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1.
J Biol Chem ; 299(7): 104845, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-37209826

RESUMO

The increase in antibiotic resistance calls for accelerated molecular engineering strategies to diversify natural products for drug discovery. The incorporation of non-canonical amino acids (ncAAs) is an elegant strategy for this purpose, offering a diverse pool of building blocks to introduce desired properties into antimicrobial lanthipeptides. We here report an expression system using Lactococcus lactis as a host for non-canonical amino acid incorporation with high efficiency and yield. We show that incorporating the more hydrophobic analog ethionine (instead of methionine) into nisin improves its bioactivity against several Gram-positive strains we tested. New-to-nature variants were further created by click chemistry. By azidohomoalanine (Aha) incorporation and subsequent click chemistry, we obtained lipidated variants at different positions in nisin or in truncated nisin variants. Some of them show improved bioactivity and specificity against several pathogenic bacterial strains. These results highlight the ability of this methodology for lanthipeptide multi-site lipidation, to create new-to-nature antimicrobial products with diverse features, and extend the toolbox for (lanthi)peptide drug improvement and discovery.


Assuntos
Química Click , Lactococcus lactis , Metionina , Nisina , Aminoácidos/metabolismo , Peptídeos Antimicrobianos/síntese química , Peptídeos Antimicrobianos/farmacologia , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Metionina/química , Metionina/metabolismo , Nisina/síntese química , Nisina/farmacologia , Bactérias/efeitos dos fármacos , Farmacorresistência Bacteriana/efeitos dos fármacos
2.
J Nat Prod ; 87(6): 1548-1555, 2024 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-38888620

RESUMO

Antimicrobial peptides (AMPs) have raised significant interest, forming a potential new class of antibiotics in the fight against multi-drug-resistant bacteria. Various AMPs are ribosomally synthesized and post-translationally modified peptides (RiPPs). One post-translational modification found in AMPs is the halogenation of Trp residues. This modification has, for example, been shown to be critical for the activity of the potent AMP NAI-107 from Actinoallomurus. Due to the importance of organohalogens, establishing methods for facile and selective halogen atom installation into AMPs is highly desirable. In this study, we introduce an expression system utilizing the food-grade strain Lactococcus lactis, facilitating the efficient incorporation of bromo-Trp (BrTrp) into (modified) peptides, exemplified by the lantibiotic nisin with a single Trp residue or analogue incorporated at position 1. This provides an alternative to the challenges posed by halogenase enzymes, such as poor substrate selectivity. Our method yields expression levels comparable to that of wild-type nisin, while BrTrp incorporation does not interfere with the post-translational modifications of nisin (dehydration and cyclization). One brominated nisin variant exhibits a 2-fold improvement in antimicrobial activity against two tested pathogens, including a WHO priority pathogen, while maintaining the same lipid II binding and bactericidal activity as wild-type nisin. The work presented here demonstrates the potential of this methodology for peptide halogenation, offering a new avenue for the development of diverse antimicrobial products labeled with BrTrp.


Assuntos
Antibacterianos , Peptídeos Antimicrobianos , Halogenação , Testes de Sensibilidade Microbiana , Nisina , Nisina/farmacologia , Nisina/química , Peptídeos Antimicrobianos/farmacologia , Peptídeos Antimicrobianos/química , Antibacterianos/farmacologia , Antibacterianos/química , Triptofano/química , Lactococcus lactis , Estrutura Molecular
3.
J Am Chem Soc ; 144(30): 13815-13822, 2022 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-35868012

RESUMO

In proteins, the amino acids Phe, Tyr, and especially Trp are frequently involved in π interactions such as π-π, cation-π, and CH-π bonds. These interactions are often crucial for protein structure and protein-ligand binding. A powerful means to study these interactions is progressive fluorination of these aromatic residues to modulate the electrostatic component of the interaction. However, to date no protein expression platform is available to produce milligram amounts of proteins labeled with such fluorinated amino acids. Here, we present a Lactococcus lactis Trp auxotroph-based expression system for efficient incorporation (≥95%) of mono-, di-, tri-, and tetrafluorinated, as well as a methylated Trp analog. As a model protein we have chosen LmrR, a dimeric multidrug transcriptional repressor protein from L. lactis. LmrR binds aromatic drugs, like daunomycin and riboflavin, between Trp96 and Trp96' in the dimer interface. Progressive fluorination of Trp96 decreased the affinity for the drugs 6- to 70-fold, clearly establishing the importance of electrostatic π-π interactions for drug binding. Presteady state kinetic data of the LmrR-drug interaction support the enthalpic nature of the interaction, while high resolution crystal structures of the labeled protein-drug complexes provide for the first time a structural view of the progressive fluorination approach. The L. lactis expression system was also used to study the role of Trp68 in the binding of riboflavin by the membrane-bound riboflavin transport protein RibU from L. lactis. Progressive fluorination of Trp68 revealed a strong electrostatic component that contributed 15-20% to the total riboflavin-RibU binding energy.


