Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 304
Filtrar
Mais filtros










Base de dados
Tipo de estudo
Intervalo de ano de publicação
1.
Nat Commun ; 10(1): 2613, 2019 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-31197182

RESUMO

Kistamicin is a divergent member of the glycopeptide antibiotics, a structurally complex class of important, clinically relevant antibiotics often used as the last resort against resistant bacteria. The extensively crosslinked structure of these antibiotics that is essential for their activity makes their chemical synthesis highly challenging and limits their production to bacterial fermentation. Kistamicin contains three crosslinks, including an unusual 15-membered A-O-B ring, despite the presence of only two Cytochrome P450 Oxy enzymes thought to catalyse formation of such crosslinks within the biosynthetic gene cluster. In this study, we characterise the kistamicin cyclisation pathway, showing that the two Oxy enzymes are responsible for these crosslinks within kistamicin and that they function through interactions with the X-domain, unique to glycopeptide antibiotic biosynthesis. We also show that the kistamicin OxyC enzyme is a promiscuous biocatalyst, able to install multiple crosslinks into peptides containing phenolic amino acids.


Assuntos
Actinobacteria/metabolismo , Antibacterianos/metabolismo , Vias Biossintéticas/genética , Glicopeptídeos/biossíntese , Peptídeos/metabolismo , Actinobacteria/genética , Antibacterianos/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biocatálise , Ciclização/genética , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Glicopeptídeos/química , Família Multigênica , Peptídeos/química
2.
Nat Commun ; 10(1): 1115, 2019 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-30846700

RESUMO

The genome of the thermophilic bacterium, Aeribacillus pallidus 8, encodes the bacteriocin pallidocin. It belongs to the small class of glycocins and is posttranslationally modified, containing an S-linked glucose on a specific Cys residue. In this study, the pallidocin biosynthetic machinery is cloned and expressed in Escherichia coli to achieve its full biosynthesis and modification. It targets other thermophilic bacteria with potent activity, demonstrated by a low minimum inhibitory concentration (MIC) value. Moreover, the characterized biosynthetic machinery is employed to produce two other glycopeptides Hyp1 and Hyp2. Pallidocin and Hyp1 exhibit antibacterial activity against closely related thermophilic bacteria and some Bacillus sp. strains. Thus, heterologous expression of a glycocin biosynthetic gene cluster including an S-glycosyltransferase provides a good tool for production of hypothetical glycocins encoded by various bacterial genomes and allows rapid in vivo screening.


Assuntos
Bacillaceae/metabolismo , Bacteriocinas/biossíntese , Sequência de Aminoácidos , Antibacterianos/biossíntese , Antibacterianos/química , Antibacterianos/farmacologia , Bacillaceae/genética , Bacteriocinas/genética , Bacteriocinas/farmacologia , Clonagem Molecular , Dissulfetos/química , Escherichia coli/genética , Escherichia coli/metabolismo , Genes Bacterianos , Glicopeptídeos/biossíntese , Glicopeptídeos/genética , Glicopeptídeos/farmacologia , Testes de Sensibilidade Microbiana , Família Multigênica , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos
3.
Redox Biol ; 20: 130-145, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30308476

RESUMO

Low molecular weight (LMW) thiols play an important role as thiol-cofactors for many enzymes and are crucial to maintain the reduced state of the cytoplasm. Most Gram-negative bacteria utilize glutathione (GSH) as major LMW thiol. However, in Gram-positive Actinomycetes and Firmicutes alternative LMW thiols, such as mycothiol (MSH) and bacillithiol (BSH) play related roles as GSH surrogates, respectively. Under conditions of hypochlorite stress, MSH and BSH are known to form mixed disulfides with protein thiols, termed as S-mycothiolation or S-bacillithiolation that function in thiol-protection and redox regulation. Protein S-thiolations are widespread redox-modifications discovered in different Gram-positive bacteria, such as Bacillus and Staphylococcus species, Mycobacterium smegmatis, Corynebacterium glutamicum and Corynebacterium diphtheriae. S-thiolated proteins are mainly involved in cellular metabolism, protein translation, redox regulation and antioxidant functions with some conserved targets across bacteria. The reduction of protein S-mycothiolations and S-bacillithiolations requires glutaredoxin-related mycoredoxin and bacilliredoxin pathways to regenerate protein functions. In this review, we present an overview of the functions of mycothiol and bacillithiol and their physiological roles in protein S-bacillithiolations and S-mycothiolations in Gram-positive bacteria. Significant progress has been made to characterize the role of protein S-thiolation in redox-regulation and thiol protection of main metabolic and antioxidant enzymes. However, the physiological roles of the pathways for regeneration are only beginning to emerge as well as their interactions with other cellular redox systems. Future studies should be also directed to explore the roles of protein S-thiolations and their redox pathways in pathogenic bacteria under infection conditions to discover new drug targets and treatment options against multiple antibiotic resistant bacteria.


