scholarly journals The Glycosyltransferase Domain of Penicillin-Binding Protein 2a from Streptococcus pneumoniae Catalyzes the Polymerization of Murein Glycan Chains

2003 ◽  
Vol 185 (15) ◽  
pp. 4418-4423 ◽  
Author(s):  
Anne Marie Di Guilmi ◽  
Andréa Dessen ◽  
Otto Dideberg ◽  
Thierry Vernet
Keyword(s):  
Streptococcus Pneumoniae ◽  
Pathogenic Bacteria ◽  
Catalytic Domain ◽  
Soluble Form ◽  
Lipid Ii ◽  
Extracellular Region ◽  
Catalytically Active ◽  
Bacterial Peptidoglycan

ABSTRACT The bacterial peptidoglycan consists of glycan chains of repeating β-1,4-linked N-acetylglucosaminyl-N-acetylmuramyl units cross-linked through short peptide chains. The polymerization of the glycans, or glycosyltransfer (GT), and transpeptidation (TP) are catalyzed by bifunctional penicillin-binding proteins (PBPs). The β-lactam antibiotics inhibit the TP reaction, but their widespread use led to the development of drug resistance in pathogenic bacteria. In this context, the GT catalytic domain represents a potential target in the antibacterial fight. In this work, the in vitro polymerization of glycan chains by the extracellular region of recombinant Streptococcus pneumoniae PBP2a, namely, PBP2a* (the asterisk indicates the soluble form of the protein) is presented. Dansylated lipid II was used as the substrate, and the kinetic parameters K m and k cat/K m were measured at 40.6 μM (± 15.5) and 1 × 10−3 M−1 s−1, respectively. The GT reaction catalyzed by PBP2a* was inhibited by moenomycin and vancomycin. Furthermore, the sequence between Lys 78 and Ser 156 is required for enzymatic activity, whereas it is dispensable for lipid II binding. In addition, we confirmed that this region of the protein is also involved in membrane interaction, independently of the transmembrane anchor. The characterization of the catalytically active GT domain of S. pneumoniae PBP2a may contribute to the development of new inhibitors, which are urgently needed to renew the antibiotic arsenal.

scholarly journals Functional Characterization of Penicillin-Binding Protein 1b from Streptococcus pneumoniae

2003 ◽  
Vol 185 (5) ◽  
pp. 1650-1658 ◽  
Author(s):  
Anne Marie Di Guilmi ◽  
Andréa Dessen ◽  
Otto Dideberg ◽  
Thierry Vernet
Keyword(s):  
Streptococcus Pneumoniae ◽  
Pathogenic Bacteria ◽  
Biochemical Characterization ◽  
Functional Characterization ◽  
Limited Proteolysis ◽  
Fluorescence Signal ◽  
Binding Studies ◽  
Lipid Ii ◽  
Extracellular Region

ABSTRACT The widespread use of antibiotics has encouraged the development of drug resistance in pathogenic bacteria. In order to overcome this problem, the modification of existing antibiotics and/or the identification of targets for the design of new antibiotics is currently being undertaken. Bifunctional penicillin-binding proteins (PBPs) are membrane-associated molecules whose transpeptidase (TP) activity is irreversibly inhibited by β-lactam antibiotics and whose glycosyltransferase (GT) activity represents a potential target in the antibacterial fight. In this work, we describe the expression and the biochemical characterization of the soluble extracellular region of Streptococcus pneumoniae PBP1b (PBP1b*). The acylation efficiency for benzylpenicillin and cefotaxime was characterized by stopped-flow fluorometry and a 40-kDa stable TP domain was generated after limited proteolysis. In order to analyze the GT activity of PBP1b*, we developed an electrophoretic assay which monitors the fluorescence signal from PBP1b*-bound dansylated lipid II. This binding was inhibited by the antibiotic moenomycin and was specific for the GT domain, since no signal was observed in the presence of the purified functional TP domain. Binding studies performed with truncated forms of PBP1b* demonstrated that the first conserved motif of the GT domain is not required for the recognition of lipid II, whereas the second motif is necessary for such interaction.