Assuntos
Lactococcus lactis , Triptofano , Aminoácidos/metabolismo , Cátions , Proteínas de Membrana Transportadoras/metabolismo , Riboflavina/metabolismo , Triptofano/química
4.
Int J Mol Sci ; 23(13)2022 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-35806299

RESUMO

Thimet oligopeptidase (TOP) is a metallopeptidase involved in the metabolism of oligopeptides inside and outside cells of various tissues. It has been proposed that substrate or inhibitor binding in the TOP active site induces a large hinge-bending movement leading to a closed structure, in which the bound ligand is enclosed. The main goal of the present work was to study this conformational change, and fluorescence techniques were used. Four active TOP mutants were created, each equipped with a single-Trp residue (fluorescence donor) and a p-nitro-phenylalanine (pNF) residue as fluorescence acceptor at opposite sides of the active site. pNF was biosynthetically incorporated with high efficiency using the amber codon suppression technology. Inhibitor binding induced shorter Donor-Acceptor (D-A) distances in all mutants, supporting the view that a hinge-like movement is operative in TOP. The activity of TOP is known to be dependent on the ionic strength of the assay buffer and D-A distances were measured at different ionic strengths. Interestingly, a correlation between the D-A distance and the catalytic activity of TOP was observed: the highest activities corresponded to the shortest D-A distances. In this study for the first time the hinge-bending motion of a metallopeptidase in solution could be studied, yielding insight about the position of the equilibrium between the open and closed conformation. This information will contribute to a more detailed understanding of the mode of action of these enzymes, including therapeutic targets like neurolysin and angiotensin-converting enzyme 2 (ACE2).


Assuntos
Metaloendopeptidases , Oligopeptídeos , Domínio Catalítico , Ligantes , Metaloendopeptidases/química , Oligopeptídeos/metabolismo , Especificidade por Substrato
5.
Biochem Biophys Res Commun ; 492(3): 343-348, 2017 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-28859987

RESUMO

Biosynthetic incorporation of non-canonic amino acids is an attractive strategy to introduce new properties in recombinant proteins. Trp analogs can be incorporated in recombinant proteins replacing regular Trp during protein translation into a Trp-auxotrophic cell host. This straightforward method however, is limited to few analogs recognized and accepted by the cellular protein production machinery. 5-hydroxy-tryptophan (5OH-Trp) can be bio-incorporated using E. coli as expression host however; we have experienced very low incorporation yields - amount of protein containing regular Trp/amount of protein containing the Trp analog - during expressions of 5OH-Trp labeled proteins. Furthermore, this low incorporation yield were verified especially when the widely-used vectors based on the T7 RNA polymerase were used. Testing different 5OH-Trp incorporation protocols we verified that in these T7-based systems, the production of the T7 RNA polymerase is driven by the same elements - lac promoter/IPTG - as the target protein. Consequently, the bio-incorporation of the 5OH-Trp residues also occurs in this crucial enzyme, but, the produced T7 RNA polymerase labeled with 5OH-Trp is inactive or much less active. In the present work, we describe an efficient method to overcome this mentioned problem and bio-incorporate 5OH-Trp in proteins expressed in E. coli., using vectors based on the T7 RNA polymerase-T7 promoter. The two-step induction protocol here described showed incorporation efficiencies of 5OH-Trp higher than 90%.


Assuntos
5-Hidroxitriptofano/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/genética , Vetores Genéticos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Virais/metabolismo , Escherichia coli/metabolismo , Proteínas Recombinantes/biossíntese
6.
Amino Acids ; 48(5): 1309-18, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26872656

