric J. Chan ◽  
Jack M. Harrowfield ◽  
Brian W. Skelton ◽  
Allan H. White
Keyword(s):  
Single Crystal ◽  
Structural Studies ◽  
X Ray ◽  
Structural Characterisation ◽  
The Third ◽  
Lanthanide Series ◽  
Binuclear Species ◽  
Lighter Lanthanides

Reactions of lanthanide chlorides or trifluoracetates (tfa) or picrates with trimethylphosphate alone in the first two cases or trimethylphosphate plus 1,10-phenanthroline or 2,2′;6′,2′′-terpyridine in the third, result in the formation of crystalline products containing dimethylphosphate (dmp–). Single crystal X-ray structural characterisation of these materials has shown that the stoichiometrically simple Ln(dmp)3 species obtained with chloride reactants and the lighter lanthanides are polymeric and commonly dimorphic, while the stoichiometrically more variable mixed dmp/tfa complexes have structures closely related to one phase of the Ln(dmp)3 family, and the presence of picrate and aza-aromatic ligands enables the isolation of Y and Lu derivatives containing binuclear species. In all, the dmp– ligands adopt exclusively the κ1O;κ1O′ bridging mode, the overall results indicating that this should apply to the complete lanthanide series.

Fractional isolation and structural characterisation of hemicellulosic polymers from delignified and ultrasonic irradiated sugarcane bagasse

e-Polymers ◽  
2006 ◽  
Vol 6 (1) ◽  
Author(s):  
Jui-Li Ren ◽  
Zeng-Chao Geng ◽  
Chan-Fu Liu ◽  
Feng Xu ◽  
Jin-Xia Sun ◽  
...  
Keyword(s):  
Sugarcane Bagasse ◽  
Glucuronic Acid ◽  
Structural Studies ◽  
Sugar Composition ◽  
Total Sugars ◽  
Structural Characterisation ◽  
Molecular Weights ◽  
Second Stage ◽  
Total Dissolution ◽  
C Nmr

AbstractHemicellulose-type polysaccharides were isolated from the delignified and ultrasonic irradiated sugarcane bagasse by a sequential two-step alkaline extraction. It was found that the successive extractions with 15% and 18% KOH for 2 h, 15% and 18% NaOH for 2 h, 8% and 10% KOH for 15 h, and with 8% and 10% NaOH for 15 h resulted in a total dissolution of 89.6%, 92.8%, 94.9%, and 97.3% of the original hemicelluloses, respectively. Sugar analysis revealed that xylose was the predominant sugar composition of all the hemicelluloses, comprising 57.4- 68.6% of the total sugars. Arabinose (12.3-18.4%) and glucose (10.8- 14.6%) appeared as the second and third major sugar constituents. Galactose (3.9-8.7%), uronic acids, mainly 4-O-methyl-glucuronic acid (2.7-5.8%), rhamnose (1.2-2.6%), and mannose (0.2-1.3%) were observed as minor constituents. The structural studies by 13C-NMR spectroscopy showed that L-arabino (4-O-methyl-D-glucurono) xylans were the major constituents of the hemicellulosic polymers. Furthermore, the current results also showed that the four alkali-soluble hemicellulosic fractions, isolated during the first step treatment with relatively lower concentrations of alkalis, were more branched and acidic, and had larger molecular weights (Mw, 23100-34500), but lower thermal stability than the other four alkali-soluble hemicellulosic preparations (Mw, 21700-28700), extracted during the second stage treatment with relatively higher concentrations of alkalis.

scholarly journals Structure of the unusual Sinorhizobium fredii HH103 lipopolysaccharide and its role in symbiosis

2020 ◽  
Vol 295 (32) ◽  
pp. 10969-10987 ◽  
Author(s):  
Flaviana Di Lorenzo ◽  
Immacolata Speciale ◽  
Alba Silipo ◽  
Cynthia Alías-Villegas ◽  
Sebastián Acosta-Jurado ◽  
...  
Keyword(s):  
Acid Derivative ◽  
Lipid A ◽  
Core Oligosaccharide ◽  
Sinorhizobium Fredii ◽  
Symbiotic Associations ◽  
Nonulosonic Acid ◽  
Surface Polysaccharides ◽  
Pseudaminic Acid ◽  
Macroptilium Atropurpureum ◽  
K Antigen