Assuntos
Bactérias Gram-Positivas/genética , Bactérias Gram-Positivas/metabolismo , Oxirredução , Processamento de Proteína Pós-Traducional , Animais , Cisteína/análogos & derivados , Cisteína/biossíntese , Cisteína/química , Cisteína/farmacologia , Glucosamina/análogos & derivados , Glucosamina/biossíntese , Glucosamina/química , Glucosamina/farmacologia , Glicopeptídeos/biossíntese , Glicopeptídeos/química , Glicopeptídeos/farmacologia , Bactérias Gram-Positivas/efeitos dos fármacos , Humanos , Inositol/biossíntese , Inositol/química , Inositol/farmacologia , Modelos Biológicos , Oxirredução/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Relação Estrutura-Atividade , Compostos de Sulfidrila/química , Compostos de Sulfidrila/metabolismo
4.
J Biomed Sci ; 25(1): 55, 2018 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-30001196

RESUMO

BACKGROUND: Three low molecular weight thiols are synthesized by Mycobacterium tuberculosis (M.tb), namely ergothioneine (ERG), mycothiol (MSH) and gamma-glutamylcysteine (GGC). They are able to counteract reactive oxygen species (ROS) and/or reactive nitrogen species (RNS). In addition, the production of ERG is elevated in the MSH-deficient M.tb mutant, while the production of MSH is elevated in the ERG-deficient mutants. Furthermore, the production of GGC is elevated in the MSH-deficient mutant and the ERG-deficient mutants. The propensity of one thiol to be elevated in the absence of the other prompted further investigations into their interplay in M.tb. METHODS: To achieve that, we generated two M.tb mutants that are unable to produce ERG nor MSH but are able to produce a moderate (ΔegtD-mshA) or significantly high (ΔegtB-mshA) amount of GGC relative to the wild-type strain. In addition, we generated an M.tb mutant that is unable to produce GGC nor MSH but is able to produce a significantly low level of ERG (ΔegtA-mshA) relative to the wild-type strain. The susceptibilities of these mutants to various in vitro and ex vivo stress conditions were investigated and compared. RESULTS: The ΔegtA-mshA mutant was the most susceptible to cellular stress relative to its parent single mutant strains (ΔegtA and ∆mshA) and the other double mutants. In addition, it displayed a growth-defect in vitro, in mouse and human macrophages suggesting; that the complete inhibition of ERG, MSH and GGC biosynthesis is deleterious for the growth of M.tb. CONCLUSIONS: This study indicates that ERG, MSH and GGC are able to compensate for each other to maximize the protection and ensure the fitness of M.tb. This study therefore suggests that the most effective strategy to target thiol biosynthesis for anti-tuberculosis drug development would be the simultaneous inhibition of the biosynthesis of ERG, MSH and GGC.


Assuntos
Cisteína/biossíntese , Dipeptídeos/biossíntese , Ergotioneína/biossíntese , Glicopeptídeos/biossíntese , Inositol/biossíntese , Tuberculose/microbiologia , Animais , Cisteína/antagonistas & inibidores , Cisteína/genética , Dipeptídeos/antagonistas & inibidores , Dipeptídeos/genética , Ergotioneína/antagonistas & inibidores , Ergotioneína/genética , Glicopeptídeos/antagonistas & inibidores , Glicopeptídeos/genética , Humanos , Inositol/antagonistas & inibidores , Inositol/genética , Camundongos , Peso Molecular , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/patogenicidade , Estresse Oxidativo , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Compostos de Sulfidrila/química , Compostos de Sulfidrila/metabolismo , Tuberculose/tratamento farmacológico , Tuberculose/genética , Tuberculose/patologia
5.
Appl Microbiol Biotechnol ; 102(16): 6791-6798, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29876605

RESUMO

The bleomycins (BLMs) belong to a subfamily of glycopeptide antibiotics and are clinically applied in combination chemotherapy regimens to treat various malignancies. But the therapeutic applications of BLMs are restricted by the accompanied dose-dependent lung toxicity and potential incidence of lung fibrosis. Many efforts have been devoted to develop novel BLM analogues, for seeking of drug leads with improved antitumor activity and/or reduced lung toxicity. The progresses in the biosynthetic studies of BLMs have greatly expedited the process to achieve such goals. This review highlights the discovery and development of microbial BLM analogues in the past two decades, especially those derived from engineered biosynthesis. Moreover, the summarized structure-activity relationship, which is specifically focusing on the sugar moiety, shall shed new insights into the prospective development of BLM analogues.


