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1.
Chem Commun (Camb) ; 54(90): 12718-12721, 2018 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-30357150

RESUMO

Supplemented with synthetic surrogates of their natural cosubstrate S-adenosyl-l-methione (AdoMet), methyltransferases represent a powerful toolbox for the functionalization of biomolecules. By employing novel cosubstrate derivatives in combination with protein engineering, we show that this chemo-enzymatic method can be used to introduce photolabile protecting groups into DNA even in the presence of AdoMet. This approach enables optochemical control of gene expression in a straight-forward manner and we have termed it reversible methyltransferase directed transfer of photoactivatable groups (re-mTAG).


Assuntos
DNA/química , DNA/metabolismo , Metiltransferases/química , Metiltransferases/metabolismo , Metiltransferases/genética , Modelos Moleculares , Estrutura Molecular , Fotólise , Engenharia de Proteínas , Especificidade por Substrato
2.
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
3.
Nat Commun ; 9(1): 1686, 2018 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-29703974

RESUMO

Bacterial toxin-antitoxin complexes are emerging as key players modulating bacterial physiology as activation of toxins induces stasis or programmed cell death by interference with vital cellular processes. Zeta toxins, which are prevalent in many bacterial genomes, were shown to interfere with cell wall formation by perturbing peptidoglycan synthesis in Gram-positive bacteria. Here, we characterize the epsilon/zeta toxin-antitoxin (TA) homologue from the Gram-negative pathogen Neisseria gonorrhoeae termed ng_ɛ1 / ng_ζ1. Contrary to previously studied streptococcal epsilon/zeta TA systems, ng_ɛ1 has an epsilon-unrelated fold and ng_ζ1 displays broader substrate specificity and phosphorylates multiple UDP-activated sugars that are precursors of peptidoglycan and lipopolysaccharide synthesis. Moreover, the phosphorylation site is different from the streptococcal zeta toxins, resulting in a different interference with cell wall synthesis. This difference most likely reflects adaptation to the individual cell wall composition of Gram-negative and Gram-positive organisms but also the distinct involvement of cell wall components in virulence.


Assuntos
Toxinas Bacterianas/metabolismo , Parede Celular/metabolismo , Neisseria gonorrhoeae/fisiologia , Peptidoglicano/biossíntese , Sistemas Toxina-Antitoxina/fisiologia , Adaptação Fisiológica , Neisseria gonorrhoeae/patogenicidade , Fosforilação , Especificidade por Substrato , Virulência/fisiologia
4.
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
5.
ACS Chem Biol ; 13(1): 110-120, 2018 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-29192758

RESUMO

The biosynthesis of the glycopeptide antibiotics (GPAs)-which include teicoplanin and vancomycin-is a complex enzymatic process relying on the interplay of nonribosomal peptide synthesis and a cytochrome P450-mediated cyclization cascade. This unique cyclization cascade generates the highly cross-linked state of these nonribosomal peptides, which is crucial for their antimicrobial activity. Given that these essential oxidative transformations occur while the peptide remains bound to the terminal module of the nonribosomal peptide synthetase (NRPS) machinery, it is important to assess the selectivity of the terminal thioesterase (TE) domain and how this domain contributes to the maintenance of an efficient biosynthetic pathway while at the same time ensuring GPA maturation is completed. In this study, we report the in vitro characterization of the thioesterase domain from teicoplanin biosynthesis, the first GPA thioesterase to be characterized. Our results show that the activity of this TE domain relies on the presence of an unusual extended N-terminal linker region that appears to be unique to the NRPS machineries found in GPA biosynthesis. In addition, we show that the activity of this domain against carrier protein bound substrates is dramatically enhanced for mature GPA aglycones as opposed to linear peptides and partially cyclized intermediates. These results demonstrate how the interplay between NRPS and P450s during late stage GPA biosynthesis is not only maintained but also leads to the efficient production of mature GPA aglycones. Thus, GPA TE domains represent another impressive example of the ability of TE domains to act as logic gates during NRPS biosynthesis, ensuring that essential late-stage peptide modifications are completed before catalyzing the release of the mature, bioactive peptide product.


