Structure-Activity Study of the Lantibiotic Mutacin II from Streptococcus mutans T8 by a Gene Replacement Strategy

ABSTRACT Mutacin II, elaborated by group II Streptococcus mutans, is a ribosomally synthesized and posttranslationally modified polypeptide antibiotic containing unusual thioether and didehydro amino acids. To ascertain the role of specific amino acid residues in mutacin II antimicrobial activity, we developed a streptococcal expression system that facilitates the replacement of themutA gene with a single copy of a mutated variant gene. As a result, variants of mutacin II can be designed and expressed. The system was tested by constructing the following mutant peptides: ΔN1, V7A, P9A, T10A, T10S, C15A, C26A, and C27A. All of these mutacin II variants except ΔN1 and T10A, which were not secreted, were isolated, and their identities were verified by mass spectrometry. Variants P9A, C15A, C26A, and C27A failed to exert antimicrobial activity. Because the P9A and T10A variants comprise the “hinge” region of mutacin II, these observations suggest that in addition to the thioether and didehydro amino acids, the hinge region is essential for biological activity and biosynthesis or export of the peptide. Tandem mass spectrometry of the N-terminal part of the wild-type molecule and its C15A variant confirmed that the threonine at position 10 is dehydrated and present as a didehydrobutyrine residue. This analysis of the active T10S variant further suggested that a didehydro amino acid at this position is specific for antimicrobial activity and that the biosynthetic machinery does not discriminate between threonine and serine. In contrast, the lack of production of mutacin variants with alanine substituted for threonine at position 10, as well as the deletion of asparagine at the N terminus (ΔN1), indicates that specific residues in the propeptide may be crucial for certain steps in the biosynthetic pathway of this lantibiotic.

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Genetic Analysis of Azole Resistance in the Darlington Strain of Candida albicans

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.44.11.2985-2990.2000 ◽

2000 ◽

Vol 44 (11) ◽

pp. 2985-2990 ◽

Cited By ~ 54

Author(s):

Hiroshi Kakeya ◽

Yoshitsugu Miyazaki ◽

Haruko Miyazaki ◽

Katherine Nyswaner ◽

Brian Grimberg ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acids ◽

Candida Albicans ◽

Amino Acid ◽

Genetic Analysis ◽

Expression System ◽

Single Copy ◽

Gene Replacement ◽

Azole Resistance ◽

High Level

ABSTRACT High-level azole resistance in the Darlington strain ofCandida albicans was investigated by gene replacement inC. albicans and expression in Saccharomyces cerevisiae. We sequenced the ERG11 gene, which encodes the sterol C14α-demethylase, from our copy of the Darlington strain. Both alleles contained the histidine for tyrosine substitution at position 132 (Y132H) reported in Darlington by others, but we also found a threonine-for-isoleucine substitution (I471T) not previously reported in the C. albicans ERG11. The encoded I471T change in amino acids conferred azole resistance when overexpressed alone and increased azole resistance when added to the Y132H amino acid sequence in an S. cerevisiae expression system. Replacement of one copy of ERG11 in an azole-susceptible strain of C. albicans with a single copy of the Darlington ERG11 resulted in expression of the integrated copy and a modest increase in azole resistance. The profound azole resistance of the Darlington strain is the result of multiple mutations.

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E. coli GyrA Tower Domain Interacts with QnrB1 Loop B and Plays an Important Role in QnrB1 Protection from Quinolone Inhibition

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00402-21 ◽

2021 ◽

Author(s):

Chunhui Chen ◽

Yin Wang ◽

Hidemasa Nakaminami ◽

Eu Suk Kim ◽

George A. Jacoby ◽

...

Keyword(s):

Mass Spectrometry ◽

Amino Acids ◽

Amino Acid ◽

Dna Gyrase ◽

Amino Acid Residues ◽

Site Specific ◽

E Coli ◽

Small Deletion ◽

Interaction Sites ◽

Repeat Proteins

The Qnr pentapeptide repeat proteins interact with DNA gyrase and protect it from quinolone inhibition. The two external loops, particularly the larger loop B, of Qnr proteins are essential for quinolone protection of DNA gyrase. The specific QnrB1 interaction sites on DNA gyrase are not known. In this study, we investigated the interaction between GyrA and QnrB1 using site-specific photo crosslinking of QnrB1 loop B combined with mass spectrometry. We found that amino acid residues 286-298 on the Tower domain of GyrA interact with QnrB1 and play a key role in QnrB1 protection of gyrase from quinolone inhibition. Alanine replacement of arginine at residue 293 and a small deletion of amino acids 286-289 of GyrA resulted in a decrease in the QnrB1-mediated increase in quinolone MICs and also abolished the QnrB1 protection of purified DNA gyrase from ciprofloxacin inhibition.