Assuntos
Bleomicina/análogos & derivados , Bleomicina/toxicidade , Fermentação , Glicopeptídeos/biossíntese , Humanos , Neoplasias/tratamento farmacológico , Estudos Prospectivos , Engenharia de Proteínas , Relação Estrutura-Atividade
6.
Sci Rep ; 8(1): 8730, 2018 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-29880804

RESUMO

Many therapeutic enzymes for lysosomal storage diseases require a high content of mannose-6-phosphate (M6P) glycan, which is important for cellular uptake and lysosomal targeting. We constructed glyco-engineered yeast harboring a high content of mannosylphosphorylated glycans, which can be converted to M6P glycans by uncapping of the outer mannose residue. In this study, the cell wall of this yeast was employed as a natural M6P glycan source for conjugation to therapeutic enzymes. The extracted cell wall mannoproteins were digested by pronase to generate short glycopeptides, which were further elaborated by uncapping and α(1,2)-mannosidase digestion steps. The resulting glycopeptides containing M6P glycans (M6PgPs) showed proper cellular uptake and lysosome targeting. The purified M6PgPs were successfully conjugated to a recombinant acid α-glucosidase (rGAA), used for the treatment of Pompe disease, by two-step reactions using two hetero-bifunctional crosslinkers. First, rGAA and M6PgPs were modified with crosslinkers containing azide and dibenzocyclooctyne, respectively. In the second reaction using copper-free click chemistry, the azide-functionalized rGAA was conjugated with dibenzocyclooctyne-functionalized M6PgPs without the loss of enzyme activity. The M6PgP-conjugated rGAA had a 16-fold higher content of M6P glycan than rGAA, which resulted in greatly increased cellular uptake and efficient digestion of glycogen accumulated in Pompe disease patient fibroblasts.


Assuntos
Glicopeptídeos/biossíntese , Lisossomos/metabolismo , Manosefosfatos/metabolismo , Engenharia Metabólica , Microrganismos Geneticamente Modificados/metabolismo , Saccharomyces cerevisiae/metabolismo , Glicopeptídeos/genética , Glicosilação , Humanos , Lisossomos/genética , Manosefosfatos/genética , Microrganismos Geneticamente Modificados/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/genética , alfa-Glucosidases/biossíntese , alfa-Glucosidases/genética
7.
J Inorg Biochem ; 185: 43-51, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29751197

RESUMO

Cytochrome P450 enzymes perform an impressive range of oxidation reactions against diverse substrate scaffolds whilst generally maintaining a conserved tertiary structure and active site chemistry. Within secondary metabolism, P450 enzymes play widespread and important roles in performing crucial modifications of precursor molecules, with one example of the importance of such reactions being found in the biosynthesis of the glycopeptide antibiotics (GPAs). In GPA biosynthesis P450s, known as Oxy enzymes, are key players in the cyclization of the linear GPA peptide precursor, which is a process that is both essential for their antibiotic activity and is the source of the synthetic challenge of these important antibiotics. In this work, we developed chimeric P450 enzymes from GPA biosynthesis based on two homologues from different GPA biosynthesis pathways - vancomycin and teicoplanin - as an approach to explore the divergent catalytic behavior of the two parental homologues. We could generate, crystalize and explore the activity of new hybrid P450 enzymes from GPA biosynthesis and show that the unusual in vitro behavior of the vancomycin OxyB homologue does not stem from the major regions of the P450 active site, and that additional regions in and around the P450 active site must contribute to the unusual properties of this P450 enzyme. Our results further show that it is possible to successfully transplant entire regions of secondary structure between such P450s and retain P450 expression and activity, which opens the door to use such targeted approaches to generate and explore novel biosynthetic P450 enzymes.


