scholarly journals Functional Characterization of the Initiation Enzyme of S-Layer Glycoprotein Glycan Biosynthesis in Geobacillus stearothermophilus NRS 2004/3a

2007 ◽  
Vol 189 (7) ◽  
pp. 2590-2598 ◽  
Author(s):  
Kerstin Steiner ◽  
René Novotny ◽  
Kinnari Patel ◽  
Evgenij Vinogradov ◽  
Chris Whitfield ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Biochemical Characterization ◽  
Functional Characterization ◽  
Geobacillus Stearothermophilus ◽  
Polysaccharide Biosynthesis ◽  
Glycan Chain ◽  
Galactose Residue ◽  
Serovar Typhimurium

ABSTRACTThe glycan chain of the S-layer glycoprotein ofGeobacillus stearothermophilusNRS 2004/3a is composed of repeating units [→2)-α-l-Rhap-(1→3)-β-l-Rhap-(1→2)-α-l-Rhap-(1→], with a 2-O-methyl modification of the terminal trisaccharide at the nonreducing end of the glycan chain, a core saccharide composed of two or three α-l-rhamnose residues, and a β-d-galactose residue as a linker to the S-layer protein. In this study, we report the biochemical characterization of WsaP of the S-layer glycosylation gene cluster as a UDP-Gal:phosphoryl-polyprenol Gal-1-phosphate transferase that primes the S-layer glycoprotein glycan biosynthesis ofGeobacillus stearothermophilusNRS 2004/3a. Our results demonstrate that the enzyme transfers in vitro a galactose-1-phosphate from UDP-galactose to endogenous phosphoryl-polyprenol and that the C-terminal half of WsaP carries the galactosyltransferase function, as already observed for the UDP-Gal:phosphoryl-polyprenol Gal-1-phosphate transferase WbaP fromSalmonella enterica. To confirm the function of the enzyme, we show that WsaP is capable of reconstituting polysaccharide biosynthesis in WbaP-deficient strains ofEscherichia coliandSalmonella entericaserovar Typhimurium.

scholarly journals Structural and Functional Characterization of ScsC, a Periplasmic Thioredoxin-Like Protein from Salmonella enterica Serovar Typhimurium

2013 ◽  
Vol 19 (13) ◽  
pp. 1494-1506 ◽  
Author(s):  
Mark Shepherd ◽  
Begoña Heras ◽  
Maud E. S. Achard ◽  
Gordon J. King ◽  
M. Pilar Argente ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Salmonella Enterica Serovar Typhimurium ◽  
Functional Characterization ◽  
Serovar Typhimurium

scholarly journals The capsular polysaccharide biosynthesis of Streptococcus pneumoniae serotype 8: functional identification of the glycosyltransferase WciS (Cap8H)

2005 ◽  
Vol 389 (1) ◽  
pp. 63-72 ◽  
Author(s):  
Nehmé SAKSOUK ◽  
Ludovic PELOSI ◽  
Pierre COLIN-MOREL ◽  
Manel BOUMEDIENNE ◽  
Patricia L. ABDIAN ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Capsular Polysaccharide ◽  
Biochemical Characterization ◽  
Repeat Unit ◽  
Sugar Residue ◽  
Functional Identification ◽  
Polysaccharide Biosynthesis ◽  
Sugar Addition ◽  
Galactose Residue

CPS (capsular polysaccharide) is a major virulence factor in Streptococcus pneumoniae. Biosynthesis of CPS RU (repeat unit) proceeds by sequential transfer of sugar residues from the appropriate sugar donor to an activated lipid carrier by committed GTs (glycosyltransferases). While the nucleotide sequence of many cps loci is already known, the real substrate specificity of the hypothetical GTs, as well as the sequence of sugar addition is unclear. In the present paper, we report the biochemical characterization of one α-galactosyltransferase, WciS (Cap8H), a member of GT family 4. This enzyme is implicated in the tetrasaccharide RU biosynthetic pathway of Strep. pneumoniae CPS 8 ([→4)-α-D-Glcp-(1→4)-α-D-Galp-(1→4)-β-D-GlcAp-(1→4)-β-D-Glcp-(1→]n). Expression of WciS–His6 in Escherichia coli BL21 (DE3) strains or BL21 (DE3)/ΔgalU strain resulted in synthesis of a 39 kDa membrane-associated protein identified by N-terminal sequencing and recognized by anti-His6-tag antibody. This protein was capable of adding a galactose residue cellobiuronic acid [β-D-GlcAp-(1→4)-D-Glcp]-pyrophosphate-polyprenol from UDP-Gal. The newly added galactose residue is removed by α-galactosidase, indicating that WciS is a retaining GT. Our results suggest that WciS catalyses the addition of the third sugar residue of the CPS 8 RU. The recombinant WciS–His6 was solubilized and purified as a soluble multimer, opening the way for structural studies.

