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1.
Org Lett ; 21(21): 8635-8640, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31603691

RESUMO

Natural products are the greatest source of antimicrobial agents, although their structural complexity often renders synthetic production and diversification of key classes impractical. One pertinent example is the glycopeptide antibiotics (GPAs), which are highly challenging to synthesize due to their heavily cross-linked structures. Here, we report an optimized method that generates >75% tricyclic peptides from synthetic precursors in order to explore the acceptance of novel GPA precursor peptides by these key existent biosynthetic enzymes.

2.
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
3.
Chem Sci ; 10(1): 118-133, 2019 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-30713624

RESUMO

Non-ribosomal peptide synthesis is a highly important biosynthetic pathway for the formation of many secondary metabolites of medical relevance. Due to the challenges associated with the chemical synthesis of many of the products of these assembly lines, understanding the activity and selectivity of non-ribosomal peptide synthetase (NRPS) machineries is an essential step towards the redesign of such machineries to produce new bioactive peptides. Whilst the selectivity of the adenylation domains responsible for amino acid activation during NRPS synthesis has been widely studied, the selectivity of the essential peptide bond forming domains - known as condensation domains - is not well understood. Here, we present the results of a combination of in vitro and in vivo investigations into the final condensation domain from the NRPS machinery that produces the glycopeptide antibiotics (GPAs). Our results show that this condensation domain is tolerant for a range of peptide substrates and even those with unnatural stereochemistry of the peptide C-terminus, which is in contrast to the widely ascribed role of these domains as a stereochemical gatekeeper during NRPS synthesis. Furthermore, we show that this condensation domain has a significant preference for linear peptide substrates over crosslinked peptides, which indicates that the GPA crosslinking cascade targets the heptapeptide bound to the final module of the NRPS machinery and reinforces the role of the unique GPA X-domain in this process. Finally, we demonstrate that the peptide bond forming activity of this condensation domain is coupled to the rate of amino acid activation performed by the subsequent adenylation domain. This is a significant result with implications for NRPS redesign, as it indicates that the rate of amino acid activation of modified adenylation domains must be maintained to prevent unwanted peptide hydrolysis from the NRPS due to a loss of the productive coupling of amino acid selection and peptide bond formation. Taken together, our results indicate that assessing condensation domain activity is a vital step in not only understanding the biosynthetic logic and timing of NRPS-mediated peptide assembly, but also the rules which redesign efforts must obey in order to successfully produce functional, modified NRPS assembly lines.

4.
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
5.
Chem Commun (Camb) ; 54(17): 2146-2149, 2018 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-29423498

RESUMO

Non-ribosomal peptides contain an array of amino acid building blocks that can present challenges for the synthesis of important intermediates. Here, we report the synthesis of glycopeptide antibiotic (GPA) thioester peptides that retains the crucial stereochemical purity of the terminal phenylglycine residue, which we show is essential for the enzymatic GPA cyclisation cascade.


Assuntos
Antibacterianos/síntese química , Glicina/análogos & derivados , Glicopeptídeos/síntese química , Antibacterianos/química , Antibacterianos/metabolismo , Vias Biossintéticas , Técnicas de Química Sintética/métodos , Ciclização , Esterificação , Glicina/síntese química , Glicina/metabolismo , Glicopeptídeos/química , Glicopeptídeos/metabolismo , Estereoisomerismo , Compostos de Sulfidrila/síntese química , Compostos de Sulfidrila/química , Compostos de Sulfidrila/metabolismo
6.
Chem Sci ; 8(9): 5992-6004, 2017 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-28989629

RESUMO

Halogenation plays a significant role in the activity of the glycopeptide antibiotics (GPAs), although up until now the timing and therefore exact substrate involved was unclear. Here, we present results combined from in vivo and in vitro studies that reveal the substrates for the halogenase enzymes from GPA biosynthesis as amino acid residues bound to peptidyl carrier protein (PCP)-domains from the non-ribosomal peptide synthetase machinery: no activity was detected upon either free amino acids or PCP-bound peptides. Furthermore, we show that the selectivity of GPA halogenase enzymes depends upon both the structure of the bound amino acid and the PCP domain, rather than being driven solely via the PCP domain. These studies provide the first detailed understanding of how halogenation is performed during GPA biosynthesis and highlight the importance and versatility of trans-acting enzymes that operate during peptide assembly by non-ribosomal peptide synthetases.

7.
Mol Biosyst ; 13(1): 9-22, 2016 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-27853778

RESUMO

The biosynthesis of complex natural products by non-ribosomal peptide synthetases (NRPSs) and the related polyketide synthases (PKSs) represents a major source of important bioactive compounds. These large, multi-domain machineries are able to produce a fascinating range of molecules due to the nature of their modular architectures, which allows natural products to be assembled and tailored in a modular, step-wise fashion. In recent years there has been significant progress in characterising the important domains and underlying mechanisms of non-ribosomal peptide synthesis. More significantly, several studies have uncovered important examples of novel activity in many NRPS domains. These discoveries not only greatly increase the structural diversity of the possible products of NRPS machineries but - possibly more importantly - they improve our understanding of what is a highly important, yet complex, biosynthetic apparatus. In this review, several recent examples of novel NRPS function will be introduced, which highlight the range of previously uncharacterised activities that have now been detected in the biosynthesis of important natural products by these mega-enzyme synthetases.


Assuntos
Peptídeo Sintases/metabolismo , Biossíntese de Proteínas , Antibacterianos/biossíntese , Antibacterianos/química , Biossíntese Peptídica , Peptídeos/química , Peptídeos/metabolismo , Policetídeo Sintases/metabolismo , Engenharia de Proteínas , Domínios e Motivos de Interação entre Proteínas
8.
Chembiochem ; 17(7): 576-84, 2016 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-26751610

RESUMO

Nonribosomal peptide synthetases (NRPSs) produce many important and structurally complex natural products. Because of their architectures, reprogramming NRPSs has long been attempted to access new bioactive compounds. However, detailed characterization of NRPS catalysis and substrate selectivity by adenylation (A) domains is needed to support such efforts. We present a simple coupled NADH/pyrophosphate (PPi ) detection assay for analyzing A domain catalysis in vitro. PPi formation is coupled to the consumption of NADH by four enzymatic steps and is detected spectroscopically (λ=340 nm) for simple analysis. We demonstrate the effectiveness of this assay with several adenylation domains, including a stand-alone A domain (DltA, cell wall biosynthesis) and an embedded A domain (Tcp10, teicoplanin biosynthesis). Substrate acceptance of the Tcp10 A domain was explored for the first time, thus demonstrating the applicability of the assay for complex, multi-domain NRPSs.


Assuntos
Domínio Catalítico , Ensaios Enzimáticos/métodos , Peptídeo Sintases/química , Peptídeo Sintases/metabolismo , Monofosfato de Adenosina/análise , Monofosfato de Adenosina/química , Difosfatos/química , Cinética , Proteínas/química , Teicoplanina/química
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