Assuntos
Antibacterianos/biossíntese , Sistema Enzimático do Citocromo P-450/metabolismo , Glicopeptídeos/biossíntese , Sequência de Aminoácidos , Antibacterianos/química , Antibacterianos/metabolismo , Catálise , Cristalização , Sistema Enzimático do Citocromo P-450/química , Glicopeptídeos/química , Glicopeptídeos/metabolismo , Conformação Proteica , Homologia de Sequência de Aminoácidos , Especificidade por Substrato
8.
J Org Chem ; 83(13): 7206-7214, 2018 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-29708747

RESUMO

Natural products such as the glycopeptide antibiotics (GPAs, including vancomycin and teicoplanin) are of great pharmaceutical importance due to their use against Gram-positive bacteria such as methicillin-resistant Staphylococcus aureus. GPAs are assembled in a complex process based on nonribosomal peptide synthesis and late-stage, multistep cross-linking of the linear heptapeptide performed by cytochrome P450 monooxygenases. These P450 enzymes demonstrate varying degrees of substrate selectivity toward the linear peptide precursor, with limited information available about their tolerance regarding modifications to amino acid residues within the essential antibiotic core of the GPA. In order to test the acceptance of altered residues by the P450-catalyzed cyclization cascade, we have explored the use of ß-amino acids in both variable and highly conserved positions within GPA peptides. Our results indicate that the incorporation of ß-amino acids at the C-terminus of the peptide leads to a dramatic reduction in the efficiency of peptide cyclization by the P450s during GPA biosynthesis, whereas replacement of residue 3 is well tolerated by the same enzymes. These results show that maintaining the C-terminal 3,5-dihydroxyphenylglycine residue is of key importance to maintain the efficiency of this complex and essential enzymatic cross-linking process.


Assuntos
Aminoácidos/química , Antibacterianos/biossíntese , Glicopeptídeos/biossíntese , Ciclização , Oxirredução
9.
Chem Soc Rev ; 46(16): 5128-5146, 2017 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-28681051

RESUMO

The endo-ß-N-acetylglucosaminidases (ENGases) are an enzyme class (EC 3.2.1.96) produced by a range of organisms, ranging from bacteria, through fungi, to higher order species, including humans, comprising two-sub families of glycosidases which all cleave the chitobiose core of N-linked glycans. Synthetic applications of these enzymes, i.e. to catalyse the reverse of their natural hydrolytic mode of action, allow the attachment of N-glycans to a wide variety of substrates which contain an N-acetylglucosamine (GlcNAc) residue to act as an 'acceptor' handle. The use of N-glycan oxazolines, high energy intermediates on the hydrolytic pathway, as activated donors allows their high yielding attachment to almost any amino acid, peptide or protein that contains a GlcNAc residue as an acceptor. The synthetic effectiveness of these biocatalysts has been significantly increased by the production of mutant glycosynthases; enzymes which can still catalyse synthetic processes using oxazolines as donors, but which do not hydrolyse the reaction products. ENGase biocatalysts are now finding burgeoning application for the production of biologically active glycopeptides and glycoproteins, including therapeutic monoclonal antibodies (mAbs) for which the oligosaccharides have been remodelled to optimise effector functions.


Assuntos
Glicopeptídeos/biossíntese , Glicoproteínas/biossíntese , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase/metabolismo , Biocatálise , Configuração de Carboidratos , Glicopeptídeos/química , Glicoproteínas/química , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase/química , Modelos Moleculares
10.
Anal Chem ; 89(13): 6992-6999, 2017 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-28590120

RESUMO

The peptide hormone calcitonin is intimately connected with human cancer development and proliferation. Its biosynthesis is reasoned to proceed via glycine-, α-hydroxyglycine-, glycyllysine-, and glycyllysyllysine-extended precursors; however, as a result of the limitations of current analytical methods, until now, there has been no procedure capable of detecting these individual species in cell or tissue samples. Therefore, their presence and dynamics in cancer had not been established. Here, we report the first methodology for the separation, detection, and quantification of calcitonin and each of its precursors in human cancer cells. We also report the discovery and characterization of O-glycosylated calcitonin and its analogous biosynthetic precursors. Through direct and simultaneous analysis of the glycosylated and nonglycosylated species, we interrogate the hormone biosynthesis. This shows that the cellular calcitonin level is maintained to mitigate effects of biosynthetic enzyme inhibitors that substantially change the proportions of calcitonin-related species released into the culture medium.