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Identification and Characterisation of Two 1-Aminocyclopropane- 1-Carboxylate (ACC) Synthase cDNAs Expressed during Papaya (Carica papaya) Fruit Ripening

Functional Plant Biology ◽

10.1071/pp96111 ◽

1997 ◽

Vol 24 (2) ◽

pp. 239 ◽

Cited By ~ 3

Author(s):

Michael Glenn Mason ◽

José Ramón Botella

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Fruit Ripening ◽

Active Site ◽

Carica Papaya ◽

Expression Patterns ◽

Acc Synthase ◽

Single Copy ◽

Mrna Levels ◽

Amino Acid Residues

The cloning and characterisation of two cDNAs (capacs1 and capacs2) encoding ACC synthase in papaya (Carica papaya L.) is described. capacs1 is 1104 bp long encoding 368 amino acids. capacs2 is 1098 bp long encoding 366 amino acids. The proteins encoded by both cDNAs contain the highly conserved active site of ACC synthases as well as 10 (capacs1) or 11 (capacs2) of the 12 amino acid residues conserved in most aminotransferases. Southern analyses indicate that capacs1 and capacs2 are present in the papaya genome as single copy genes. Both genes show very different expression patterns during fruit ripening. capacs1 mRNA levels are high in mature green fruits, decreasing steadily during ripening, whereas capacs2 mRNA levels are undetectable in mature green fruits but experience a dramatic increase with the onset of ripening and remain high throughout the various stages of ripening.

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Assignment of Amino Acid Residues of the AVR9 Peptide of Cladosporium fulvum That Determine Elicitor Activity

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.1997.10.7.821 ◽

1997 ◽

Vol 10 (7) ◽

pp. 821-829 ◽

Cited By ~ 43

Author(s):

Miriam Kooman-Gersmann ◽

Ralph Vogelsang ◽

Erwin C. M. Hoogendijk ◽

Pierre J. G. M. De Wit

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Resistance Gene ◽

Expression System ◽

Potato Virus X ◽

Cladosporium Fulvum ◽

Amino Acid Residues ◽

Tomato Plants ◽

Loop Region ◽

Relationship Of

The AVR9 peptide of Cladosporium fulvum is an elicitor of the hypersensitive response in tomato plants carrying the Cf-9 resistance gene (MM-Cf9). To determine the structure-activity relationship of the AVR9 peptide, amino acids important for AVR9 elicitor activity were identified by independently substituting each amino acid of AVR9 by alanine. In addition, surface-exposed amino acid residues of AVR9 were substituted by other amino acids. Activity of the mutant Avr9 constructs was studied by expressing the constructs in MM-Cf9 tomato plants, using the potato virus X (PVX) expression system and assessing the severity of necrosis induced by each PVX∷Avr9 construct. This allowed direct identification of amino acid residues of AVR9 that are essential for elicitor activity. We identified amino acid substitutions that resulted in AVR9 mutants with higher, similar, or lower elicitor activity compared to the wild-type AVR9 peptide. Some mutants had completely lost elicitor activity. A selection of peptides, representing different categories, was isolated and injected into leaves of MM-Cf9 plants. The necrosis-inducing activity of the isolated peptides correlated well with the necrosis induced by the corresponding PVX∷Avr9 derivatives. Based on the necrosis-inducing activity of the mutant AVR9 peptides and the global structure of AVR9, we assigned sites in AVR9 that are important for its necrosis-inducing activity. We postulate that the “hydrophobic β-loop” region of the AVR9 peptide is crucial for necrosis-inducing activity in tomato plants that carry the Cf-9 resistance gene.

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Statistical Force-Field for Structural Modeling Using Chemical Cross-Linking/mass Spectrometry Distance Constraints

10.26434/chemrxiv.6030563 ◽

2018 ◽

Author(s):

Allan J. R. Ferrari ◽

Fabio C. Gozzo ◽

Leandro Martinez

Keyword(s):

Mass Spectrometry ◽

Amino Acid ◽

Force Field ◽

Protein Structures ◽

Protein Structure Determination ◽

Structural Modeling ◽

Cross Linking ◽

Amino Acid Residues ◽

Distance Constraints ◽

Chemical Cross Linking

<div><p>Chemical cross-linking/Mass Spectrometry (XLMS) is an experimental method to obtain distance constraints between amino acid residues, which can be applied to structural modeling of tertiary and quaternary biomolecular structures. These constraints provide, in principle, only upper limits to the distance between amino acid residues along the surface of the biomolecule. In practice, attempts to use of XLMS constraints for tertiary protein structure determination have not been widely successful. This indicates the need of specifically designed strategies for the representation of these constraints within modeling algorithms. Here, a force-field designed to represent XLMS-derived constraints is proposed. The potential energy functions are obtained by computing, in the database of known protein structures, the probability of satisfaction of a topological cross-linking distance as a function of the Euclidean distance between amino acid residues. The force-field can be easily incorporated into current modeling methods and software. In this work, the force-field was implemented within the Rosetta ab initio relax protocol. We show a significant improvement in the quality of the models obtained relative to current strategies for constraint representation. This force-field contributes to the long-desired goal of obtaining the tertiary structures of proteins using XLMS data. Force-field parameters and usage instructions are freely available at http://m3g.iqm.unicamp.br/topolink/xlff <br></p></div><p></p><p></p>