RESUMO

Lantibiotics are posttranslationally modified peptides with efficient inhibitory activity against various Gram-positive bacteria. In addition to the original modifications, incorporation of non-canonical amino acids can render new properties and functions to lantibiotics. Nisin is the most studied lantibiotic and contains no tryptophan residues. In this study, a system was constructed to incorporate tryptophan analogues into nisin, which included the modification machinery (NisBTC) and the overexpression of tryptophanyl-tRNA synthetase (TrpRS). Tryptophan and three different tryptophan analogues (5-fluoroTrp (5FW), 5-hydroxyTrp (5HW) and 5-methylTrp (5MeW)) were successfully incorporated at four different positions of nisin (I1W, I4W, M17W and V32W). The incorporation efficiency of tryptophan analogues into mutants I1W, M17W and V32W was over 97 %, while the mutant I4W showed relatively low incorporation efficiency (69-93 %). The variants with 5FW showed relatively higher production yield, while 5MeW-containing variants showed the lowest yield. The dehydration efficiency of serines or threonines was affected by the tryptophan mutants of I4W and V32W. The affinity of the peptides for the cation-ion exchange and reverse phase chromatography columns was significantly reduced when 5HW was incorporated. The antimicrobial activity of IIW and its 5FW analogue both decreased two times compared to that of nisin, while that of its 5HW analogue decreased four times. The 5FW analogue of I4W also showed two times decreased activity than nisin. However, the mutant M17W and its 5HW analogue both showed 32 times reduced activity relative to that of nisin.


Assuntos
Bacteriocinas/química , Nisina/química , Nisina/farmacologia , Triptofano/farmacologia , Bacteriocinas/genética , Bacteriocinas/farmacologia , Lactococcus lactis/efeitos dos fármacos , Estrutura Molecular , Nisina/genética , Triptofano/análogos & derivados , Triptofano/genética
7.
Amino Acids ; 47(1): 213-6, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25399056

RESUMO

Biosynthetic incorporation of ß-(1-azulenyl)-L-alanine, an isostere of tryptophan, is reported using a tryptophan auxotroph expression host. The azulene moiety introduced this way in proteins features many attractive spectroscopic properties, particularly suitable for in vivo studies.


Assuntos
Alanina/metabolismo , Azulenos/metabolismo , Proteínas de Bactérias/biossíntese , Lactococcus lactis/metabolismo , Biossíntese de Proteínas , Proteínas de Bactérias/genética , Lactococcus lactis/genética , Triptofano/metabolismo
8.
J Membr Biol ; 247(9-10): 1019-30, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24952466

RESUMO

Amphipols (APols) are short amphipathic polymers that keep integral membrane proteins water-soluble while stabilizing them as compared to detergent solutions. In the present work, we have carried out functional and structural studies of a membrane transporter that had not been characterized in APol-trapped form yet, namely EII(mtl), a dimeric mannitol permease from the inner membrane of Escherichia coli. A tryptophan-less and dozens of single-tryptophan (Trp) mutants of this transporter are available, making it possible to study the environment of specific locations in the protein. With few exceptions, the single-Trp mutants show a high mannitol-phosphorylation activity when in membranes, but, as variance with wild-type EII(mtl), some of them lose most of their activity upon solubilization by neutral (PEG- or maltoside-based) detergents. Here, we present a protocol to isolate these detergent-sensitive mutants in active form using APol A8-35. Trapping with A8-35 keeps EII(mtl) soluble and functional in the absence of detergent. The specific phosphorylation activity of an APol-trapped Trp-less EII(mtl) mutant was found to be ~3× higher than the activity of the same protein in dodecylmaltoside. The preparations are suitable both for functional and for fluorescence spectroscopy studies. A fluorescein-labeled version of A8-35 has been synthesized and characterized. Exploratory studies were conducted to examine the environment of specific Trp locations in the transmembrane domain of EII(mtl) using Trp fluorescence quenching by water-soluble quenchers and by the fluorescein-labeled APol. This approach has the potential to provide information on the transmembrane topology of MPs.


Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/isolamento & purificação , Escherichia coli/enzimologia , Fluoresceína/química , Precipitação Fracionada/métodos , Proteínas de Transporte de Monossacarídeos/química , Proteínas de Transporte de Monossacarídeos/isolamento & purificação , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/química , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/isolamento & purificação , Polímeros/química , Propilaminas/química , Tensoativos/química , Proteínas de Escherichia coli/ultraestrutura , Fluoresceína/análise , Corantes Fluorescentes/análise , Corantes Fluorescentes/química , Interações Hidrofóbicas e Hidrofílicas , Proteínas de Transporte de Monossacarídeos/ultraestrutura , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/ultraestrutura , Solubilidade , Soluções , Manejo de Espécimes/métodos , Coloração e Rotulagem
9.
Peptides ; 174: 171152, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38220092