Assuntos
Calcitonina/análogos & derivados , Calcitonina/análise , Cromatografia Líquida de Alta Pressão/métodos , Glicopeptídeos/análise , Precursores de Proteínas/análise , Amidina-Liases/antagonistas & inibidores , Calcitonina/biossíntese , Calcitonina/metabolismo , Carboxipeptidase H/antagonistas & inibidores , Linhagem Celular Tumoral , Ácidos Graxos Monoinsaturados/farmacologia , Glicopeptídeos/biossíntese , Glicopeptídeos/química , Glicopeptídeos/metabolismo , Glicosilação , Humanos , Oxigenases de Função Mista/antagonistas & inibidores , Precursores de Proteínas/biossíntese , Precursores de Proteínas/química , Precursores de Proteínas/metabolismo , Extração em Fase Sólida/métodos , Succinatos/farmacologia
11.
ACS Chem Biol ; 12(7): 1796-1804, 2017 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-28489343

RESUMO

Cyanobacteria produce a wide range of natural products with antifungal bioactivity. The cyclic glycosylated lipopeptides of the hassallidin family have potent antifungal activity and display a great degree of chemical diversity. Here, we report the discovery of a hassallidin biosynthetic gene cluster from the filamentous cyanobacterium Planktothrix serta PCC 8927. The hassallidin gene cluster showed heavy rearrangement and marks of genomic plasticity. Nucleotide bias, differences in GC content, and phylogenetic incongruence suggested the acquisition of the hassallidin biosynthetic gene cluster in Planktothrix serta PCC 8927 by horizontal gene transfer. Chemical analyses by liquid chromatography and mass spectrometry demonstrated that this strain produced hassallidin E, a new glycosylated hassallidin variant. Hassallidin E was the only structural variant produced by Planktothrix serta PCC 8927 in all tested conditions. Further evaluated on human pathogenic fungi, hassallidin E showed an antifungal bioactivity. Hassallidin production levels correlated with nitrogen availability, in the only nitrogen-fixing Planktothrix described so far. Our results provide insights into the distribution and chemical diversity of cyanobacterial antifungal compounds as well as raise questions on their ecological relevance.


Assuntos
Cianobactérias/genética , Glicopeptídeos/biossíntese , Glicopeptídeos/genética , Família Multigênica , Peptídeos Cíclicos/química , Antibacterianos/biossíntese , Antibacterianos/farmacologia , Cianobactérias/metabolismo , Fungos/efeitos dos fármacos , Transferência Genética Horizontal , Glicopeptídeos/química , Glicopeptídeos/isolamento & purificação , Glicopeptídeos/farmacologia , Peptídeos Cíclicos/biossíntese
12.
Org Biomol Chem ; 15(17): 3775-3782, 2017 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-28406517

RESUMO

Mycothiol (MSH) is the predominant low molecular weight thiol produced by actinomycetes, and it plays a pivotal role in the bacterial detoxication process. 1L-myo-Inositol-1-phosphate (1L-Ins-1-P) α-N-acetylglucosaminyltransferase (GlcNAc-T), known as MshA, is the only glycosyltransferase involved in MSH biosynthesis. In this work, the MshA from Corynebacterium diphtheria, named as CdMshA, was expressed, purified, and studied in detail. Its enzymatic activity to transfer GlcNAc to 1L-Ins-1-P was confirmed by the isolation and rigorous characterization of its reaction product 3-phospho-1-d-myo-inositol-2-acetamido-2-deoxy-α-d-glucopyranoside. CdMshA was shown to accept only UDP-GlcNAc and 1L-Ins-1-P as its substrates among various tested glycosyl donors, such as UDP-GlcNAc, UDP-Gal, UDP-Glc, UDP-GalNAc and UDP-GlcA, and glycosyl acceptors, such as myo-inositol, 1L-Ins-1-P and 1D-Ins-1-P. The results have demonstrated the strict substrate selectivity of CdMshA. Furthermore, its reaction kinetics with UDP-GlcNAc and 1L-Ins-1-P as substrates were characterized, while site-directed mutagenesis of CdMshA disclosed that its amino acid residues N28, K81 and R157 were essential for its enzymatic activity.


Assuntos
Corynebacterium diphtheriae/metabolismo , Cisteína/biossíntese , Glicopeptídeos/biossíntese , Inositol/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Domínio Catalítico , Inositol/biossíntese , Cinética , Modelos Moleculares , Mutagênese , N-Acetilglucosaminiltransferases/química , N-Acetilglucosaminiltransferases/genética , Relação Estrutura-Atividade , Especificidade por Substrato
13.
Microbiology ; 163(3): 343-354, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28073401