Assuntos
Peptídeo Sintases/química , Peptídeo Sintases/metabolismo , Teicoplanina/biossíntese , Tioléster Hidrolases/química , Peptídeo Sintases/genética , Peptídeos/química , Peptídeos/metabolismo , Domínios Proteicos , Especificidade por Substrato , Tioléster Hidrolases/metabolismo
6.
Biochemistry ; 56(9): 1239-1247, 2017 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-28218515

RESUMO

The activity of glycopeptide antibiotics (GPAs) depends upon important structural modifications to their precursor heptapeptide backbone: specifically, the cytochrome P450-catalyzed oxidative cross-linking of aromatic side chains as well as the halogenation of specific residues within the peptide. The timing of halogenation and its effect on the cyclization of the peptide are currently unclear. Our results show that chlorination of peptide precursors improves their processing by P450 enzymes in vitro, which provides support for GPA halogenation occurring prior to peptide cyclization during nonribosomal peptide synthesis. We could also determine that the activity of the second enzyme in the oxidative cyclization cascade, OxyA, remains higher for chlorinated peptide substrates even when the biosynthetic GPA product possesses an altered chlorination pattern, which supports the role of the chlorine atoms in orienting the peptide substrate in the active site of these enzymes.


Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Biocatálise/efeitos dos fármacos , Sistema Enzimático do Citocromo P-450/metabolismo , Glicopeptídeos/química , Glicopeptídeos/farmacologia , Halogenação , Domínio Catalítico , Ciclização/efeitos dos fármacos , Sistema Enzimático do Citocromo P-450/química , Oxirredução
7.
Chem Commun (Camb) ; 52(94): 13679-13682, 2016 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-27812569

RESUMO

We show that two α-N-methyltransferases involved in the biosynthesis of glycopeptide antibiotics (GPAs) already recognise partly crosslinked precursor peptides of teicoplanin aglycone indicating that in vivo N-methylation can occur as an early tailoring step during GPA biosynthesis. This relaxed substrate specificity is accompanied by a remarkable promiscuity regarding the co-substrate enabling modulation of biological activity and the introduction of reactive handles which could be further modified using bio-orthogonal chemistry.


Assuntos
Antibacterianos/biossíntese , Glicopeptídeos/metabolismo , Metiltransferases/metabolismo , Coloração e Rotulagem , Antibacterianos/química , Biocatálise , Conformação Molecular
8.
Sci Rep ; 6: 35584, 2016 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-27752135

RESUMO

The glycopeptide antibiotics are peptide-based natural products with impressive antibiotic function that derives from their unique three-dimensional structure. Biosynthesis of the glycopeptide antibiotics centres of the combination of peptide synthesis, mediated by a non-ribosomal peptide synthetase, and the crosslinking of aromatic side chains of the peptide, mediated by the action of a cascade of Cytochrome P450s. Here, we report the first example of in vitro activity of OxyE, which catalyses the F-O-G ring formation reaction in teicoplanin biosynthesis. OxyE was found to only act after an initial C-O-D crosslink is installed by OxyB and to require an interaction with the unique NRPS domain from glycopeptide antibiotic - the X-domain - in order to display catalytic activity. We could demonstrate that OxyE displays limited stereoselectivity for the peptide, which mirrors the results from OxyB-catalysed turnover and is in sharp contrast to OxyA. Furthermore, we show that activity of a three-enzyme cascade (OxyB/OxyA/OxyE) in generating tricyclic glycopeptide antibiotic peptides depends upon the order of addition of the OxyA and OxyE enzymes to the reaction. This work demonstrates that complex enzymatic cascades from glycopeptide antibiotic biosynthesis can be reconstituted in vitro and provides new insights into the biosynthesis of these important antibiotics.