RESUMO

Nisin serves as the prototype within the lantibiotic group of antimicrobial peptides, exhibiting a broad-spectrum inhibition against Gram-positive bacteria, including important food-borne pathogens and clinically relevant antibiotic-resistant strains. The gene-encoded nature of nisin allows for gene-based bioengineering, enabling the generation of novel derivatives. It has been demonstrated that nisin mutants can be produced with improved functional properties. Here, we particularly focus on the uncommon amino acid residues dehydroalanine (Dha) and dehydrobutyrin (Dhb), whose functions are not yet fully elucidated. Prior to this study, we developed a new expression system that utilizes the nisin modification machinery NisBTC to advance expression, resulting in enhanced peptide dehydration efficiency. Through this approach, we discovered that the dehydrated amino acid Dhb at position 18 in the peptide rombocin, a short variant of nisin, displayed four times higher activity compared to the non-dehydrated peptide against the strain Lactococcus lactis. Furthermore, we observed that in the peptides nisin and rombocin, the dehydrated amino acid Dha at residue positon 18 exhibited superior activity compared to the dehydrated amino acid Dhb. Upon purifying the wild-type nisin and its variant nisinG18/Dha to homogeneity, the minimum inhibitory concentration (MIC) indicated that the variant exhibited activity similar to that of wild-type nisin in inhibiting the growth of Bacillus cereus but showed twice the MIC values against the other four tested Gram-positive strains. Further stability tests demonstrated that the dehydrated peptide exhibited properties similar to wild-type nisin under different temperatures but displayed higher resistance to proteolytic enzymes compared to wild-type nisin.


Assuntos
Bacteriocinas , Lactococcus lactis , Nisina , Nisina/genética , Nisina/farmacologia , Aminoácidos/genética , Peptídeos Antimicrobianos , Antibacterianos/farmacologia , Antibacterianos/química , Bacteriocinas/química , Lactococcus lactis/metabolismo
10.
Microbiol Res ; 282: 127640, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38350171

RESUMO

Antimicrobial peptides (AMPs) show promise as alternatives to traditional antibiotics for treating drug-resistant infections. Their adaptability and diverse sequence possibilities allow for rational design by modulating physicochemical determinants to achieve desired biological properties, transforming them into peptides for potential new therapies. Nisin, one of the best-studied AMPs, is believed to have potential to be used as a therapeutic, particularly against antibiotic-resistant bacteria. However, its instability in physiological conditions limits its use in clinical applications and pharmaceutical development. Exploration of new natural variants of nisin has uncovered diverse properties using different domains. Shuffling peptide modules can fine-tune the chemical properties of these molecules, potentially enhancing stability while maintaining or improving antimicrobial activity. In this study, hybrid AMPs were created by combining domains from three unique nisin variants, i.e. nisin A, cesin and rombocin, leading to the identification of a promising variant, named cerocin A, which harbours only 25 amino acids compared to the typical 31-35 amino acid length of nisin. Cerocin A demonstrates potent antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA), approaching that of nisin itself. Cerocin A's mode of action involves a dual mechanism through the combination of two domains, consisting of a small ring/domain (6 amino acids) from the C-terminal end of rombocin attached to the preceding peptide of cesin, changing it from a bacteriostatic to a bactericidal peptide. Further mutation studies identified a new variant, cerocin V, with significantly improved resistance against trypsin degradation, while maintaining high potency. Importantly, cerocin V showed no undesired toxic effects on human red blood cells and remained stable in human plasma. In conclusion, we demonstrate that peptide construction using domain engineering is an effective strategy for manipulating both biological and physicochemical aspects, leading to the creation of novel bioactive molecules with desired properties. These constructs are appealing candidates for further optimization and development as novel antibiotics.


Assuntos
Bacteriocinas , Staphylococcus aureus Resistente à Meticilina , Nisina , Humanos , Antibacterianos/farmacologia , Antibacterianos/química , Bacteriocinas/genética , Bacteriocinas/farmacologia , Nisina/genética , Nisina/farmacologia , Staphylococcus aureus Resistente à Meticilina/genética , Aminoácidos , Testes de Sensibilidade Microbiana
11.
ACS Synth Biol ; 13(1): 370-383, 2024 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-38194633