RESUMO

Rhodococcus jostii RHA1 is able to degrade toxic compounds and accumulate high amounts of triacylglycerols (TAG) upon nitrogen starvation. These NADPH-dependent processes are essential for the adaptation of rhodococci to fluctuating environmental conditions. In this study, we used an MS-based, label-free and quantitative proteomic approach to better understand the integral response of R. jostii RHA1 to the presence of methyl viologen (MV) in relation to the synthesis and accumulation of TAG. The addition of MV promoted a decrease of TAG accumulation in comparison to cells cultivated under nitrogen-limiting conditions in the absence of this pro-oxidant. Proteomic analyses revealed that the abundance of key proteins of fatty acid biosynthesis, the Kennedy pathway, glyceroneogenesis and methylmalonyl-CoA pathway, among others, decreased in the presence of MV. In contrast, some proteins involved in lipolysis and ß-oxidation of fatty acids were upregulated. Some metabolic pathways linked to the synthesis of NADPH remained activated during oxidative stress as well as under nitrogen starvation conditions. Additionally, exposure to MV resulted in the activation of complete antioxidant machinery comprising superoxide dismutases, catalases, mycothiol biosynthesis, mycothione reductase and alkyl hydroperoxide reductases, among others. Our study suggests that oxidative stress response affects TAG accumulation under nitrogen-limiting conditions through programmed molecular mechanisms when both stresses occur simultaneously.


Assuntos
Nitrogênio/deficiência , Estresse Oxidativo/fisiologia , Paraquat/metabolismo , Rhodococcus/metabolismo , Triglicerídeos/biossíntese , Acil Coenzima A/metabolismo , Adaptação Fisiológica , Catalase/metabolismo , Cisteína/biossíntese , Ácidos Graxos/biossíntese , Glicopeptídeos/biossíntese , Inositol/biossíntese , NADP/metabolismo , Nitrogênio/metabolismo , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Oxirredutases/biossíntese , Peroxirredoxinas/biossíntese , Proteoma , Rhodococcus/crescimento & desenvolvimento , Superóxido Dismutase/metabolismo
14.
J Pept Sci ; 23(1): 16-27, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27910178

RESUMO

Understanding the structure and function of protein complexes and multi-domain proteins is highly important in biology, although the in vitro characterization of these systems is often complicated by their size or the transient nature of protein/protein interactions. To assist in the characterization of such protein complexes, we have developed a modular approach to fusion protein generation that relies upon Sortase-mediated and Native chemical ligation using synthetic Peptide linkers (SNaPe) to link two separately expressed proteins. In this approach, we utilize two separate linking steps - sortase-mediated and native chemical ligation - together with a library of peptide linkers to generate libraries of fusion proteins. We have demonstrated the viability of SNaPe to generate libraries from fusion protein constructs taken from the biosynthetic enzymes responsible for late stage aglycone assembly during glycopeptide antibiotic biosynthesis. Crucially, SNaPe was able to generate fusion proteins that are inaccessible via direct expression of the fusion construct itself. This highlights the advantages of SNaPe to not only access fusion proteins that have been previously unavailable for biochemical and structural characterization but also to do so in a manner that enables the linker itself to be controlled as an experimental parameter of fusion protein generation. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.


Assuntos
Glicopeptídeos/biossíntese , Peptídeos Cíclicos/biossíntese , Peptídeos/química , Proteínas Recombinantes de Fusão/biossíntese , Sequência de Aminoácidos , Aminoaciltransferases/genética , Aminoaciltransferases/metabolismo , Antibacterianos/química , Técnicas de Química Sintética , Clorofenóis/química , Reagentes para Ligações Cruzadas/química , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Biblioteca Gênica , Glicopeptídeos/química , Glicopeptídeos/genética , Peptídeo Sintases/genética , Peptídeo Sintases/metabolismo , Peptídeos/síntese química , Peptídeos Cíclicos/química , Peptídeos Cíclicos/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética
15.
J Phys Chem B ; 121(3): 471-478, 2017 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-27935720

RESUMO

Understanding enzymatic reactions with atomic resolution has proven in recent years to be of tremendous interest for biochemical research, and thus, the use of QM/MM methods for the study of reaction mechanisms is experiencing a continuous growth. Glycosyltransferases (GTs) catalyze the formation of glycosidic bonds, and are important for many biotechnological purposes, including drug targeting. Their reaction product may result with only one of the two possible stereochemical outcomes for the reacting anomeric center, and therefore, they are classified as either inverting or retaining GTs. While the inverting GT reaction mechanism has been widely studied, the retaining GT mechanism has always been controversial and several questions remain open to this day. In this work, we take advantage of our recent GPU implementation of a pure QM(DFT-PBE)/MM approach to explore the reaction and inhibition mechanism of MshA, a key retaining GT responsible for the first step of mycothiol biosynthesis, a low weight thiol compound found in pathogens like Mycobacterium tuberculosis that is essential for its survival under oxidative stress conditions. Our results show that the reaction proceeds via a front-side SNi-like concerted reaction mechanism (DNAN in IUPAC nomenclature) and has a 17.5 kcal/mol free energy barrier, which is in remarkable agreement with experimental data. Detailed analysis shows that the key reaction step is the diphosphate leaving group dissociation, leading to an oxocarbenium-ion-like transition state. In contrast, fluorinated substrate analogues increase the reaction barrier significantly, rendering the enzyme effectively inactive. Detailed analysis of the electronic structure along the reaction suggests that this particular inhibition mechanism is associated with fluorine's high electronegative nature, which hinders phosphate release and proper stabilization of the transition state.