Assuntos
Antibacterianos/química , Bactérias/enzimologia , Proteínas de Bactérias/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Glicopeptídeos/química , Peptídeo Sintases/metabolismo , Teicoplanina/biossíntese , Aminoácidos Aromáticos/química , Antibacterianos/metabolismo , Fenômenos Fisiológicos Bacterianos , Proteínas de Bactérias/genética , Catálise , Sistema Livre de Células , Clonagem Molecular , Ciclização , Sistema Enzimático do Citocromo P-450/genética , Glicopeptídeos/genética , Glicopeptídeos/metabolismo , Peptídeo Sintases/química , Conformação Proteica , Estereoisomerismo , Especificidade por Substrato
9.
J Biol Chem ; 291(44): 22868-22880, 2016 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-27621317

RESUMO

An arsenal of effector proteins is injected by bacterial pathogens into the host cell or its vicinity to increase virulence. The commonly used top-down approaches inferring the toxic mechanism of individual effector proteins from the host's phenotype are often impeded by multiple targets of different effectors as well as by their pleiotropic effects. Here we describe our bottom-up approach, showing that the bacterial type III effector AvrRxo1 of plant pathogens is an authentic phosphotransferase that produces two novel metabolites by phosphorylating nicotinamide/nicotinic acid adenine dinucleotide at the adenosine 3'-hydroxyl group. Both products of AvrRxo1, 3'-NADP and 3'-nicotinic acid adenine dinucleotide phosphate (3'-NAADP), are substantially different from the ubiquitous co-enzyme 2'-NADP and the calcium mobilizer 2'-NAADP. Interestingly, 3'-NADP and 3'-NAADP have previously been used as inhibitors or signaling molecules but were regarded as "artificial" compounds so far. Our findings now necessitate a shift in thinking about the biological importance of 3'-phosphorylated NAD derivatives.


Assuntos
Proteínas de Bactérias/metabolismo , NADP/análogos & derivados , NADP/metabolismo , Xanthomonas/metabolismo , Proteínas de Bactérias/genética , Xanthomonas/genética
10.
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
11.
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
12.
Methods Mol Biol ; 1401: 85-102, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26831703

RESUMO

The glycopeptide antibiotics are an important class of complex, medically relevant peptide natural products. Given that the production of such compounds all stems from in vivo biosynthesis, understanding the mechanisms of the natural assembly system--consisting of a nonribosomal-peptide synthetase machinery (NRPS) and further modifying enzymes--is vital. In order to address the later steps of peptide biosynthesis, which are catalyzed by Cytochrome P450s that interact with the peptide-producing nonribosomal peptide synthetase, peptide substrates are required: these peptides must also be in a form that can be conjugated to carrier protein domains of the nonribosomal peptide synthetase machinery. Here, we describe a practical and effective route for the solid phase synthesis of glycopeptide antibiotic precursor peptides as their Coenzyme A (CoA) conjugates to allow enzymatic conjugation to carrier protein domains. This route utilizes Fmoc-chemistry suppressing epimerization of racemization-prone aryl glycine derivatives and affords high yields and excellent purities, requiring only a single step of simple solid phase extraction for chromatographic purification. With this, comprehensive investigations of interactions between various NRPS-bound substrates and Cytochrome P450s are enabled.


Assuntos
Antibacterianos/síntese química , Bactérias/enzimologia , Coenzima A/química , Glicopeptídeos/síntese química , Peptídeo Sintases/metabolismo , Técnicas de Síntese em Fase Sólida/métodos , Sequência de Aminoácidos , Antibacterianos/química , Antibacterianos/metabolismo , Bactérias/química , Bactérias/metabolismo , Coenzima A/síntese química , Coenzima A/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Glicopeptídeos/química , Glicopeptídeos/metabolismo , Dados de Sequência Molecular , Teicoplanina/síntese química , Teicoplanina/química , Teicoplanina/metabolismo
13.
Beilstein J Org Chem ; 12: 2849-2864, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-28144358