RESUMO

Nisin, with its unique mode of action and potent antimicrobial activity, serves as a remarkable inspiration for the design of novel antibiotics. However, peptides possess inherent weaknesses, particularly their susceptibility to proteolytic degradation, such as by trypsin, which limits their broader applications. This led us to speculate that natural variants of nisin produced by underexplored bacterial species can potentially overcome these limitations. We carried out genome mining of two Romboutsia sedimentorum strains, RC001 and RC002, leading to the discovery of rombocin A, which is a 25 amino acid residue short nisin variant that is predicted to have only four macrocycles compared to the known 31-35 amino acids long nisin variants with five macrocycles. Using the nisin-controlled expression system, we heterologously expressed fully modified and functional rombocin A in Lactococcus lactis and demonstrated its selective antimicrobial activity against Listeria monocytogenes. Rombocin A uses a dual mode of action involving lipid II binding activity and dissipation of the membrane potential to kill target bacteria. Stability tests confirmed its high stability at different pH values, temperatures, and in particular, against enzymatic degradation. With its gene-encoded characteristic, rombocin A is amenable to bioengineering to generate novel derivatives. Further mutation studies led to the identification of rombocin K, a mutant with enhanced bioactivity against L. monocytogenes. Our findings suggest that rombocin A and its bioengineered variant, rombocin K, are promising candidates for development as food preservatives or antibiotics against L. monocytogenes.


Assuntos
Lactococcus lactis , Listeria monocytogenes , Nisina , Nisina/genética , Nisina/farmacologia , Nisina/química , Listeria monocytogenes/genética , Listeria monocytogenes/metabolismo , Antibacterianos/metabolismo , Mutação , Lactococcus lactis/genética , Lactococcus lactis/metabolismo
12.
Biochim Biophys Acta ; 1818(3): 861-8, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22100747

RESUMO

The mannitol transporter EII(mtl) from Escherichia coli is responsible for the uptake of mannitol over the inner membrane and its concomitant phosphorylation. EII(mtl) is functional as a dimer and its membrane-embedded C domain, IIC(mtl), harbors one high affinity mannitol binding site. To characterize this domain in more detail the microenvironments of thirteen residue positions were explored by 5-fluorotryptophan (5-FTrp) fluorescence spectroscopy. Because of the simpler photophysics of 5-FTrp compared to Trp, one can distinguish between the two 5-FTrp probes present in dimeric IIC(mtl). At many labeled positions, the microenvironment of the 5-FTrps in the two protomers differs. Spectroscopic properties of three mutants labeled at positions 198, 251, and 260 show that two conserved motifs (Asn194-His195 and Gly254-Ile255-His256-Glu257) are located in well-structured parts of IIC(mtl). Mannitol binding has a large impact on the structure around position 198, while only minor changes are induced at positions 251 and 260. Phosphorylation of the cytoplasmic B domain of EII(mtl) is sensed by 5-FTrp at positions 30, 42, 251 and 260. We conclude that many parts of the IIC(mtl) structure are involved in the sugar translocation. The structure of EII(mtl), as investigated in this work, differs from the recently solved structure of a IIC protein transporting diacetylchitobiose, ChbC, and also belonging to the glucose superfamily of EII sugar transporters. In EII(mtl), the sugar binding site is more close to the periplasmic face and the structure of the 2 protomers in the dimer is different, while both protomers in the ChbC dimer are essentially the same.


Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/química , Manitol/química , Proteínas de Transporte de Monossacarídeos/química , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/química , Triptofano/análogos & derivados , Motivos de Aminoácidos , Transporte Biológico Ativo/fisiologia , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Manitol/metabolismo , Proteínas de Transporte de Monossacarídeos/genética , Proteínas de Transporte de Monossacarídeos/metabolismo , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/genética , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/metabolismo , Fosforilação , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Espectrometria de Fluorescência/métodos , Triptofano/química
13.
Amino Acids ; 44(5): 1329-36, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23404517

RESUMO

Biosynthetic incorporation of tryptophan (Trp) analogues in recombinant proteins using an E. coli Trp auxotroph expression host is limited to analogues modified with a small substituent like a fluoro atom or a hydroxyl or amine group. We report here the efficient incorporation (>89 %) of chloro- and bromo atoms containing Trp analogues in alloproteins at high expression levels using a Lactococcus lactis Trp auxotroph strain. This result was only obtained after coexpression of the enzyme tryptophanyl-tRNA synthetase (TrpRS) of L. lactis, an enzyme believed to show a more relaxed substrate specificity than TrpRS from E. coli. Chloro- and bromo-Trps are attractive intrinsic phosphorescence probes as these Trp analogues are much less sensitive for quenchers in the medium, like oxygen, than Trp. Coexpression of TrpRS was also essential for the biosynthetic incorporation (94 %) of the Trp analogue 5,6 difluoroTrp. This makes our expression system ideally suited to generate a set of methyl- and fluoro-substituted Trp analogue-containing alloproteins in high yield for investigating the involvement of the Trp residue in cation-pi or pi-pi interactions. Taken together, the presented Trp auxotroph expression system features the most relaxed specificity for Trp analogue structures reported to date and gives a high alloprotein yield.


