scholarly journals A single point mutation converts a glutaryl-7-aminocephalosporanic acid acylase into an N-acyl-homoserine lactone acylase

2021 ◽  
Author(s):  
Shereen A. Murugayah ◽  
Gary B. Evans ◽  
Joel D. A. Tyndall ◽  
Monica L. Gerth
Keyword(s):  
Single Point ◽  
Quorum Quenching ◽  
Homoserine Lactone ◽  
Site Directed Mutagenesis ◽  
Saturation Mutagenesis ◽  
Single Amino Acid ◽  
Single Point Mutation ◽  
Acyl Homoserine Lactone ◽  
Signalling Molecules ◽  
Single Amino Acid Residue

Abstract Objective To change the specificity of a glutaryl-7-aminocephalosporanic acid acylase (GCA) towards N-acyl homoserine lactones (AHLs; quorum sensing signalling molecules) by site-directed mutagenesis. Results Seven residues were identified by analysis of existing crystal structures as potential determinants of substrate specificity. Site-saturation mutagenesis libraries were created for each of the seven selected positions. High-throughput activity screening of each library identified two variants—Arg255Ala, Arg255Gly—with new activities towards N-acyl homoserine lactone substrates. Structural modelling of the Arg255Gly mutation suggests that the smaller side-chain of glycine (as compared to arginine in the wild-type enzyme) avoids a key clash with the acyl group of the N-acyl homoserine lactone substrate. Conclusions Mutation of a single amino acid residue successfully converted a GCA (with no detectable activity against AHLs) into an AHL acylase. This approach may be useful for further engineering of ‘quorum quenching’ enzymes.

Modulation of keratin intermediate filament assembly by single amino acid exchanges in the consensus sequence at the C-terminal end of the rod domain

1991 ◽  
Vol 99 (2) ◽  
pp. 351-362 ◽  
Author(s):  
M. Hatzfeld ◽  
K. Weber
Keyword(s):  
Intermediate Filament ◽  
Single Point ◽  
Consensus Sequence ◽  
Site Directed Mutagenesis ◽  
Single Amino Acid ◽  
Single Point Mutation ◽  
Wild Type ◽  
Filament Assembly ◽  
Elongation Process

All known intermediate filament (IF) proteins display -8 -4 -1 a consensus sequence TYRKLLEGE at the carboxyl end of the rod domain. To analyse the contribution of this sequence to the formation of IF we have changed two of the invariant positions of this motif by site-directed mutagenesis. We produced three mutant keratins, each containing a single point mutation. Tyrosine at position -8 was changed to alanine in keratin K8 (K8Y----A-8) and keratin K18 (K18Y----A-8) and leucine at position -4 was changed to glycine in keratin K18 (K18L----G-4). Mutant keratins were expressed in Escherichia coli, purified and analysed for their filament-forming capacity in vitro using either the complementary wild-type keratin or the corresponding mixture of mutant keratins. In standard filament buffer (50 mM Tris-HCl, pH7.5), assembly involving any of the mutants leads to large electron-dense aggregates instead of normal IF. In order to explain this effect, we studied the process of filament formation in more detail. Whereas the formation of tetramers in buffers containing 4M urea is unaffected, the elongation process seems slowed down. In buffer of lower ionic strength (10 mM Tris-HCl, pH7.5) mutant keratins K8Y----A-8 plus K18Y----A-8 become able to form long filaments, although short filaments and protofilamentous material are still detected. The filaments formed differ from normal keratin IF by their remarkable tendency to aggregate into thick cables. Assemblies involving K18L----G-4 can only form short IF lengths. The dense aggregates formed in standard filament buffer are able to dissociate into IF and their fragments upon dialysis into 10 mM Tris-HCl, pH7.5. The results show that the consensus sequence is needed for IF formation under normal conditions and that already one mutation per heterodimer affects the assembly.

scholarly journals A Single Point Mutation, Asn16→Lys, Dictates the Temperature-Sensitivity of the Reovirus tsG453 Mutant