RESUMO

The chemical complexity and biological activity of the glycopeptide antibiotics (GPAs) stems from their unique crosslinked structure, which is generated by the actions of cytochrome P450 (Oxy) enzymes that affect the crosslinking of aromatic side chains of amino acid residues contained within the GPA heptapeptide precursor. Given the crucial role peptide cyclisation plays in GPA activity, the characterisation of this process is of great importance in understanding the biosynthesis of these important antibiotics. Here, we report the cyclisation activity and crystal structure of StaF, the D-O-E ring forming Oxy enzyme from A47934 biosynthesis. Our results show that the specificity of StaF is reduced when compared to Oxy enzymes catalysing C-O-D ring formation and that this activity relies on interactions with the non-ribosomal peptide synthetase via the X-domain. Despite the interaction of StaF with the A47934 X-domain being weaker than for the preceding Oxy enzyme StaH, StaF retains higher levels of in vitro activity: we postulate that this is due to the ability of the StaF/X-domain complex to allow substrate reorganisation after initial complex formation has occurred. These results highlight the importance of testing different peptide/protein carrier constructs for in vitro GPA cyclisation assays and show that different Oxy homologues can display significantly different catalytic propensities despite their overall similarities.

14.
Angew Chem Int Ed Engl ; 54(52): 15715-9, 2015 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-26549530

RESUMO

The biosynthesis of the glycopeptide antibiotics, which include vancomycin and teicoplanin, relies on the interplay between the peptide-producing non-ribosomal peptide synthetase (NRPS) and Cytochrome P450 enzymes (P450s) that catalyze side-chain crosslinking of the peptide. We demonstrate that sequential in vitro P450-catalyzed cyclization of peptide substrates is enabled by the use of an NRPS peptide carrier protein (PCP)-X di-domain as a P450 recruitment platform. This study reveals that whilst the precursor peptide sequence influences the installation of the second crosslink by the P450 OxyAtei , activity is not restricted to the native teicoplanin peptide. Initial peptide cyclization is possible with teicoplanin and vancomycin OxyB homologues, and the latter displays excellent activity with all substrate combinations tested. By using non-natural X-domain substrates, bicyclization of hexapeptides was also shown, which demonstrates the utility of this method for the cyclization of varied peptide substrates in vitro.


Assuntos
Antibacterianos/química , Sistema Enzimático do Citocromo P-450/química , Glicopeptídeos/química , Biossíntese Peptídica , Ciclização
15.
Chemistry ; 21(49): 17870-6, 2015 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-26489532

RESUMO

Herein, we report the reversible light-regulated destabilization of DNA duplexes by using azobenzene C-nucleoside photoswitches. The incorporation of two different azobenzene residues into DNA and their photoswitching properties are described. These new residues demonstrate a photoinduced destabilization effect comparable to the widely applied D-threoninol-linked azobenzene switch, which is currently the benchmark. The photoswitches presented herein show excellent photoswitching efficiencies in DNA duplexes - even at room temperature - which are superior to commonly used azobenzene-based nucleic acid photoswitches. In addition, these photoswitching residues exhibit high thermal stability and excellent fatigue resistance, thus rendering them one of the most efficient candidates for the regulation of duplex stability with light.


Assuntos
Amino Álcoois/química , Compostos Azo/química , Butileno Glicóis/química , DNA/química , Nucleosídeos/química , Pareamento de Bases , DNA/metabolismo , Hibridização Genética , Luz , Nucleosídeos/metabolismo , Fotoquímica , Temperatura Ambiente
16.
Nat Prod Rep ; 32(8): 1207-35, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25940955

RESUMO

Phenylglycine-type amino acids occur in a wide variety of peptide natural products, including glycopeptide antibiotics and biologically active linear and cyclic peptides. Sequencing of biosynthesis gene clusters of chloroeremomycin, balhimycin and pristinamycin paved the way for intensive investigations on the biosynthesis of 4-hydroxyphenylglycine (Hpg), 3,5-dihydroxyphenylglycine (Dpg) and phenylglycine (Phg) in recent years. The significance and importance of this type of unusual non-proteinogenic aromatic amino acids also for medicinal chemistry has drawn the attention of many research groups and pharmaceutical companies. Herein structures and properties of phenylglycine containing natural products as well as the biosynthetic origin and incorporation of phenylglycines are discussed.