scholarly journals Discovery and Characterization of Low-Molecular Weight Inhibitors of Erwinia tracheiphila

2020 ◽  
Vol 110 (5) ◽  
pp. 989-998
Author(s):  
Cláudio M. Vrisman ◽  
Loïc Deblais ◽  
Yosra A. Helmy ◽  
Reed Johnson ◽  
Gireesh Rajashekara ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
High Throughput Screening ◽  
Pathogenic Bacteria ◽  
Bacterial Species ◽  
Bacillus Species ◽  
Erwinia Tracheiphila ◽  
Static Activity ◽  
Low Toxicity

Plant pathogenic bacteria in the genus Erwinia cause economically important diseases, including bacterial wilt of cucurbits caused by Erwinia tracheiphila. Conventional bactericides are insufficient to control this disease. Using high-throughput screening, 464 small molecules (SMs) with either cidal or static activity at 100 µM against a cucumber strain of E. tracheiphila were identified. Among them, 20 SMs (SM1 to SM20), composed of nine distinct chemical moiety structures, were cidal to multiple E. tracheiphila strains at 100 µM. These lead SMs had low toxicity to human cells and honey bees at 100 µM. No phytotoxicity was observed on melon plants at 100 µM, except when SM12 was either mixed with Silwet L-77 and foliar sprayed or when delivered through the roots. Lead SMs did not inhibit the growth of beneficial Pseudomonas and Enterobacter species but inhibited the growth of Bacillus species. Nineteen SMs were cidal to Xanthomonas cucurbitae and showed >50% growth inhibition against Pseudomonas syringae pv. lachrymans. In addition, 19 SMs were cidal or static against Erwinia amylovora in vitro. Five SMs demonstrated potential to suppress E. tracheiphila when foliar sprayed on melon plants at 2× the minimum bactericidal concentration. Thirteen SMs reduced Et load in melon plants when delivered via roots. Temperature and light did not affect the activity of SMs. In vitro cidal activity was observed after 3 to 10 h of exposure to these five SMs. Here, we report 19 SMs that provide chemical scaffolds for future development of bactericides against plant pathogenic bacterial species.

scholarly journals THE PRODUCTION OF MONOCLONAL ANTIBODIES TO TETRASACCHARIDE - SYNTHETIC ANALOGUE OF THE CAPSULAR POLYSACCHARIDE OF STREPTOCOCCUS PNEUMONIAE OF SEROTYPE 14 AND THEIR IMMUNOCHEMICAL CHARACTERIZATION

2018 ◽  
pp. 26-31
Author(s):  
I. V. Yakovleva ◽  
E. A. Kurbatova ◽  
E. A. Akhmatova ◽  
E. V. Sukhova ◽  
D. V. Yashunsky ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Streptococcus Pneumoniae ◽  
Capsular Polysaccharide ◽  
Solid Phase ◽  
Synthetic Analogue ◽  
Immunochemical Characterization ◽  
Carbohydrate Epitopes ◽  
First Time

Aim. Production of monoclonal antibodies (mAb) to synthetic tetrasaccharide - repeating unit of the capsular polysaccharide (CP) of Streptococcus pneumoniae serotype 14 and their immunochemical characterization. Materials and methods. In order to generate the hybridoma producing mAb, mice were immunized with synthetic tetrasaccharide conjugated with bovine serum albumin (BSA) with following hybridization of B lymphocytes with mouse myeloma cells. Antibodies were obtained in vitro andin vivo. Immunochemical characterization of mAb to tetrasaccharide was carried out using a variety of ELISA options. Results. For the first time obtained mouse hybridoma, producing IgM to tetrasacchride. The IgM titer of anti-tetrasacharide antibodies in supernatants of clones and in the ascitic fluid of mice in ELISA detected by biotinylated tetrasaccharide and synthetic CP adsorbed on the solid phase was higher compared to the use of bacterial CP as well cover antigen. In the reaction of inhibition of the ELISA, the mAb recognized the corresponding carbohydrate epitopes of the bacterial CP of S. pneumoniae serotype 14 dissolved in the liquid phase better than tetrasaccharide ligand and synthetic CP. Conclusion. To detect mAb to tetrasaccharide in ELISA preferably to use synthetic analogues of the CP as solid phase antigens. The obtained mAb to tetrasaccharide can be used to determine the representation of the protective tetrasaccharide epitope of CP in the development of pneumococcal vaccines.

scholarly journals Insight into the Lytic Functions of the Lactococcal Prophage TP712

Viruses ◽  
2019 ◽  
Vol 11 (10) ◽  
pp. 881 ◽  
Author(s):  
Escobedo ◽  
Campelo ◽  
Wegmann ◽  
García ◽  
Rodríguez ◽  
...  
Keyword(s):  
Cell Wall ◽  
Signal Peptide ◽  
Catalytic Domain ◽  
Experimental Conditions ◽  
Binding Domains ◽  
Cell Wall Binding ◽  
Additional Cell ◽  
Translational Start ◽  
Bacterial Peptidoglycan

The lytic cassette of Lactococcus lactis prophage TP712 contains a putative membrane protein of unknown function (Orf54), a holin (Orf55), and a modular endolysin with a N-terminal glycoside hydrolase (GH_25) catalytic domain and two C-terminal LysM domains (Orf56, LysTP712). In this work, we aimed to study the mode of action of the endolysin LysTP712. Inducible expression of the holin-endolysin genes seriously impaired growth. The growth of lactococcal cells overproducing the endolysin LysTP712 alone was only inhibited upon the dissipation of the proton motive force by the pore-forming bacteriocin nisin. Processing of a 26-residues signal peptide is required for LysTP712 activation, since a truncated version without the signal peptide did not impair growth after membrane depolarization. Moreover, only the mature enzyme displayed lytic activity in zymograms, while no lytic bands were observed after treatment with the Sec inhibitor sodium azide. LysTP712 might belong to the growing family of multimeric endolysins. A C-terminal fragment was detected during the purification of LysTP712. It is likely to be synthesized from an alternative internal translational start site located upstream of the cell wall binding domain in the lysin gene. Fractions containing this fragment exhibited enhanced activity against lactococcal cells. However, under our experimental conditions, improved in vitro inhibitory activity of the enzyme was not observed upon the supplementation of additional cell wall binding domains in. Finally, our data pointed out that changes in the lactococcal cell wall, such as the degree of peptidoglycan O-acetylation, might hinder the activity of LysTP712. LysTP712 is the first secretory endolysin from a lactococcal phage described so far. The results also revealed how the activity of LysTP712 might be counteracted by modifications of the bacterial peptidoglycan, providing guidelines to exploit the biotechnological potential of phage endolysins within industrially relevant lactococci and, by extension, other bacteria.

scholarly journals The interaction of small domains between the subunits of phosphatidylinositol 3-kinase determines enzyme activity.

1994 ◽  
Vol 14 (4) ◽  
pp. 2675-2685 ◽  
Author(s):  
A Klippel ◽  
J A Escobedo ◽  
M Hirano ◽  
L T Williams
Keyword(s):  
Amino Acids ◽  
Catalytic Domain ◽  
Specific Interaction ◽  
Sh2 Domains ◽  
Catalytically Active ◽  
Functional Complex ◽  
Src Homology ◽  
Carboxy Terminal

Previous studies have suggested that the two subunits of phosphatidylinositol (PI) 3-kinase, p85 and p110, function as localizing and catalytic subunits, respectively. Using recombinant p85 and p110 molecules, we have reconstituted the specific interaction between the two subunits of mouse PI 3-kinase in cells and in vitro. We have previously shown that the region between the two Src homology 2 (SH2) domains of p85 is able to form a functional complex with the 110-kDa subunit in vivo. In this report, we identify the corresponding domain in p110 which directs the binding to p85. We demonstrate that the interactive domains in p85 and p110 are less than 103 and 124 amino acids, respectively, in size. We also show that the association of p85 and p110 mediated by these domains is critical for PI 3-kinase activity. Surprisingly, a complex between a 102-amino-acid segment of p85 and the full-length p110 molecule is catalytically active, whereas p110 alone has no activity. In addition to the catalytic domain in the carboxy-terminal region, 123 amino acids at the amino terminus of p110 were required for catalytic activity and were sufficient for the interaction with p85. These results indicate that the 85-kDa subunit, previously thought to have only a linking role in localizing the p110 catalytic subunit, is an important component of the catalytic complex.