Viruses ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 289
Author(s):  
Kathleen K. M. Glover ◽  
Danica M. Sutherland ◽  
Terence S. Dermody ◽  
Kevin M. Coombs
Keyword(s):  
Amino Acid ◽  
Temperature Sensitivity ◽  
Reverse Genetics ◽  
Core Protein ◽  
Single Point ◽  
Single Amino Acid ◽  
Single Point Mutation ◽  
Temperature Sensitive ◽  
Amino Acid Substitutions ◽  
Gene Encoding

Studies of conditionally lethal mutants can help delineate the structure-function relationships of biomolecules. Temperature-sensitive (ts) mammalian reovirus (MRV) mutants were isolated and characterized many years ago. Two of the most well-defined MRV ts mutants are tsC447, which contains mutations in the S2 gene encoding viral core protein σ2, and tsG453, which contains mutations in the S4 gene encoding major outer-capsid protein σ3. Because many MRV ts mutants, including both tsC447 and tsG453, encode multiple amino acid substitutions, the specific amino acid substitutions responsible for the ts phenotype are unknown. We used reverse genetics to recover recombinant reoviruses containing the single amino acid polymorphisms present in ts mutants tsC447 and tsG453 and assessed the recombinant viruses for temperature-sensitivity by efficiency-of-plating assays. Of the three amino acid substitutions in the tsG453 S4 gene, Asn16-Lys was solely responsible for the tsG453ts phenotype. Additionally, the mutant tsC447 Ala188-Val mutation did not induce a temperature-sensitive phenotype. This study is the first to employ reverse genetics to identify the dominant amino acid substitutions responsible for the tsC447 and tsG453 mutations and relate these substitutions to respective phenotypes. Further studies of other MRV ts mutants are warranted to define the sequence polymorphisms responsible for temperature sensitivity.

Tailoring GalNAcα1-3Galβ-specific lectins from a multi-specific fungal galectin: dramatic change of carbohydrate specificity by a single amino-acid substitution

2013 ◽  
Vol 453 (2) ◽  
pp. 261-270 ◽  
Author(s):  
Dan Hu ◽  
Hiroaki Tateno ◽  
Takashi Sato ◽  
Hisashi Narimatsu ◽  
Jun Hirabayashi
Keyword(s):  
Dissociation Constants ◽  
Multiple Roles ◽  
Site Directed Mutagenesis ◽  
Saturation Mutagenesis ◽  
Single Amino Acid ◽  
Dramatic Improvement ◽  
Carbohydrate Specificity ◽  
Cell Staining ◽  
Group A

Galectins exhibit multiple roles through recognition of diverse structures of β-galactosides. However, this broad specificity often hinders their practical use as probes. In the present study we report a dramatic improvement in the carbohydrate specificity of a multi-specific fungal galectin from the mushroom Agrocybe cylindricea, which binds not only to simple β-galactosides, but also to their derivatives. Site-directed mutagenesis targeting five residues involved in β-galactose binding revealed that replacement of Asn46 with alanine (N46A) increased the binding to GalNAcα1-3Galβ-containing glycans, while eliminating binding to all other β-galactosides, as shown by glycoconjugate microarray analysis. Quantitative analysis by frontal affinity chromatography showed that the mutant N46A had enhanced affinity towards blood group A tetraose (type 2), A hexaose (type 1) and Forssman pentasaccharide with dissociation constants of 5.0×10−6 M, 3.8×10−6 M and 1.0×10−5 M respectively. Surprisingly, all the other mutants generated by saturation mutagenesis of Asn46 exhibited essentially the same specificity as N46A. Moreover, alanine substitution for Pro45, which forms the cis-conformation upon β-galactose binding, exhibited the same specificity as N46A. From a practical viewpoint, the derived N46A mutant proved to be unique as a specific probe to detect GalNAcα1-3Galβ-containing glycans by methods such as flow cytometry, cell staining and lectin microarray.