Assuntos
Proteínas de Bactérias/biossíntese , Lactococcus lactis/enzimologia , Triptofano-tRNA Ligase/biossíntese , Triptofano/análogos & derivados , Triptofano/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Clonagem Molecular , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Lactococcus lactis/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Espectrometria de Fluorescência , Coloração e Rotulagem , Especificidade por Substrato
14.
Microbiol Spectr ; : e0531922, 2023 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-37754751

RESUMO

Nisin is a widely used lantibiotic owing to its potent antimicrobial activity and its food-grade status. Its mode of action includes cell wall synthesis inhibition and pore formation, which are attributed to the lipid II binding and pore-forming domains, respectively. We discovered cesin, a short natural variant of nisin, produced by the psychrophilic anaerobe Clostridium estertheticum. Unlike other natural nisin variants, cesin lacks the two terminal macrocycles constituting the pore-forming domain. The current study aimed at heterologous expression and characterization of the antimicrobial activity and physicochemical properties of cesin. Following the successful heterologous expression of cesin in Lactococcus lactis, the lantibiotic demonstrated a broad and potent antimicrobial profile comparable to that of nisin. Determination of its mode of action using lipid II and lipoteichoic acid binding assays linked the potent antimicrobial activity to lipid II binding and electrostatic interactions with teichoic acids. Fluorescence microscopy showed that cesin lacks pore-forming ability in its natural form. Stability tests have shown the lantibiotic is highly stable at different pH values and temperature conditions, but that it can be degraded by trypsin. However, a bioengineered analog, cesin R15G, overcame the trypsin degradation, while keeping full antimicrobial activity. This study shows that cesin is a novel (small) nisin variant that efficiently kills target bacteria by inhibiting cell wall synthesis without pore formation. IMPORTANCE The current increase in antibiotic-resistant pathogens necessitates the discovery and application of novel antimicrobials. In this regard, we recently discovered cesin, which is a short natural variant of nisin produced by the psychrophilic Clostridium estertheticum. However, its suitability as an antimicrobial compound was in doubt due to its structural resemblance to nisin(1-22), a bioengineered short variant of nisin with low antimicrobial activity. Here, we show by heterologous expression, purification, and characterization that the potency of cesin is not only much higher than that of nisin(1-22), but that it is even comparable to the full-length nisin, despite lacking two C-terminal rings that are essential for nisin's activity. We show that cesin is a suitable scaffold for bioengineering to improve its applicability, such as resistance to trypsin. This study demonstrates the suitability of cesin for future application in food and/or for health as a potent and stable antimicrobial compound.

15.
Anal Biochem ; 428(2): 111-8, 2012 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-22713342

RESUMO

The lysin motif (LysM) is a peptidoglycan binding protein domain found in a wide range of prokaryotes and eukaryotes. Various techniques have been used to study the LysM-ligand interaction, but a sensitive spectroscopic method to directly monitor this interaction has not been reported. Here a tryptophan analog fluorescence spectroscopy approach is presented to monitor the LysM-ligand interaction using the LysM of the N-acetylglucosaminidase enzyme of Lactococcus lactis. A three-dimensional model of this LysM protein was built based on available structural information of a homolog. This model allowed choosing the amino acid positions to be labeled with a Trp analog. Four functional single-Trp LysM mutants and one double-Trp LysM mutant were constructed and biosynthetically labeled with 7-azatryptophan or 5-hydroxytryptophan. These Trp analogs feature red-shifted absorption spectra, enabling the monitoring of the LysM-ligand interaction in media with a Trp background. The emission intensities of four of the five LysM constructs were found to change markedly on exposure to either L. lactis bacterium-like particles or peptidoglycan as ligands. The method reported here is suitable to monitor LysM-ligand interactions at (sub)micromolar LysM concentrations and can be used for the detection of low levels of peptidoglycan or microbes in solutions.


Assuntos
Lactococcus lactis/enzimologia , Muramidase/química , Espectrometria de Fluorescência/métodos , Triptofano/análogos & derivados , Motivos de Aminoácidos , Sequência de Aminoácidos , Carboidratos/química , Biologia Computacional , Ensaio de Imunoadsorção Enzimática , Ligantes , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Muramidase/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Alinhamento de Sequência , Especificidade por Substrato , Titulometria , Triptofano/metabolismo
16.
J Biol Chem ; 285(33): 25324-31, 2010 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-20522557

RESUMO

The mannitol transporter from Escherichia coli, EII(mtl), belongs to a class of membrane proteins coupling the transport of substrates with their chemical modification. EII(mtl) is functional as a homodimer, and it harbors one high affinity mannitol-binding site in the membrane-embedded C domain (IIC(mtl)). To localize this binding site, 19 single Trp-containing mutants of EII(mtl) were biosynthetically labeled with 5-fluorotryptophan (5-FTrp) and mixed with azi-mannitol, a substrate analog acting as a Förster resonance energy transfer (FRET) acceptor. Typically, for mutants showing FRET, only one 5-FTrp was involved, whereas the 5-FTrp from the other monomer was too distant. This proves that the mannitol-binding site is asymmetrically positioned in dimeric IIC(mtl). Combined with the available two-dimensional projection maps of IIC(mtl), it is concluded that a second resting binding site is present in this transporter. Active transport of mannitol only takes place when EII(mtl) becomes phosphorylated at Cys(384) in the cytoplasmic B domain. Stably phosphorylated EII(mtl) mutants were constructed, and FRET experiments showed that the position of mannitol in IIC(mtl) remains the same. We conclude that during the transport cycle, the phosphorylated B domain has to move to the mannitol-binding site, located in the middle of the membrane, to phosphorylate mannitol.


Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Proteínas de Transporte de Monossacarídeos/química , Proteínas de Transporte de Monossacarídeos/metabolismo , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/química , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/metabolismo , Sítios de Ligação/genética , Sítios de Ligação/fisiologia , Proteínas de Escherichia coli/genética , Transferência Ressonante de Energia de Fluorescência , Manitol/análogos & derivados , Manitol/metabolismo , Modelos Biológicos , Proteínas de Transporte de Monossacarídeos/genética , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/genética , Fosforilação , Ligação Proteica/genética , Ligação Proteica/fisiologia , Multimerização Proteica/genética , Multimerização Proteica/fisiologia , Espectrometria de Fluorescência
17.
Biochim Biophys Acta ; 1788(2): 581-6, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19013424

RESUMO

This paper presents domain complementation studies in the mannitol transporter, EIImtl, from Escherichia coli. EIImtl is responsible for the transport and concomitant phosphorylation of mannitol over the cytoplasmic membrane. By using tryptophan-less EIImtl as a basis, each of the four phenylalanines located in the cytoplasmic loop between putative transmembrane helices II and III in the membrane-embedded C domain were replaced by tryptophan, yielding the mutants W97, W114, W126, and W133. Except for W97, these single-tryptophan mutants exhibited a high, wild-type-like, binding affinity for mannitol. Of the four mutants, only W114 showed a high mannitol phosphorylation activity. EIImtl is functional as a dimer and the effect of these mutations on the oligomeric activity was investigated via heterodimer formation (C/C domain complementation studies). The low phosphorylation activities of W126 and W133 could be increased 7-28 fold by forming heterodimers with either the C domain of W97 (IICmtlW97) or the inactive EIImtl mutant G196D. W126 and W133, on the other hand, did not complement each other. This study points towards a role of positions 97, 126 and 133 in the oligomeric activation of EIImtl. The involvement of specific residue positions in the oligomeric functioning of a sugar-translocating EII protein has not been presented before.


Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Proteínas de Transporte de Monossacarídeos/química , Proteínas de Transporte de Monossacarídeos/metabolismo , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/química , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/metabolismo , Sequência de Aminoácidos , Ativação Enzimática , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Manitol/metabolismo , Dados de Sequência Molecular , Proteínas de Transporte de Monossacarídeos/genética , Mutação/genética , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/genética , Fosforilação , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
18.
Biochemistry ; 48(23): 5284-90, 2009 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-19402710

RESUMO

In this work, four single tryptophan (Trp) mutants of the dimeric mannitol transporter of Escherichia coli, EII(mtl), are characterized using Trp and 5-fluoroTrp (5-FTrp) fluorescence spectroscopy. The four positions, 97, 114, 126, and 133, are located in a region shown by recent studies to be involved in the mannitol translocation process. To spectroscopically distinguish between the Trp positions in each subunit of dimeric EII(mtl), 5-FTrp was biosynthetically incorporated because of its much simpler photophysics compared to those of Trp. The steady-state and time-resolved fluorescence methodologies used point out that all four positions are in structured environments, both in the absence and in the presence of a saturating concentration of mannitol. The fluorescence decay of all 5-FTrp-containing mutants was highly homogeneous, suggesting similar microenvironments for both probes per dimer. However, Stern-Volmer quenching experiments using potassium iodide indicate different solvent accessibilities for the two probes at positions 97 and 133. A 5 A two-dimensional (2D) projection map of the membrane-embedded IIC(mtl) dimer showing 2-fold symmetry is available. The results of this work are in better agreement with a 7 A projection map from a single 2D crystal on which no symmetry was imposed.


Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Proteínas de Transporte de Monossacarídeos/química , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/química , Triptofano/análogos & derivados , Triptofano/química , Sequência de Aminoácidos , Citoplasma/química , Citoplasma/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Manitol/química , Manitol/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Proteínas de Transporte de Monossacarídeos/genética , Proteínas de Transporte de Monossacarídeos/metabolismo , Mutação , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/genética , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/metabolismo , Conformação Proteica , Espectrometria de Fluorescência , Relação Estrutura-Atividade , Triptofano/metabolismo
19.
Biochem J ; 409(1): 193-8, 2008 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-17910535

RESUMO

Incorporation of Trp (tryptophan) analogues into a protein may facilitate its structural analysis by spectroscopic techniques. Development of a biological system for the biosynthetic incorpor-ation of such analogues into proteins is of considerable importance. The Gram-negative Escherichia coli is the only prokaryotic expression host regularly used for the incorporation of Trp analogues into recombinant proteins. Here, we present the use of the versatile Gram-positive expression host Lactococcus lactis for the incorporation of Trp analogues. The availability of a tightly regulated expression system for this organism, the potential to secrete modified proteins into the growth medium and the construction of the trp-synthetase deletion strain PA1002 of L. lactis rendered this organism potentially an efficient tool for the incorporation of Trp analogues into recombinant proteins. The Trp analogues 7-azatryptophan, 5-fluorotryptophan and 5-hydroxytryptophan were incorporated with efficiencies of >97, >97 and 89% respectively. Interestingly, 5-methylTrp (5-methyltryptophan) could be incorporated with 92% efficiency. Successful biosynthetical incorporation of 5-methylTrp into recombinant proteins has not been reported previously.


Assuntos
Bioquímica/métodos , Lactococcus lactis/metabolismo , Proteínas Recombinantes/química , Triptofano/análogos & derivados , Triptofano/química , Proteínas de Bactérias/química , Clonagem Molecular , Escherichia coli/metabolismo , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Glicina/análogos & derivados , Glicina/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Glifosato
20.
Biophys J ; 95(7): 3419-28, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18599622

RESUMO

The dI component of Rhodospirillum rubrum transhydrogenase has a single Trp residue (Trp(72)), which has distinctive optical properties, including short-wavelength fluorescence emission with clear vibrational fine structure, and long-lived, well-resolved phosphorescence emission. We have made a set of mutant dI proteins in which residues contacting Trp(72) are conservatively substituted. The room-temperature fluorescence-emission spectra of our three Met(97) mutants are blue shifted by approximately 4 nm, giving them a shorter-wavelength emission than any other protein described in the literature, including azurin from Pseudomonas aeruginosa. Fluorescence spectra in low-temperature glasses show equivalent well-resolved vibrational bands in wild-type and the mutant dI proteins, and in azurin. Substitution of Met(97) in dI changes the relative intensities of some of these vibrational bands. The analysis supports the view that fluorescence from the Met(97) mutants arises predominantly from the (1)L(b) excited singlet state of Trp(72), whereas (1)L(a) is the predominant emitting state in wild-type dI. It is suggested that the sulfur atom of Met(97) promotes greater stabilization of (1)L(a) than either (1)L(b) or the ground state. The phosphorescence spectra of Met(97) mutants are also blue-shifted, indicating that the sulfur atom decreases the transition energy between the (3)L(a) state of the Trp and the ground state.


Assuntos
Fluorescência , Mutação , NADP Trans-Hidrogenases/química , NADP Trans-Hidrogenases/genética , Rhodospirillum rubrum/enzimologia , Triptofano , Substituição de Aminoácidos , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , NADP Trans-Hidrogenases/metabolismo , Espectrometria de Fluorescência , Fatores de Tempo
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