Cell wall substrate specificity of six different lysozymes and lysozyme inhibitory activity of bacterial extracts

(+)-10-Methyldodecanoic acid and 12-methyltridecanoic acid have been isolated from the acid fraction of wool wax. These acids have a high inhibitory activity against Gram positive bacteria, but not against Gram negatives or fungi. A steric relationship with the Gram positive cell wall surface is suggested.

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Increase in the in vitro susceptibility of Staphylococcus aureus to antimicrobial agents in the presence of Candida albicans

Canadian Journal of Microbiology ◽

10.1139/m79-066 ◽

1979 ◽

Vol 25 (4) ◽

pp. 429-435 ◽

Cited By ~ 2

Author(s):

J. deRepentigny ◽

R. Lévesque ◽

L. G. Mathieu

Keyword(s):

Staphylococcus Aureus ◽

Cell Wall ◽

Candida Albicans ◽

Inhibitory Activity ◽

Antimicrobial Agents ◽

Mixed Cultures ◽

Alpha Toxin ◽

In Vitro Susceptibility ◽

Pure Cultures

In experiments with mixed cultures of Staphylococcus aureus and Candida albicans both in the absence and in the presence of 5-fluorocytosine (5-FC), we have observed that (1) there is an inhibition of S. aureus growth in mixed cultures with C. albicans in media supplemented with 1 μg/mL of 5-FC and that 5-FC has no effect on staphylococci in pure cultures; (2) this inhibition occurred with clinically isolated and laboratory strains and could be reversed by specific metabolites; (3) Staphylococcus aureus was inhibited by filtrates of C. albicans cultures treated with 5-FC and this seemed to be favored by some C. albicans filterable product which can affect the cell wall and the permeability of the staphylococcal cells since they become sensitive to 5-FC; (4) nine other commonly used antimicrobials showed an increased inhibitory activity against S. aureus in mixed cultures with C. albicans; and (5) there is a decrease in the number of precipitating antigens of S. aureus and of the activity of alpha toxin when this species was grown with both C. albicans and 5-FC. Our results indicate that the susceptibility of some species to antimicrobials could be significantly modified in the presence of other species. One cannot exclude that a similar phenomenon could happen in hosts under treatment with antibiotics against infection.

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Preliminary crystallographic study of theStreptococcus agalactiaesortases, sortase A and sortase C1

Acta Crystallographica Section F Structural Biology and Crystallization Communications ◽

10.1107/s1744309110031106 ◽

2010 ◽

Vol 66 (9) ◽

pp. 1096-1100 ◽

Cited By ~ 4

Author(s):

Baldeep Khare ◽

Alexandra Samal ◽

Krishnan Vengadesan ◽

K. R. Rajashankar ◽

Xin Ma ◽

...

Keyword(s):

Cell Wall ◽

Cell Surface ◽

Substrate Specificity ◽

Streptococcus Agalactiae ◽

Crystal Form ◽

Sortase A ◽

Crystal Forms ◽

Gram Positive Bacteria ◽

Crystallographic Study ◽

Surface Adhesins

Sortases are cysteine transpeptidases that are essential for the assembly and anchoring of cell-surface adhesins in Gram-positive bacteria. InStreptococcus agalactiae(GBS), the pilin-specific sortase SrtC1 catalyzes the polymerization of pilins encoded by pilus island 1 (PI-1) and the housekeeping sortase SrtA is necessary for cell-wall anchoring of the resulting pilus polymers. These sortases are known to utilize different substrates for pilus polymerization and cell-wall anchoring; however, the structural correlates that dictate their substrate specificity have not yet been clearly defined. This report presents the expression, purification and crystallization of SrtC1 (SAG0647) and SrtA (SAG0961) fromS. agalactiaestrain 2603V/R. The GBS SrtC1 has been crystallized in three crystal forms and the GBS SrtA has been crystallized in one crystal form.

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Functional analysis of Clostridium difficile sortase B reveals key residues for catalytic activity and substrate specificity

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.011322 ◽

2020 ◽

Vol 295 (11) ◽

pp. 3734-3745

Author(s):

Chia-Yu Kang ◽

I-Hsiu Huang ◽

Chi-Chi Chou ◽

Tsai-Yu Wu ◽

Jyun-Cyuan Chang ◽

...

Keyword(s):