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Mass spectrometry imaging of L-[ring-13C6]-labeled phenylalanine and tyrosine kinetics in non-small cell lung carcinoma

Cancer & Metabolism ◽

10.1186/s40170-021-00262-9 ◽

2021 ◽

Vol 9 (1) ◽

Author(s):

Jianhua Cao ◽

Benjamin Balluff ◽

Martijn Arts ◽

Ludwig J. Dubois ◽

Luc J. C. van Loon ◽

...

Keyword(s):

Mass Spectrometry ◽

Amino Acids ◽

Amino Acid ◽

Mass Spectrometry Imaging ◽

Tumor Tissue ◽

Small Cell Lung Carcinoma ◽

Small Cell ◽

Small Cell Lung ◽

Tracer Injection ◽

Cell Lung Carcinoma

Abstract Background Metabolic reprogramming is a common phenomenon in tumorigenesis and tumor progression. Amino acids are important mediators in cancer metabolism, and their kinetics in tumor tissue are far from being understood completely. Mass spectrometry imaging is capable to spatiotemporally trace important endogenous metabolites in biological tissue specimens. In this research, we studied L-[ring-13C6]-labeled phenylalanine and tyrosine kinetics in a human non-small cell lung carcinoma (NSCLC) xenografted mouse model using matrix-assisted laser desorption/ionization Fourier-transform ion cyclotron resonance mass spectrometry imaging (MALDI-FTICR-MSI). Methods We investigated the L-[ring-13C6]-Phenylalanine (13C6-Phe) and L-[ring-13C6]-Tyrosine (13C6-Tyr) kinetics at 10 min (n = 4), 30 min (n = 3), and 60 min (n = 4) after tracer injection and sham-treated group (n = 3) at 10 min in mouse-xenograft lung tumor tissues by MALDI-FTICR-MSI. Results The dynamic changes in the spatial distributions of 19 out of 20 standard amino acids are observed in the tumor tissue. The highest abundance of 13C6-Phe was detected in tumor tissue at 10 min after tracer injection and decreased progressively over time. The overall enrichment of 13C6-Tyr showed a delayed temporal trend compared to 13C6-Phe in tumor caused by the Phe-to-Tyr conversion process. Specifically, 13C6-Phe and 13C6-Tyr showed higher abundances in viable tumor regions compared to non-viable regions. Conclusions We demonstrated the spatiotemporal intra-tumoral distribution of the essential aromatic amino acid 13C6-Phe and its de-novo synthesized metabolite 13C6-Tyr by MALDI-FTICR-MSI. Our results explore for the first time local phenylalanine metabolism in the context of cancer tissue morphology. This opens a new way to understand amino acid metabolism within the tumor and its microenvironment.

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Development of Antimicrobial Stapled Peptides Based on Magainin 2 Sequence

Molecules ◽

10.3390/molecules26020444 ◽

2021 ◽

Vol 26 (2) ◽

pp. 444

Author(s):

Motoharu Hirano ◽

Chihiro Saito ◽

Hidetomo Yokoo ◽

Chihiro Goto ◽

Ryuji Kawano ◽

...

Keyword(s):

Antimicrobial Activity ◽

Amino Acid ◽

Hemolytic Activity ◽

Helical Structure ◽

Antimicrobial Activities ◽

Side Chain ◽

Membrane Disruption ◽

Amino Acid Residues ◽

Electrophysiological Measurements ◽

Cell Membrane Disruption

Magainin 2 (Mag2), which was isolated from the skin of the African clawed frog, is a representative antimicrobial peptide (AMP) that exerts antimicrobial activity via microbial membrane disruption. It has been reported that the helicity and amphipathicity of Mag2 play important roles in its antimicrobial activity. We investigated and recently reported that 17 amino acid residues of Mag2 are required for its antimicrobial activity, and accordingly developed antimicrobial foldamers containing α,α-disubstituted amino acid residues. In this study, we further designed and synthesized a set of Mag2 derivatives bearing the hydrocarbon stapling side chain for helix stabilization. The preferred secondary structures, antimicrobial activities, and cell-membrane disruption activities of the synthesized peptides were evaluated. Our analyses revealed that hydrocarbon stapling strongly stabilized the helical structure of the peptides and enhanced their antimicrobial activity. Moreover, peptide 2 stapling between the first and fifth position from the N-terminus showed higher antimicrobial activity than that of Mag2 against both gram-positive and gram-negative bacteria without exerting significant hemolytic activity. To investigate the modes of action of tested peptides 2 and 8 in antimicrobial and hemolytic activity, electrophysiological measurements were performed.

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