scholarly journals Double Mutations in Succinate Dehydrogenase Are Involved in SDHI Resistance in Corynespora cassiicola

2022 ◽  
Vol 10 (1) ◽  
pp. 132
Author(s):  
Bingxue Sun ◽  
Guangxue Zhu ◽  
Xuewen Xie ◽  
Ali Chai ◽  
Lei Li ◽  
...  
Keyword(s):  
Succinate Dehydrogenase ◽  
Point Mutation ◽  
Single Point ◽  
Resistance Management ◽  
Site Directed Mutagenesis ◽  
Single Point Mutation ◽  
Single Mutation ◽  
Corynespora Cassiicola ◽  
Double Mutation ◽  
Mutation Analyses

With the further application of succinate dehydrogenase inhibitors (SDHI), the resistance caused by double mutations in target gene is gradually becoming a serious problem, leading to a decrease of control efficacy. It is important to assess the sensitivity and fitness of double mutations to SDHI in Corynespora cassiicola and analysis the evolution of double mutations. We confirmed, by site-directed mutagenesis, that all double mutations (B-I280V+D-D95E/D-G109V/D-H105R, B-H278R+D-D95E/D-G109V, B-H278Y+D-D95E/D-G109V) conferred resistance to all SDHI and exhibited the increased resistance to at least one fungicide than single point mutation. Analyses of fitness showed that all double mutations had lower fitness than the wild type; most of double mutations suffered more fitness penalties than the corresponding single mutants. We also further found that double mutations (B-I280V+D-D95E/D-G109V/D-H105R) containing low SDHI-resistant single point mutation (B-I280V) exhibited higher resistance to SDHI and low fitness penalty than double mutations (B-H278Y+D-D95E/D-G109V) containing high SDHI-resistant single mutations (B-H278Y). Therefore, we may infer that a single mutation conferring low resistance is more likely to evolve into a double mutation conferring higher resistance under the selective pressure of SDHI. Taken together, our results provide some important reference for resistance management.

Detection of Diverse N-Acyl Homoserine Lactone Signalling Molecules Among Bacteria Associated with Rice Rhizosphere

2020 ◽  
Vol 77 (11) ◽  
pp. 3480-3491
Author(s):  
Ganga Viswanath ◽  
Jegan Sekar ◽  
Prabavathy Vaiyapuri Ramalingam
Keyword(s):  
Homoserine Lactone ◽  
Acyl Homoserine Lactone ◽  
Signalling Molecules ◽  
Rice Rhizosphere

scholarly journals Diversity of Acyl Homoserine Lactone Molecules in Anaerobic Membrane Bioreactors Treating Sewage at Psychrophilic Temperatures

Membranes ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 320
Author(s):  
Shamas Tabraiz ◽  
Burhan Shamurad ◽  
Evangelos Petropoulos ◽  
Alex Charlton ◽  
Obaidullah Mohiudin ◽  
...  
Keyword(s):  
Membrane Bioreactor ◽  
Quorum Quenching ◽  
Homoserine Lactone ◽  
Membrane Bioreactors ◽  
Acyl Homoserine Lactone ◽  
Mixed Liquor ◽  
Anaerobic Membrane Bioreactor ◽  
Anaerobic Bioreactors ◽  
New Information ◽  
Anaerobic Membrane Bioreactors