scholarly journals Genetic and Biochemical Characterization of Salmonella enterica Serovar Typhi Deoxyribokinase

2000 ◽  
Vol 182 (4) ◽  
pp. 869-873 ◽  
Author(s):  
Lise Tourneux ◽  
Nadia Bucurenci ◽  
Cosmin Saveanu ◽  
Pierre Alexandre Kaminski ◽  
Madeleine Bouzon ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Biochemical Characterization ◽  
Site Directed Mutagenesis ◽  
Gene Encoding ◽  
E Coli ◽  
Salmonella Enterica Serovar Typhi ◽  
Acid Protein ◽  
Serovar Typhimurium ◽  
Amino Acid Protein

ABSTRACT We identified in the genome of Salmonella entericaserovar Typhi the gene encoding deoxyribokinase, deoK. Two other genes, vicinal to deoK, were determined to encode the putative deoxyribose transporter (deoP) and a repressor protein (deoQ). This locus, located between theuhpA and ilvN genes, is absent inEscherichia coli. The deoK gene inserted on a plasmid provides a selectable marker in E. coli for growth on deoxyribose-containing medium. Deoxyribokinase is a 306-amino-acid protein which exhibits about 35% identity with ribokinase from serovar Typhi, S. enterica serovar Typhimurium, or E. coli. The catalytic properties of the recombinant deoxyribokinase overproduced in E. colicorrespond to those previously described for the enzyme isolated from serovar Typhimurium. From a sequence comparison between serovar Typhi deoxyribokinase and E. coliribokinase, whose crystal structure was recently solved, we deduced that a key residue differentiating ribose and deoxyribose is Met10, which in ribokinase is replaced by Asn14. Replacement by site-directed mutagenesis of Met10 with Asn decreased theV max of deoxyribokinase by a factor of 2.5 and increased the K m for deoxyribose by a factor of 70, compared to the parent enzyme.

scholarly journals Identification and Functional Characterization of Chicken Toll-Like Receptor 5 Reveals a Fundamental Role in the Biology of Infection with Salmonella enterica Serovar Typhimurium

2005 ◽  
Vol 73 (4) ◽  
pp. 2344-2350 ◽  
Author(s):  
Muhammad Iqbal ◽  
Victoria J. Philbin ◽  
G. S. K. Withanage ◽  
Paul Wigley ◽  
Richard K. Beal ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Salmonella Enterica Serovar Typhimurium ◽  
Functional Characterization ◽  
Systemic Infection ◽  
Structural Similarity ◽  
Conserved Sequence ◽  
Receptor Repertoire ◽  
Serovar Typhimurium ◽  
Toll Like Receptor 5

ABSTRACT Toll-like receptors (TLRs) are a major component of the pattern recognition receptor repertoire that detect invading microorganisms and direct the vertebrate immune system to eliminate infection. In chickens, the differential biology of Salmonella serovars (systemic versus gut-restricted localization) correlates with the presence or absence of flagella, a known TLR5 agonist. Chicken TLR5 (chTLR5) exhibits conserved sequence and structural similarity with mammalian TLR5 and is expressed in tissues and cell populations of immunological and stromal origin. Exposure of chTLR5+ cells to flagellin induced elevated levels of chicken interleukin-1β (chIL-1β) but little upregulation of chIL-6 mRNA. Consistent with the flagellin-TLR5 hypothesis, an aflagellar Salmonella enterica serovar Typhimurium fliM mutant exhibited an enhanced ability to establish systemic infection. During the early stages of infection, the fliM mutant induced less IL-1β mRNA and polymorphonuclear cell infiltration of the gut. Collectively, the data represent the identification and functional characterization of a nonmammalian TLR5 and indicate a role in restricting the entry of flagellated Salmonella into systemic sites of the chicken.

scholarly journals Structural and functional characterization of Salmonella enterica serovar Typhimurium YcbL: An unusual Type II glyoxalase