Assuntos
Amidoidrolases/antagonistas & inibidores , Amidoidrolases/metabolismo , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Cisteína/biossíntese , Glicopeptídeos/biossíntese , Glicosiltransferases/metabolismo , Inositol/biossíntese , Metais/metabolismo , Teoria Quântica , Biocatálise , Cisteína/química , Glicopeptídeos/química , Inositol/química , Mycobacterium tuberculosis/metabolismo
16.
ACS Infect Dis ; 2(9): 642-650, 2016 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-27759388

RESUMO

Modification of natural product backbones is a proven strategy for the development of clinically useful antibiotics. Such modifications have traditionally been achieved through medicinal chemistry strategies or via in vitro enzymatic activities. In an orthogonal approach, engineering of biosynthetic pathways using synthetic biology techniques can generate chemical diversity. Here we report the use of a minimal teicoplanin class glycopeptide antibiotic (GPA) scaffold expressed in a production-optimized Streptomyces coelicolor strain to expand GPA chemical diversity. Thirteen scaffold-modifying enzymes from 7 GPA biosynthetic gene clusters in different combinations were introduced into S. coelicolor, enabling us to explore the criteria for in-cell GPA modification. These include identifying specific isozymes that tolerate the unnatural GPA scaffold and modifications that prevent or allow further elaboration by other enzymes. Overall, 15 molecules were detected, 9 of which have not been reported previously. Some of these compounds showed activity against GPA-resistant bacteria. This system allows us to observe the complex interplay between substrates and both non-native and native tailoring enzymes in a cell-based system and establishes rules for GPA synthetic biology and subsequent expansion of GPA chemical diversity.


Assuntos
Antibacterianos/biossíntese , Glicopeptídeos/biossíntese , Streptomyces coelicolor/metabolismo , Antibacterianos/química , Bactérias/efeitos dos fármacos , Vias Biossintéticas , Glicopeptídeos/química , Família Multigênica , Streptomyces coelicolor/química , Streptomyces coelicolor/genética , Biologia Sintética , Teicoplanina/química , Teicoplanina/metabolismo
17.
Mol Biosyst ; 12(10): 2992-3004, 2016 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-27477788

RESUMO

Glycopeptide antibiotic biosynthesis involves a complex cascade of reactions centred on a non-ribosomal peptide synthetase and modifiying proteins acting in trans, such as Cytochrome P450 enzymes. These P450s are responsible for cyclisation of the peptide via cross-linking aromatic amino acid side chains, which are a hallmark of the glycopeptide antibiotics. Here, we analysed the first cyclisation reaction in the biosynthesis of the glycopeptide antibiotic A47934. Our results demonstrate that the P450 StaH is recruited to the NRPS machinery through interaction with the X-domain present in the last A47934 NRPS module. We determined the crystal structure of StaH and showed that it is responsible for the first cyclisation in A47934 biosynthesis and additionally exhibits flexible substrate specificity. Our results further point out that the X-domain has an impact on the efficiency of the in vitro cyclisation reaction: hybrid PCP-X constructs obtained by domain exchange between A47934 and teicoplanin biosynthesis NRPS modules reveal that the X-domain from A47934 leads to decreased P450 activity and alternate stereochemical preference for the substrate peptide. We determined that a tight interaction between StaH and the A47934 X-domain correlates with decreased in vitro P450 activity: this highlights the need for glycopeptide antibiotic cyclisation to be a dynamic system, with an overly tight interaction interfering with substrate turnover in vitro.