scholarly journals MYST Family Histone Acetyltransferases in the Protozoan Parasite Toxoplasma gondii

2005 ◽  
Vol 4 (12) ◽  
pp. 2057-2065 ◽  
Author(s):  
Aaron T. Smith ◽  
Samantha D. Tucker-Samaras ◽  
Alan H. Fairlamb ◽  
William J. Sullivan
Keyword(s):  
Toxoplasma Gondii ◽  
Catalytic Domain ◽  
Regulation Of Gene Expression ◽  
Protozoan Parasite ◽  
Opportunistic Pathogen ◽  
Covalent Modification ◽  
Histone H4 ◽  
Genomic Locus

ABSTRACT The restructuring of chromatin precedes tightly regulated events such as DNA transcription, replication, and repair. One type of chromatin remodeling involves the covalent modification of nucleosomes by histone acetyltransferase (HAT) complexes. The observation that apicidin exerts antiprotozoal activity by targeting a histone deacetyltransferase has prompted our search for more components of the histone modifying machinery in parasitic protozoa. We have previously identified GNAT family HATs in the opportunistic pathogen Toxoplasma gondii and now describe the first MYST (named for members MOZ, Ybf2/Sas3, Sas2, and Tip60) family HATs in apicomplexa (TgMYST-A and -B). The TgMYST-A genomic locus is singular and generates a ∼3.5-kb transcript that can encode two proteins of 411 or 471 amino acids. TgMYST-B mRNA is ∼7.0 kb and encodes a second MYST homologue. In addition to the canonical MYST HAT catalytic domain, both TgMYST-A and -B possess an atypical C2HC zinc finger and a chromodomain. Recombinant TgMYST-A exhibits a predilection to acetylate histone H4 in vitro at lysines 5, 8, 12, and 16. Antibody generated to TgMYST-A reveals that both the long and short (predominant) versions are present in the nucleus and are also plentiful in the cytoplasm. Moreover, both TgMYST-A forms are far more abundant in rapidly replicating parasites (tachyzoites) than encysted parasites (bradyzoites). A bioinformatics survey of the Toxoplasma genome reveals numerous homologues known to operate in native MYST complexes. The characterization of TgMYST HATs represents another important step toward understanding the regulation of gene expression in pathogenic protozoa and provides evolutionary insight into how these processes operate in eukaryotic cells in general.

scholarly journals Characterization of the interaction domains of Ure2p, a prion-like protein of yeast

1999 ◽  
Vol 338 (2) ◽  
pp. 403-407 ◽  
Author(s):  
Eric FERNANDEZ-BELLOT ◽  
Elisabeth GUILLEMET ◽  
Agnès BAUDIN-BAILLIEU ◽  
Sébastien GAUMER ◽  
Anton A. KOMAR ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Deletion Mutants ◽  
Loss Of Function ◽  
Hybrid Analysis ◽  
Yeast Saccharomyces Cerevisiae ◽  
Interaction Domains ◽  
Two Hybrid

In the yeast Saccharomyces cerevisiae, the non-Mendelian inherited genetic element [URE3] behaves as a prion. A hypothesis has been put forward which states that [URE3] arises spontaneously from its cellular isoform Ure2p (the product of the URE2 gene), and propagates through interactions of the N-terminal domain of the protein, thus leading to its aggregation and loss of function. In the present study, various N- and C-terminal deletion mutants of Ure2p were constructed and their cross-interactions were tested in vitro and in vivo using affinity binding and a two-hybrid analysis. We show that the self-interaction of the protein is mediated by at least two domains, corresponding to the first third of the protein (the so-called prion-forming domain) and the C-terminal catalytic domain.