This study explores the types of acyl homoserine lactone (AHL) and their concentrations in different compartments of different conventional anaerobic bioreactors: (i) an upflow anaerobic membrane bioreactor (UAnMBR, biofilm/mixed liquor (sludge)); (ii) an anaerobic membrane bioreactor (AnMBR, biofilm/mixed liquor (sludge)); and (iii) an upflow sludge blanket (UASB, sludge only), all operating at 15 °C. Ten types of the AHL, namely C4-HSL, 3-oxo-C4-HSL, C6-HSL, 3-oxo-C6-HSL, C8-HSL, 3-oxo-C8-HSL, C10-HSL, 3-oxo-C10-HSL, C12-HSL, and 3-oxo-C12-HSL, which were investigated in this study, were found in UAnMBR and UASB, whilst only six of them (C4-HSL, 3-oxo-C4-HSL, C8-HSL, C10-HSL, 3-oxo-C10-HSL, and C12-HSL) were found in AnMBR. Concentrations of total AHL were generally higher in the biofilm than the sludge for both membrane bioreactors trialed. C10-HSL was the predominant AHL found in all reactors (biofilm and sludge) followed by C4-HSL and C8-HSL. Overall, the UAnMBR biofilm and sludge had 10-fold higher concentrations of AHL compared to the AnMBR. C10-HSL was only correlated with bacteria (p < 0.05), whilst other types of AHL were correlated with both bacteria and archaea. This study improves our understanding of AHL-mediated Quorum Sensing (QS) in the biofilms/sludge of UAnMBR and AnMBR, and provides new information that could contribute to the development of quorum quenching anti-fouling strategies in such systems.

Acyl-homoserine lactone-based quorum sensing and quorum quenching hold promise to determine the performance of biological wastewater treatments: An overview

Chemosphere ◽  
2016 ◽  
Vol 157 ◽  
pp. 137-151 ◽  
Author(s):  
Jinhui Huang ◽  
Yahui Shi ◽  
Guangming Zeng ◽  
Yanling Gu ◽  
Guiqiu Chen ◽  
...  
Keyword(s):  
Quorum Sensing ◽  
Quorum Quenching ◽  
Homoserine Lactone ◽  
Acyl Homoserine Lactone

Factor VIII:E113A Represents a High Specific Activity Factor VIII Arising from a Single Point Mutation within a Ca2+ Binding Site.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1725-1725 ◽  
Author(s):  
Hironao Wakabayashi ◽  
Philip J. Fay
Keyword(s):  
Factor Viii ◽  
Specific Activity ◽  
High Specific Activity ◽  
Factor Xa ◽  
Saturation Mutagenesis ◽  
Single Amino Acid ◽  
Single Point Mutation ◽  
Wild Type ◽  
Activity Increase ◽  
Wide Range

Abstract We recently identified an acidic-rich segment in the A1 domain of factor VIII (residues 110-126) that functions in the coordination of Ca2+, an ion necessary for cofactor activity (Wakabayashi et al., J. Biol. Chem.279:12677–12684, 2004). Using Ala-scanning mutagenesis, it was determined that replacement of residue E113 with Ala yielded a factor VIII point mutant that possessed an ~2-fold increased affinity for Ca2+ as compared with wild type, suggesting that this residue did not directly contribute to Ca2+ coordination but rather modulated the affinity of the ion at this site. Furthermore, the E113A factor VIII possessed twice the specific activity of wild type as determined by a one-stage clotting assay. This increased activity was not likely a result of increased affinity for Ca2+, since assays were performed at saturating Ca2+ levels. Saturation mutagenesis at position 113 revealed that substitution at this position with relatively small, nonpolar residues were well-tolerated, whereas replacement with a number of polar or charged residues was detrimental to activity. Ala-substitution yielded the greatest activity increase of ~2-fold and this level was observed over a wide range of factor VIII concentrations. Time course experiments of factor VIII activation following reaction with thrombin revealed similar rates of activation and inactivation of E113A as observed for the wild type. Interestingly, results from factor Xa generation assays using purified reactants showed the mutant possessed <10% greater specific activity than wild type and yielded similar values for Km for substrate factor X, kcat for factor Xa generation and Kd for factor IXa. Thus the single amino acid substitution minimally altered cofactor structure or inter-molecular interactions relating to its participation in factor Xase. These results indicate that mutations within this Ca2+ coordination site may selectively enhance cofactor specific activity as measured in a plasma-based assay compared to activity determined in a purified system. The enhanced activity observed for E113A factor VIII may derive from a subtle alteration in conformation affecting a yet to be identified functional parameter.

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