Rhizobia are soil bacteria that form important symbiotic associations with legumes, and rhizobial surface polysaccharides, such as K-antigen polysaccharide (KPS) and lipopolysaccharide (LPS), might be important for symbiosis. Previously, we obtained a mutant of Sinorhizobium fredii HH103, rkpA, that does not produce KPS, a homopolysaccharide of a pseudaminic acid derivative, but whose LPS electrophoretic profile was indistinguishable from that of the WT strain. We also previously demonstrated that the HH103 rkpLMNOPQ operon is responsible for 5-acetamido-3,5,7,9-tetradeoxy-7-(3-hydroxybutyramido)-l-glycero-l-manno-nonulosonic acid [Pse5NAc7(3OHBu)] production and is involved in HH103 KPS and LPS biosynthesis and that an HH103 rkpM mutant cannot produce KPS and displays an altered LPS structure. Here, we analyzed the LPS structure of HH103 rkpA, focusing on the carbohydrate portion, and found that it contains a highly heterogeneous lipid A and a peculiar core oligosaccharide composed of an unusually high number of hexuronic acids containing β-configured Pse5NAc7(3OHBu). This pseudaminic acid derivative, in its α-configuration, was the only structural component of the S. fredii HH103 KPS and, to the best of our knowledge, has never been reported from any other rhizobial LPS. We also show that Pse5NAc7(3OHBu) is the complete or partial epitope for a mAb, NB6-228.22, that can recognize the HH103 LPS, but not those of most of the S. fredii strains tested here. We also show that the LPS from HH103 rkpM is identical to that of HH103 rkpA but devoid of any Pse5NAc7(3OHBu) residues. Notably, this rkpM mutant was severely impaired in symbiosis with its host, Macroptilium atropurpureum.

scholarly journals Identification and Characterization of NeuB3 from Campylobacter jejuni as a Pseudaminic Acid Synthase

2005 ◽  
Vol 280 (43) ◽  
pp. 35922-35928 ◽  
Author(s):  
Wayne K. Chou ◽  
Scott Dick ◽  
Warren W. Wakarchuk ◽  
Martin E. Tanner
Keyword(s):  
Campylobacter Jejuni ◽  
Metal Ion ◽  
Bond Cleavage ◽  
Mechanistic Study ◽  
Campylobacter Coli ◽  
Duodenal Ulcers ◽  
Divalent Metal Ion ◽  
Nonulosonic Acid ◽  
Pseudaminic Acid

Campylobacter jejuni and Campylobacter coli are the main causes of bacterial diarrhea worldwide, and Helicobacter pylori is known to cause duodenal ulcers. In all of these pathogenic organisms, the flagellin proteins are heavily glycosylated with a 2-keto-3-deoxy acid, pseudaminic acid (5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-l-manno-nonulosonic acid). The presence of pseudaminic acid is required for the proper development of the flagella and is thereby necessary for motility in, and invasion of, the host. In this study we report the first characterization of NeuB3 from C. jejuni as a pseudaminic acid synthase; the enzyme directly responsible for the biosynthesis of pseudaminic acid. Pseudaminic acid synthase catalyzes the condensation of phosphoenolpyruvate (PEP) with the hexose, 2,4-diacetamido-2,4,6-trideoxy-l-altrose (6-deoxy-AltdiNAc), to form pseudaminic acid and phosphate. The enzymatic activity was monitored using 1H and 31P NMR spectroscopy, and the product was isolated and characterized. Kinetic analysis reveals that pseudaminic acid synthase requires the presence of a divalent metal ion for catalysis and that optimal catalysis occurs at pH 7.0. A coupled enzymatic assay gave the values for kcat of 0.65 ± 0.01 s–1, KmPEP of 6.5 ± 0.4 μm, and Km6-deoxy-AltdiNAc of 9.5 ± 0.7 μm. A mechanistic study on pseudaminic acid synthase, using [2-18O]PEP, shows that catalysis proceeds through a C-O bond cleavage mechanism similar to other PEP condensing synthases such as sialic acid synthase.

scholarly journals The sps Genes Encode an Original Legionaminic Acid Pathway Required for Crust Assembly in Bacillus subtilis

mBio ◽  
2020 ◽  
Vol 11 (4) ◽  
Author(s):  
Thomas Dubois ◽  
Frederic Krzewinski ◽  
Nao Yamakawa ◽  
Christelle Lemy ◽  
Audrey Hamiot ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Food Industry ◽  
Safety Concern ◽  
Superficial Layer ◽  
Biosynthesis Pathway ◽  
Spore Surface ◽  
Human Pathogens ◽  
Adhesion Properties ◽  
Content Type ◽  
Nonulosonic Acid