Cell Wall ◽

Catalytic Activity ◽

Clostridium Difficile ◽

Substrate Specificity ◽

Surface Proteins ◽

Site Directed Mutagenesis ◽

Serine Residue ◽

Peptidoglycan Cell Wall ◽

Attractive Therapeutic Target ◽

Key Residues

Most of Gram-positive bacteria anchor surface proteins to the peptidoglycan cell wall by sortase, a cysteine transpeptidase that targets proteins displaying a cell wall sorting signal. Unlike other bacteria, Clostridium difficile, the major human pathogen responsible for antibiotic-associated diarrhea, has only a single functional sortase (SrtB). Sortase's vital importance in bacterial virulence has been long recognized, and C. difficile sortase B (Cd-SrtB) has become an attractive therapeutic target for managing C. difficile infection. A better understanding of the molecular activity of Cd-SrtB may help spur the development of effective agents against C. difficile infection. In this study, using site-directed mutagenesis, biochemical and biophysical tools, LC-MS/MS, and crystallographic analyses, we identified key residues essential for Cd-SrtB catalysis and substrate recognition. To the best of our knowledge, we report the first evidence that a conserved serine residue near the active site participates in the catalytic activity of Cd-SrtB and also SrtB from Staphylococcus aureus. The serine residue indispensable for SrtB activity may be involved in stabilizing a thioacyl-enzyme intermediate because it is neither a nucleophilic residue nor a substrate-interacting residue, based on the LC-MS/MS data and available structural models of SrtB–substrate complexes. Furthermore, we also demonstrated that residues 163–168 located on the β6/β7 loop of Cd-SrtB dominate specific recognition of the peptide substrate PPKTG. The results of this work reveal key residues with roles in catalysis and substrate specificity of Cd-SrtB.

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Substrate specificity of Rv3378c, an enzyme from Mycobacterium tuberculosis, and the inhibitory activity of the bicyclic diterpenoids against macrophagephagocytosis

Organic & Biomolecular Chemistry ◽

10.1039/c0ob00884b ◽

2011 ◽

Vol 9 (7) ◽

pp. 2156-2165 ◽

Cited By ~ 23

Author(s):

Tsutomu Hoshino ◽

Chiaki Nakano ◽

Takahiro Ootsuka ◽

Yosuke Shinohara ◽

Takashi Hara

Keyword(s):

Mycobacterium Tuberculosis ◽

Substrate Specificity ◽

Inhibitory Activity

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Insights into Substrate Specificity of NlpC/P60 Cell Wall Hydrolases Containing Bacterial SH3 Domains

mBio ◽

10.1128/mbio.02327-14 ◽

2015 ◽

Vol 6 (5) ◽

Cited By ~ 20

Author(s):

Qingping Xu ◽

Dominique Mengin-Lecreulx ◽

Xueqian W. Liu ◽

Delphine Patin ◽

Carol L. Farr ◽

...

Keyword(s):

Cell Wall ◽

Amino Acid ◽

Substrate Specificity ◽

Catalytic Domain ◽

Substrate Binding ◽

Diaminopimelic Acid ◽

Single Mutation ◽

Molecular Architecture ◽

Substrate Specificities ◽

Cell Wall Hydrolases

ABSTRACTBacterial SH3 (SH3b) domains are commonly fused with papain-like Nlp/P60 cell wall hydrolase domains. To understand how the modular architecture of SH3b and NlpC/P60 affects the activity of the catalytic domain, three putative NlpC/P60 cell wall hydrolases were biochemically and structurally characterized. These enzymes all have γ-d-Glu-A2pm (A2pm is diaminopimelic acid) cysteine amidase (ordl-endopeptidase) activities but with different substrate specificities. One enzyme is a cell wall lysin that cleaves peptidoglycan (PG), while the other two are cell wall recycling enzymes that only cleave stem peptides with an N-terminall-Ala. Their crystal structures revealed a highly conserved structure consisting of two SH3b domains and a C-terminal NlpC/P60 catalytic domain, despite very low sequence identity. Interestingly, loops from the first SH3b domain dock into the ends of the active site groove of the catalytic domain, remodel the substrate binding site, and modulate substrate specificity. Two amino acid differences at the domain interface alter the substrate binding specificity in favor of stem peptides in recycling enzymes, whereas the SH3b domain may extend the peptidoglycan binding surface in the cell wall lysins. Remarkably, the cell wall lysin can be converted into a recycling enzyme with a single mutation.IMPORTANCEPeptidoglycan is a meshlike polymer that envelops the bacterial plasma membrane and bestows structural integrity. Cell wall lysins and recycling enzymes are part of a set of lytic enzymes that target covalent bonds connecting the amino acid and amino sugar building blocks of the PG network. These hydrolases are involved in processes such as cell growth and division, autolysis, invasion, and PG turnover and recycling. To avoid cleavage of unintended substrates, these enzymes have very selective substrate specificities. Our biochemical and structural analysis of three modular NlpC/P60 hydrolases, one lysin, and two recycling enzymes, show that they may have evolved from a common molecular architecture, where the substrate preference is modulated by local changes. These results also suggest that new pathways for recycling PG turnover products, such as tracheal cytotoxin, may have evolved in bacteria in the human gut microbiome that involve NlpC/P60 cell wall hydrolases.