2010 ◽  
Vol 19 (10) ◽  
pp. 1897-1905 ◽  
Author(s):  
Anna L. Stamp ◽  
Paul Owen ◽  
Kamel El Omari ◽  
Charles E. Nichols ◽  
Michael Lockyer ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Salmonella Enterica Serovar Typhimurium ◽  
Functional Characterization ◽  
Type Ii ◽  
Serovar Typhimurium ◽  
Unusual Type

scholarly journals The stm4066 Gene Product of Salmonella enterica Serovar Typhimurium Has Aminoimidazole Riboside (AIRs) Kinase Activity and Allows AIRs To Satisfy the Thiamine Requirement of pur Mutant Strains

2003 ◽  
Vol 185 (1) ◽  
pp. 332-339 ◽  
Author(s):  
Michael Dougherty ◽  
Diana M. Downs
Keyword(s):  
Gene Product ◽  
Salmonella Enterica ◽  
Kinase Activity ◽  
Salmonella Enterica Serovar Typhimurium ◽  
Mutant Strains ◽  
Serovar Typhimurium ◽  
Pyrimidine Moiety

ABSTRACT In bacteria the biosynthetic pathways for purine mononucleotides and the hydroxymethyl pyrimidine moiety of thiamine share five reactions that result in the formation of aminoimidazole ribotide, the last metabolite common to both pathways. Here we describe the characterization of a Salmonella enterica mutant strain that has gained the ability to efficiently use exogenous aminoimidazole riboside (AIRs) as a source of thiamine. The lesion responsible for this phenotype is a null mutation in a transcriptional regulator of the GntR family (encoded by stm4068). Lack of this protein derepressed transcription of an associated operon (stm4065-4067) that encoded a predicted kinase. The stm4066 gene product was purified and shown to have AIRs kinase activity in vitro. This activity was consistent with the model presented to explain the phenotype caused by the original mutation. This mutation provides a genetic means to isolate the synthesis of the hydroxymethyl pyrimidine moiety of thiamine from the pathway for purine mononucleotide biosynthesis and thus facilitate in vivo analyses.

Biochemical and functional characterization of the human tissue kallikrein 9

2017 ◽  
Vol 474 (14) ◽  
pp. 2417-2433 ◽  
Author(s):  
Panagiota S. Filippou ◽  
Sofia Farkona ◽  
Davor Brinc ◽  
Yijing Yu ◽  
Ioannis Prassas ◽  
...  
Keyword(s):  
Human Tissue ◽  
Biochemical Characterization ◽  
Functional Characterization ◽  
Squamous Carcinoma ◽  
Biochemical Properties ◽  
Tissue Kallikrein ◽  
Related Family ◽  
Squamous Carcinoma Cells

Human tissue kallikrein 9 (KLK9) is a member of the kallikrein-related family of proteases. Despite its known expression profile, much less is known about the functional roles of this protease and its implications in normal physiology and disease. We present here the first data on the biochemical characterization of KLK9, investigate parameters that affect its enzymatic activity (such as inhibitors) and provide preliminary insights into its putative substrates. We show that mature KLK9 is a glycosylated chymotrypsin-like enzyme with strong preference for tyrosine over phenylalanine at the P1 cleavage position. The enzyme activity is enhanced by Mg2+ and Ca2+, but is reversibly attenuated by Zn2+. KLK9 is inhibited in vitro by many naturally occurring or synthetic protease inhibitors. Using a combination of degradomic and substrate specificity assays, we identified candidate KLK9 substrates in two different epithelial cell lines [the non-tumorigenic human keratinocyte cells (HaCaT) and the tumorigenic tongue squamous carcinoma cells (SCC9)]. Two potential KLK9 substrates [KLK10 and midkine (MDK)] were subjected to further validation. Taken together, our data delineate some functional and biochemical properties of KLK9 for future elucidation of the role of this enzyme in health and disease.

scholarly journals Functional identification of ygiP as a positive regulator of the ttdA-ttdB-ygjE operon

Microbiology ◽  
2006 ◽  
Vol 152 (7) ◽  
pp. 2129-2135 ◽  
Author(s):  
Taku Oshima ◽  
Francis Biville
Keyword(s):  
Functional Characterization ◽  
Anaerobic Growth ◽  
Functional Identification ◽  
New Members ◽  
E Coli ◽  
Inner Membrane Protein ◽  
Tartrate Dehydratase