ABSTRACT The crust is the outermost spore layer of most Bacillus strains devoid of an exosporium. This outermost layer, composed of both proteins and carbohydrates, plays a major role in the adhesion and spreading of spores into the environment. Recent studies have identified several crust proteins and have provided insights about their organization at the spore surface. However, although carbohydrates are known to participate in adhesion, little is known about their composition, structure, and localization. In this study, we showed that the spore surface of Bacillus subtilis is covered with legionaminic acid (Leg), a nine-carbon backbone nonulosonic acid known to decorate the flagellin of the human pathogens Helicobacter pylori and Campylobacter jejuni. We demonstrated that the spsC, spsD, spsE, spsG, and spsM genes of Bacillus subtilis are required for Leg biosynthesis during sporulation, while the spsF gene is required for Leg transfer from the mother cell to the surface of the forespore. We also characterized the activity of SpsM and highlighted an original Leg biosynthesis pathway in B. subtilis. Finally, we demonstrated that Leg is required for the assembly of the crust around the spores, and we showed that in the absence of Leg, spores were more adherent to stainless steel probably because of their reduced hydrophilicity and charge. IMPORTANCE Bacillus species are a major economic and food safety concern of the food industry because of their food spoilage-causing capability and persistence. Their persistence is mainly due to their ability to form highly resistant spores adhering to the surfaces of industrial equipment. Spores of the Bacillus subtilis group are surrounded by the crust, a superficial layer which plays a key role in their adhesion properties. However, knowledge of the composition and structure of this layer remains incomplete. Here, for the first time, we identified a nonulosonic acid (Leg) at the surfaces of bacterial spores (B. subtilis). We uncovered a novel Leg biosynthesis pathway, and we demonstrated that Leg is required for proper crust assembly. This work contributes to the description of the structure and composition of Bacillus spores which has been under way for decades, and it provides keys to understanding the importance of carbohydrates in Bacillus adhesion and persistence in the food industry.

scholarly journals Novel Serine/Threonine-O-glycosylation with N-Acetylneuraminic acid and 3-Deoxy-D-manno-octulosonic acid by Maf glycosyltransferases

2020 ◽  
Author(s):  
Aasawari Khairnar ◽  
Sonali Sunsunwal ◽  
Ponnusamy Babu ◽  
T.N.C. Ramya
Keyword(s):  
Clostridium Botulinum ◽  
Nucleotide Sugar ◽  
Post Translational Modifications ◽  
Acetylneuraminic Acid ◽  
Gram Positive Bacteria ◽  
Geobacillus Kaustophilus ◽  
Nonulosonic Acid ◽  
Glycosyl Donor ◽  
Pseudaminic Acid ◽  
First Time

AbstractSome bacterial flagellins are O-glycosylated on surface-exposed Serine/Threonine residues with nonulosonic acids such as pseudaminic acid, legionaminic acid, and their derivatives by flagellin nonulosonic acid glycosyltransferases, also called Motility associated factors (Maf). We report here two new glycosidic linkages previously unknown in any organism, Serine/Threonine-O-linked N-Acetylneuraminic acid (Ser/Thr-O-Neu5Ac) and Serine/Threonine-O-linked 3-Deoxy-D-manno-octulosonic acid (Ser/Thr-O-KDO), both catalysed by Geobacillus kaustophilus Maf (putative flagellin sialyltransferase) and Clostridium botulinum Maf (putative flagellin legionaminic acid transferase). We identified these novel glycosidic linkages in recombinant G. kaustophilus and C. botulinum flagellins that were co-expressed with their cognate recombinant Maf protein in Escherichia coli strains producing the appropriate nucleotide sugar glycosyl donor. The glycosylation of G. kaustophilus flagellin with KDO, and that of C. botulinum flagellin with Neu5Ac and KDO indicates that Maf glycosyltransferases display donor substrate promiscuity. Maf glycosyltransferases have the potential to radically expand the scope of neoglycopeptide synthesis and posttranslational protein engineering.Significance StatementGlycosylation, the modification of proteins with sugars, is one of the most common post-translational modifications observed in proteins. While glycosylation is versatile, the most common forms of glycosylation are N-glycosylation, where the N atom of Asparagine is modified with a glycan, and O-glycosylation where the O atom of serine or threonine residues is modified with a glycan. Here, we report a novel type of O-glycosylation in the bacterial flagellin proteins of two Gram-positive bacteria, Geobacillus kaustophilus and Clostridium botulinum. We demonstrate for the first time that the enzyme flagellin Maf glycosyltransferase is capable of transferring the monosaccharides, N-acetylneuraminic acid and 3-Deoxy-D-manno-octulosonic acid, on to serine and threonine residues of these proteins.

Ribosome Structural Organization

1974 ◽  
Vol 32 ◽  
pp. 332-333
Author(s):  
James A. Lake
Keyword(s):  
Ribosomal Proteins ◽  
Structural Organization ◽  
Three Dimensional ◽  
Small Subunit ◽  
Structural Studies ◽  
X Ray Diffraction ◽  
Specific Antibodies ◽  
X Ray ◽  
E Coli ◽  
Complete Sequences

The understanding of ribosome structure has advanced considerably in the last several years. Biochemists have characterized the constituent proteins and rRNA's of ribosomes. Complete sequences have been determined for some ribosomal proteins and specific antibodies have been prepared against all E. coli small subunit proteins. In addition, a number of naturally occuring systems of three dimensional ribosome crystals which are suitable for structural studies have been observed in eukaryotes. Although the crystals are, in general, too small for X-ray diffraction, their size is ideal for electron microscopy.

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