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Substrate Specificity of the AmpG Permease Required for Recycling of Cell Wall Anhydro-Muropeptides

Journal of Bacteriology ◽

10.1128/jb.184.23.6434-6436.2002 ◽

2002 ◽

Vol 184 (23) ◽

pp. 6434-6436 ◽

Cited By ~ 86

Author(s):

Qiaomei Cheng ◽

James T. Park

Keyword(s):

Cell Wall ◽

Substrate Specificity ◽

Proton Motive Force ◽

Single Component ◽

Motive Force ◽

Logarithmic Growth

ABSTRACT AmpG was originally identified as a gene required for induction of β-lactamase. Subsequently, we found AmpG to be a permease required for recycling of murein tripeptide and uptake of anhydro-muropeptides. We have now studied the specificity of the AmpG permease. The principal requirement is for the presence of the disaccharide, N-acetylglucosaminyl-β-1,4-anhydro-N-acetylmuramic acid (GlcNAc-anhMurNAc). These unique substrates for AmpG, which contain murein peptides linked to GlcNAc-anhMurNAc, are produced by turnover of the cell wall during logarithmic growth. AmpG permease is sensitive to carbonylcyanide m-chlorophenylhydrazone, demonstrating that AmpG permease is a single-component permease and that transport is dependent on the proton motive force.

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Crystal Structure and Substrate Specificity Modification of Acetyl Xylan Esterase from Aspergillus luchuensis

Applied and Environmental Microbiology ◽

10.1128/aem.01251-17 ◽

2017 ◽

Vol 83 (20) ◽

Cited By ~ 8

Author(s):

Dai Komiya ◽

Akane Hori ◽

Takuya Ishida ◽

Kiyohiko Igarashi ◽

Masahiro Samejima ◽

...

Keyword(s):

Crystal Structure ◽

Cell Wall ◽

Substrate Specificity ◽

Ferulic Acid ◽

Plant Cell ◽

Plant Cell Wall ◽

Catalytic Triad ◽

Acetyl Xylan Esterase ◽

Content Type ◽

Aspergillus Luchuensis

ABSTRACT Acetyl xylan esterase (AXE) catalyzes the hydrolysis of the acetyl bonds present in plant cell wall polysaccharides. Here, we determined the crystal structure of AXE from Aspergillus luchuensis (AlAXEA), providing the three-dimensional structure of an enzyme in the Esterase_phb family. AlAXEA shares its core α/β-hydrolase fold structure with esterases in other families, but it has an extended central β-sheet at both its ends and an extra loop. Structural comparison with a ferulic acid esterase (FAE) from Aspergillus niger indicated that AlAXEA has a conserved catalytic machinery: a catalytic triad (Ser119, His259, and Asp202) and an oxyanion hole (Cys40 and Ser120). Near the catalytic triad of AlAXEA, two aromatic residues (Tyr39 and Trp160) form small pockets at both sides. Homology models of fungal FAEs in the same Esterase_phb family have wide pockets at the corresponding sites because they have residues with smaller side chains (Pro, Ser, and Gly). Mutants with site-directed mutations at Tyr39 showed a substrate specificity similar to that of the wild-type enzyme, whereas those with mutations at Trp160 acquired an expanded substrate specificity. Interestingly, the Trp160 mutants acquired weak but significant type B-like FAE activity. Moreover, the engineered enzymes exhibited ferulic acid-releasing activity from wheat arabinoxylan. IMPORTANCE Hemicelluloses in the plant cell wall are often decorated by acetyl and ferulic acid groups. Therefore, complete and efficient degradation of plant polysaccharides requires the enzymes for cleaving the side chains of the polymer. Since the Esterase_phb family contains a wide array of fungal FAEs and AXEs from fungi and bacteria, our study will provide a structural basis for the molecular mechanism of these industrially relevant enzymes in biopolymer degradation. The structure of the Esterase_phb family also provides information for bacterial polyhydroxyalkanoate depolymerases that are involved in biodegradation of thermoplastic polymers.

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Cell-Wall-Bound Proteinase of Lactobacillus delbrueckii subsp. lactis ACA-DC 178: Characterization and Specificity for β-Casein

Applied and Environmental Microbiology ◽

10.1128/aem.65.5.2035-2040.1999 ◽

1999 ◽

Vol 65 (5) ◽

pp. 2035-2040 ◽

Cited By ~ 42

Author(s):

E. Tsakalidou ◽

R. Anastasiou ◽

I. Vandenberghe ◽

J. van Beeumen ◽

G. Kalantzopoulos

Keyword(s):

Cell Wall ◽

Substrate Specificity ◽

Cell Envelope ◽

Lactobacillus Delbrueckii ◽

Phenylmethylsulfonyl Fluoride ◽

A Cell ◽

Lactobacillus Delbrueckii Subsp

ABSTRACT Lactobacillus delbrueckii subsp. lactisACA-DC 178, which was isolated from Greek Kasseri cheese, produces a cell-wall-bound proteinase. The proteinase was removed from the cell envelope by washing the cells with a Ca2+-free buffer. The crude proteinase extract shows its highest activity at pH 6.0 and 40°C. It is inhibited by phenylmethylsulfonyl fluoride, showing that the enzyme is a serine-type proteinase. Considering the substrate specificity, the enzyme is similar to the lactococcal PI-type proteinases, since it hydrolyzes β-casein mainly and α- and κ-caseins to a much lesser extent. The cell-wall-bound proteinase from L. delbrueckii subsp. lactisACA-DC 178 liberates four main peptides from β-casein, which have been identified.

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