Assuntos
Sistema Enzimático do Citocromo P-450/química , Fenóis/química , Domínios e Motivos de Interação entre Proteínas , Ristocetina/análogos & derivados , Domínio Catalítico , Ciclização , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Ativação Enzimática , Glicopeptídeos/biossíntese , Glicopeptídeos/química , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Ristocetina/biossíntese , Ristocetina/química , Análise Espectral , Especificidade por Substrato
18.
Curr Opin Struct Biol ; 41: 46-53, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27289043

RESUMO

The importance of Cytochrome P450-catalyzed modifications of natural products produced by non-ribosomal peptide synthetase machineries is most apparent during glycopeptide antibiotic biosynthesis: specifically, the formation of essential amino acid side chains crosslinks in the peptide backbone of these clinically relevant antibiotics. These cyclization reactions take place whilst the peptide substrate remains bound to the non-ribosomal peptide synthetase in a process mediated by a conserved domain of previously unknown function-the X-domain. This review addresses recent advances in understanding P450 recruitment to non-ribosomal peptide synthetase-bound substrates and highlights the importance of both carrier proteins and the X-domain in different P450-catalyzed reactions.


Assuntos
Antibacterianos/biossíntese , Sistema Enzimático do Citocromo P-450/metabolismo , Glicopeptídeos/biossíntese , Peptídeo Sintases/metabolismo , Sistema Enzimático do Citocromo P-450/química , Humanos , Ligação Proteica , Domínios Proteicos
19.
Bioelectromagnetics ; 37(5): 331-7, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-27271865

RESUMO

The effect of the extremely low frequency magnetic field (ELF-MF) on biomass and mannatide production by α-hemolytic Streptococcus in liquid-state fermentation culture medium was studied during shake flask culture. Magnetic field (MF) inductions, exposure times, and exposure periods varied in a range of 0-1.5 mT, 0-16 h, and six periods of incubation time, respectively. Results showed both biomass and mannatide production increased significantly at MF induction 0.4, 0.6, and 0.9 mT and decreased at both 1.2 and 1.5 mT. Biomass increased by exposure for initial and middle stages of fermentation. Mannatide production increased significantly at 4-8, 8-12, and 17-21 h. Peak yield of biomass and mannatide production increased by 10.7% and 14.0% at 25 and 27 h of incubation at 0.6 mT MF induction and exposure to 8-12 h of incubation time, compared with the control experiment, respectively. ELF-MF could also enhance the growth rate and mannatide production rate of α-hemolytic Streptococcus. However, ELF-MF did not alter α-hemolytic Streptococcus cell growth and mannatide metabolizing regulation or fermentation pattern. Mannatide production was not associated with cellular growth but rather only partially associated. Bioelectromagnetics. 37:331-337, 2016. © 2016 Wiley Periodicals, Inc.


Assuntos
Glicopeptídeos/biossíntese , Campos Magnéticos , Streptococcus/metabolismo , Biomassa , Fermentação , Cinética , Streptococcus/crescimento & desenvolvimento
20.
J Am Chem Soc ; 138(21): 6746-53, 2016 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-27213615

RESUMO

Glycopeptide antibiotics (GPAs) are nonribosomal peptides rich in modifications introduced by external enzymes. These enzymes act on the free peptide aglycone or intermediates bound to the nonribosomal peptide synthetase (NRPS) assembly line. In this process the terminal module of the NRPS plays a crucial role as it contains a unique recruitment platform (X-domain) interacting with three to four modifying Cytochrome P450 (P450) enzymes that are responsible for cyclizing bound peptides. However, whether these enzymes share the same binding site on the X-domain and how the order of the cyclization steps is orchestrated has remained elusive. In this study we investigate the first two reactions in teicoplanin aglycone maturation catalyzed by the enzymes OxyBtei and OxyAtei. We demonstrate that both enzymes interact with the X-domain via the identical interaction site with similar affinities, irrespective of the peptide modification stage, while their catalytic activity is restricted to the correctly cross-linked peptide. On the basis of steady state kinetics of the OxyBtei-catalyzed reaction, we propose a model for P450 recruitment and peptide modification that involves continuous association/dissociation of the P450 enzymes with the NRPS, followed by specific recognition of the peptide cyclization state by the P450 (scanning). This leads to an induced conformational change that enhances the affinity of the enzyme/substrate complex and initiates catalysis; product release then occurs, with the product itself becoming the substrate for the second enzyme in the pathway. This model rationalizes our experimental findings for this complex enzyme cascade and provides insights into the orchestration of the sequential peptide tailoring reactions on the terminal NRPS module in GPA biosynthesis.


Assuntos
Antibacterianos/biossíntese , Sistema Enzimático do Citocromo P-450/química , Glicopeptídeos/biossíntese , Oxigênio/química , Peptídeo Sintases/química , Sítios de Ligação , Ciclização , Ligação Proteica
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA