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

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.

scholarly journals Mycobacterial OtsA structures unveil substrate preference mechanism and allosteric regulation by 2-oxoglutarate and 2-phosphoglycerate

2019 ◽  
Author(s):  
Vítor Mendes ◽  
Marta Acebrón-García-de-Eulate ◽  
Nupur Verma ◽  
Michal Blaszczyk ◽  
Márcio V. B. Dias ◽  
...  
Keyword(s):  
Cell Wall ◽  
Feedback Inhibition ◽  
Structural Information ◽  
Site Directed Mutagenesis ◽  
Substrate Preference ◽  
Directed Mutagenesis ◽  
Allosteric Site ◽  
Trehalose Biosynthesis ◽  
Key Residues ◽  
Allosteric Regulators

AbstractTrehalose is an essential disaccharide for mycobacteria and a key constituent of several cell wall glycolipids with fundamental roles in pathogenesis. Mycobacteria possess two pathways for trehalose biosynthesis. However, only the OtsAB pathway was found to be essential inM. tuberculosis, with marked growth and virulence defects of OtsA mutants and strict essentiality of OtsB2. Herein, we report the first mycobacterial OtsA structures fromM. thermoresistibilein both apo and ligand-bound forms. Structural information reveals three key residues in the mechanism of substrate preference that were further confirmed by site-directed mutagenesis. Additionally, we identify 2-oxoglutarate and 2-phosphoglycerate as allosteric regulators of OtsA. The structural analysis in this work strongly contributed to define the mechanisms for feedback inhibition, show different conformational states of the enzyme and map a new allosteric site.

scholarly journals Spatial organization of Clostridium difficile S-layer biogenesis

2018 ◽  
Author(s):  
Peter Oatley ◽  
Joseph A. Kirk ◽  
Shuwen Ma ◽  
Simon Jones ◽  
Robert P. Fagan
Keyword(s):  
Cell Wall ◽  
Clostridium Difficile ◽  
Spatial Organization ◽  
Protein A ◽  
Layer Growth ◽  
Bacterial Cells ◽  
Surface Layers ◽  
Positive Bacterium ◽  
Absolute Requirement ◽  
Peptidoglycan Cell Wall

AbstractSurface layers (S-layers) are protective protein coats which form around all archaea and most bacterial cells. Clostridium difficile is a Gram-positive bacterium with an S-layer covering its peptidoglycan cell wall. The S-layer in C. difficile is constructed mainly of S-layer protein A (SlpA), which is a key virulence factor and an absolute requirement for disease. S-layer biogenesis is a complex multi-step process, disruption of which has severe consequences for the bacterium. We examined the subcellular localization of SlpA secretion and S-layer growth; observing formation of S-layer at specific sites that coincide with cell wall synthesis, while the secretion of SlpA from the cell is relatively delocalized. We conclude that this delocalized secretion of SlpA leads to a pool of precursor in the cell wall which is available to repair openings in the S-layer formed during cell growth or following damage.

scholarly journals Hepatic lipase: site-directed mutagenesis of a serine residue important for catalytic activity.

1990 ◽  
Vol 265 (11) ◽  
pp. 6291-6295 ◽  
Author(s):  
R C Davis ◽  
G Stahnke ◽  
H Wong ◽  
M H Doolittle ◽  
D Ameis ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Hepatic Lipase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Serine Residue

Biochemical study of fibrinolytic protease from Euphausia superba possessing multifunctional serine protease activity

2020 ◽  
Vol 27 ◽  
Author(s):  
Guo-Ying Qian ◽  
Gyutae Lim ◽  
Shang-Jun Yin ◽  
Jun-Mo Yang ◽  
Jinhyuk Lee ◽  
...  
Keyword(s):  
Defense Mechanisms ◽  
Fluorescence Spectra ◽  
Biochemical Study ◽  
Euphausia Superba ◽  
Md Simulations ◽  
Time Interval ◽  
Serine Residue ◽  
Catalytic Function ◽  
Serine Protease Activity ◽  
Key Residues

Background: Background: Fibrinolytic protease from Euphausia superba (EFP) was isolated. Objective: Biochemical distinctions, regulation of the catalytic function, and the key residues of EFP were investigated. Methods: The serial inhibition kinetic evaluations coupled with measurements of fluorescence spectra in the presence of 4- (2-aminoethyl) benzene sulfonyl fluoride hydrochloride (AEBSF) was conducted. The computational molecular dynamics (MD) simulations were also applied for a comparative study. Results: The enzyme behaved as a monomeric protein with a molecular mass of about 28.6 kD with Km BApNA = 0.629 ± 0.02 mM and kcat/Km BApNA = 7.08 s-1 /mM. The real-time interval measurements revealed that the inactivation was a first-order reaction, with the kinetic processes shifting from a monophase to a biphase. Measurements of fluorescence spectra showed that serine residue modification by AEBSF directly caused conspicuous changes of the tertiary structures and exposed hydrophobic surfaces. Some osmolytes were applied to find protective roles. These results confirmed that the active region of EFP is more flexible than the overall enzyme molecule and serine, as the key residue, is associated with the regional unfolding of EFP in addition to its catalytic role. The MD simulations were supportive to the kinetics data. Conclusion: Our study indicated that EFP has an essential serine residue for its catalyst function and associated folding behaviors. Also, the functional role of osmolytes such as proline and glycine that may play a role in defense mechanisms from environmental adaptation in a krill’s body was suggested.

scholarly journals Sorting out the Superbugs: Potential of Sortase A Inhibitors among Other Antimicrobial Strategies to Tackle the Problem of Antibiotic Resistance

Antibiotics ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 164 ◽  
Author(s):  
Nikita Zrelovs ◽  
Viktorija Kurbatska ◽  
Zhanna Rudevica ◽  
Ainars Leonchiks ◽  
Davids Fridmanis
Keyword(s):  
Cell Wall ◽  
Antibiotic Resistance ◽  
Pathogenic Bacteria ◽  
Resistance Mechanisms ◽  
Surface Proteins ◽  
Sortase A ◽  
Inhibitory Compounds ◽  
Alternative Means ◽  
Alternative Approaches ◽  
Conventional Antibiotics

Rapid spread of antibiotic resistance throughout the kingdom bacteria is inevitably bringing humanity towards the “post-antibiotic” era. The emergence of so-called “superbugs”—pathogen strains that develop resistance to multiple conventional antibiotics—is urging researchers around the globe to work on the development or perfecting of alternative means of tackling the pathogenic bacteria infections. Although various conceptually different approaches are being considered, each comes with its advantages and drawbacks. While drug-resistant pathogens are undoubtedly represented by both Gram(+) and Gram(−) bacteria, possible target spectrum across the proposed alternative approaches of tackling them is variable. Numerous anti-virulence strategies aimed at reducing the pathogenicity of target bacteria rather than eliminating them are being considered among such alternative approaches. Sortase A (SrtA) is a membrane-associated cysteine protease that catalyzes a cell wall sorting reaction by which surface proteins, including virulence factors, are anchored to the bacterial cell wall of Gram(+) bacteria. Although SrtA inhibition seems perspective among the Gram-positive pathogen-targeted antivirulence strategies, it still remains less popular than other alternatives. A decrease in virulence due to inactivation of SrtA activity has been extensively studied in Staphylococcus aureus, but it has also been demonstrated in other Gram(+) species. In this manuscript, results of past studies on the discovery of novel SrtA inhibitory compounds and evaluation of their potency were summarized and commented on. Here, we discussed the rationale behind the inhibition of SrtA, raised some concerns on the comparability of the results from different studies, and touched upon the possible resistance mechanisms as a response to implementation of such therapy in practice. The goal of this article is to encourage further studies of SrtA inhibitory compounds.

scholarly journals Active-site serine mutants of the Streptomyces albus G β-lactamase

1991 ◽  
Vol 277 (3) ◽  
pp. 647-652 ◽  
Author(s):  
F Jacob ◽  
B Joris ◽  
J M Frère
Keyword(s):  
Active Site ◽  
Specific Activity ◽  
Site Directed Mutagenesis ◽  
Beta Lactamase ◽  
Wild Type ◽  
Serine Residue ◽  
Wild Type Enzyme ◽  
Ph Values ◽  
Streptomyces Albus ◽  
Small Production

By using site-directed mutagenesis, the active-site serine residue of the Streptomyces albus G beta-lactamase was substituted by alanine and cysteine. Both mutant enzymes were produced in Streptomyces lividans and purified to homogeneity. The cysteine beta-lactamase exhibited a substrate-specificity profile distinct from that of the wild-type enzyme, and its kcat./Km values at pH 7 were never higher than 0.1% of that of the serine enzyme. Unlike the wild-type enzyme, the activity of the mutant increased at acidic pH values. Surprisingly, the alanine mutant exhibited a weak but specific activity for benzylpenicillin and ampicillin. In addition, a very small production of wild-type enzyme, probably due to mistranslation, was detected, but that activity could be selectively eliminated. Both mutant enzymes were nearly as thermostable as the wild-type.

scholarly journals Study of substrate specificity of human aromatase by site directed mutagenesis

2002 ◽  
Vol 269 (5) ◽  
pp. 1393-1405 ◽  
Author(s):  
P. Auvray ◽  
C. Nativelle ◽  
R. Bureau ◽  
P. Dallemagne ◽  
G.-E. Séralini ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Human Aromatase
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