scholarly journals Bacterial flavin homeostasis: the role of an FAD pyrophosphatase/FMN transferase

2014 ◽  
Vol 70 (a1) ◽  
pp. C311-C311
Author(s):  
Diana Tomchick ◽  
Ranjit Deka ◽  
Chad Brautigam ◽  
Wei Liu ◽  
Michael Norgard
Keyword(s):  
Pathogenic Bacteria ◽  
Treponema Pallidum ◽  
Covalent Attachment ◽  
Post Translational Modification ◽  
New Paradigm ◽  
Uptake Mechanism ◽  
Structural Investigations

Treponema pallidum, an obligate parasite of humans and the causative agent of syphilis, has evolved the capacity to exploit host-derived metabolites for its survival. Flavin-containing compounds are essential cofactors that are required for metabolic processes in all living organisms, and riboflavin is a direct precursor of the cofactors FMN and FAD. Unlike many pathogenic bacteria, Treponema pallidum cannot synthesize riboflavin; we recently described a flavin-uptake mechanism composed of an ABC-type transporter [1]. However, there is a paucity of information about flavin utilization in bacterial periplasms. We have identified the TP0796 lipoprotein as a previously uncharacterized Mg2+-dependent FAD pyrophosphatase/FMN transferase within the ApbE superfamily [2,3]. Biochemical and structural investigations revealed that the enzyme has a unique bimetal Mg2+ catalytic center. Furthermore, the pyrophosphatase activity is product-inhibited by AMP, indicating a possible role for this molecule in modulating FMN and FAD levels in the treponemal periplasm. The ApbE superfamily was previously thought to be involved in thiamine biosynthesis, but our characterization of TP0796 prompts a renaming of this superfamily as a periplasmic flavin-trafficking protein (Ftp). Treponemal Ftp (Ftp_Tp) is the first structurally and biochemically characterized metal-dependent FAD pyrophosphatase/FMN transferase in bacteria. We have shown in vitro and in vivo that Ftps from several types of pathogenic bacteria are capable of flavinylating proteins through covalent attachment of FMN via a phosphoester bond to threonine residues of an appropriate sequence signature. Progress on the structural characterization of a product of this post-translational modification will be presented. This new paradigm for a bacterial flavin utilization pathway may prove to be useful for future inhibitor design.

scholarly journals Characterization of protease activity of Nsp3 from SARS-CoV-2 and its in vitro inhibition by nanobodies

2020 ◽  
Author(s):  
Lee A. Armstrong ◽  
Sven M. Lange ◽  
Virginia de Cesare ◽  
Stephen P. Matthews ◽  
Raja Sekar Nirujogi ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Transmembrane Protein ◽  
Cross Reactivity ◽  
Host Cells ◽  
Alternative Methods ◽  
Structural Proteins ◽  
Active Protease ◽  
Micromolar Range

AbstractOf the 16 non-structural proteins (Nsps) encoded by SARS CoV-2, Nsp3 is the largest and plays important roles in the viral life cycle. Being a large, multidomain, transmembrane protein, Nsp3 has been the most challenging Nsp to characterize. Encoded within Nsp3 is the papain-like protease PLpro domain that cleaves not only the viral protein but also polyubiquitin and the ubiquitin-like modifier ISG15 from host cells. We here compare the interactors of PLpro and Nsp3 and find a largely overlapping interactome. Intriguingly, we find that near full length Nsp3 is a more active protease compared to the minimal catalytic domain of PLpro. Using a MALDI-TOF based assay, we screen 1971 approved clinical compounds and identify five compounds that inhibit PLpro with IC50s in the low micromolar range but showed cross reactivity with other human deubiquitinases and had no significant antiviral activity in cellular SARS-CoV-2 infection assays. We therefore looked for alternative methods to block PLpro activity and engineered competitive nanobodies that bind to PLpro at the substrate binding site with nanomolar affinity thus inhibiting the enzyme. Our work highlights the importance of studying Nsp3 and provides tools and valuable insights to investigate Nsp3 biology during the viral infection cycle.

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