Functional characterization of unknown genes is currently a major task in biology. The search for gene function involves a combination of various in silico, in vitro and in vivo approaches. Available knowledge from the study of more than 21 LysR-type regulators in Escherichia coli has facilitated the classification of new members of the family. From sequence similarities and its location on the E. coli chromosome, it is suggested that ygiP encodes a lysR regulator controlling the expression of a neighbouring operon; this operon encodes the two subunits of tartrate dehydratase (TtdA, TtdB) and YgiE, an integral inner-membrane protein possibly involved in tartrate uptake. Expression of tartrate dehydratase, which converts tartrate to oxaloacetate, is required for anaerobic growth on glycerol as carbon source in the presence of tartrate. Here, it has been demonstrated that disruption of ygiP, ttdA or ygjE abolishes tartrate-dependent anaerobic growth on glycerol. It has also been shown that tartrate-dependent induction of the ttdA-ttdB-ygjE operon requires a functional YgiP.

scholarly journals Functional Characterization of the mazEF Toxin-Antitoxin System in the Pathogenic Bacterium Agrobacterium tumefaciens

2021 ◽  
Vol 9 (5) ◽  
pp. 1107
Author(s):  
Wonho Choi ◽  
Yoshihiro Yamaguchi ◽  
Ji-Young Park ◽  
Sang-Hyun Park ◽  
Hyeok-Won Lee ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Physiological Function ◽  
Functional Characterization ◽  
Site Directed Mutagenesis ◽  
In Vitro Assays ◽  
Cell Growth Inhibition ◽  
The Right

Agrobacterium tumefaciens is a pathogen of various plants which transfers its own DNA (T-DNA) to the host plants. It is used for producing genetically modified plants with this ability. To control T-DNA transfer to the right place, toxin-antitoxin (TA) systems of A. tumefaciens were used to control the target site of transfer without any unintentional targeting. Here, we describe a toxin-antitoxin system, Atu0939 (mazE-at) and Atu0940 (mazF-at), in the chromosome of Agrobacterium tumefaciens. The toxin in the TA system has 33.3% identity and 45.5% similarity with MazF in Escherichia coli. The expression of MazF-at caused cell growth inhibition, while cells with MazF-at co-expressed with MazE-at grew normally. In vivo and in vitro assays revealed that MazF-at inhibited protein synthesis by decreasing the cellular mRNA stability. Moreover, the catalytic residue of MazF-at was determined to be the 24th glutamic acid using site-directed mutagenesis. From the results, we concluded that MazF-at is a type II toxin-antitoxin system and a ribosome-independent endoribonuclease. Here, we characterized a TA system in A. tumefaciens whose understanding might help to find its physiological function and to develop further applications.

Functional and Biochemical Characterization of Immunologically Derived Equine Platelet-Activating Factor

1985 ◽  
Vol 22 (4) ◽  
pp. 375-386 ◽  
Author(s):  
H. C. Wimberly ◽  
D. O. Slauson ◽  
N. R. Neilsen
Keyword(s):  
Functional Activity ◽  
Biochemical Characterization ◽  
Platelet Activating Factor ◽  
Biochemical Properties ◽  
Naturally Occurring ◽  
Rabbit Platelets ◽  
Rapid Reaction ◽  
Specific Challenge

Antigen-specific challenge of equine leukocytes induced the non-lytic release of a platelet-activating factor in vitro. The equine platelet-activating factor stimulated the release of serotonin from equine platelets in a dose-responsive manner, independent of the presence of cyclo-oxygenase pathway inhibitors such as indomethacin. Rabbit platelets were also responsive to equine platelet-activating factor. The release of equine platelet-activating factor was a rapid reaction with near maximal secretion taking place in 30 seconds. Addition of equine platelet-activating factor to washed equine platelets stimulated platelet aggregation which could not be inhibited by the presence of aspirin or indomethacin. Platelets preincubated with equine platelet-activating factor became specifically desensitized to equine platelet-activating factor while remaining responsive to other platelet stimuli such as collagen and epinephrine. The following biochemical properties of equine platelet-activating factor are identical to those properties of 1-0-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC): stability upon exposure to air and acid; loss of functional activity after basecatalyzed methanolysis with subsequent acylation that returned all functional activity; and identical relative mobilities on silica gel G plates developed with chloroform:methanol:water (65:35:6, volume/volume). The combined functional and biochemical characteristics of equine platelet-activating factor strongly suggest identity between this naturally occurring, immunologically derived equine factor and AGEPC.

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