Assuntos
Aminoácidos/química , Produtos Biológicos/química , Glicina/análogos & derivados , Peptídeos/química , Aminoácidos/metabolismo , Produtos Biológicos/metabolismo , Glicina/química , Glicina/metabolismo , Estrutura Molecular , Peptídeos/metabolismo
17.
Nature ; 521(7550): 105-9, 2015 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-25686610

RESUMO

Non-ribosomal peptide synthetase (NRPS) mega-enzyme complexes are modular assembly lines that are involved in the biosynthesis of numerous peptide metabolites independently of the ribosome. The multiple interactions between catalytic domains within the NRPS machinery are further complemented by additional interactions with external enzymes, particularly focused on the final peptide maturation process. An important class of NRPS metabolites that require extensive external modification of the NRPS-bound peptide are the glycopeptide antibiotics (GPAs), which include vancomycin and teicoplanin. These clinically relevant peptide antibiotics undergo cytochrome P450-catalysed oxidative crosslinking of aromatic side chains to achieve their final, active conformation. However, the mechanism underlying the recruitment of the cytochrome P450 oxygenases to the NRPS-bound peptide was previously unknown. Here we show, through in vitro studies, that the X-domain, a conserved domain of unknown function present in the final module of all GPA NRPS machineries, is responsible for the recruitment of oxygenases to the NRPS-bound peptide to perform the essential side-chain crosslinking. X-ray crystallography shows that the X-domain is structurally related to condensation domains, but that its amino acid substitutions render it catalytically inactive. We found that the X-domain recruits cytochrome P450 oxygenases to the NRPS and determined the interface by solving the structure of a P450-X-domain complex. Additionally, we demonstrated that the modification of peptide precursors by oxygenases in vitro--in particular the installation of the second crosslink in GPA biosynthesis--occurs only in the presence of the X-domain. Our results indicate that the presentation of peptidyl carrier protein (PCP)-bound substrates for oxidation in GPA biosynthesis requires the presence of the NRPS X-domain to ensure conversion of the precursor peptide into a mature aglycone, and that the carrier protein domain alone is not always sufficient to generate a competent substrate for external cytochrome P450 oxygenases.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Glicopeptídeos/biossíntese , Peptídeo Sintases/química , Peptídeo Sintases/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X , Modelos Moleculares , Estrutura Terciária de Proteína , Teicoplanina/análogos & derivados , Teicoplanina/biossíntese , Teicoplanina/química , Teicoplanina/metabolismo , Vancomicina/biossíntese
18.
Org Biomol Chem ; 13(7): 2012-21, 2015 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-25501135

RESUMO

Understanding the mechanisms underpinning glycopeptide antibiotic biosynthesis is key to the future ability to reinvent these compounds. For effective in vitro characterization of the crucial later steps of the biosynthesis, facile access to a wide range of substrate peptides as their Coenzyme A (CoA) conjugates is essential. Here we report the development of a rapid route to glycopeptide precursor CoA conjugates that affords both high yields and excellent purities. This synthesis route is applicable to the synthesis of peptide CoA-conjugates containing racemization-prone arylglycine residues: such residues are hallmarks of non-ribosomal peptide synthesis and have previously been inaccessible to peptide synthesis using Fmoc-type chemistry. We have applied this route to generate glycopeptide precursor peptides in their carrier protein-bound form as substrates to explore the specificity of the first oxygenase enzyme from vancomycin biosynthesis (OxyBvan). Our results indicate that OxyBvan is a highly promiscuous catalyst for phenolic coupling of diverse glycopeptide precursors that accepts multiple carrier protein substrates, even on carrier protein domains from alternate glycopeptide biosynthetic machineries. These results represent the first important steps in the development of an in vitro biomimetic synthesis of modified glycopeptide aglycones.


Assuntos
Antibacterianos/biossíntese , Antibacterianos/química , Biocatálise , Materiais Biomiméticos/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Glicopeptídeos/biossíntese , Materiais Biomiméticos/química , Coenzima A/química , Coenzima A/metabolismo , Glicopeptídeos/química , Conformação Molecular
19.
Chemistry ; 21(7): 2845-54, 2015 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-25537843

RESUMO

Photoregulation of RNA remains a challenging task as the introduction of a photoswitch entails changes in the shape and the stability of the duplex that strongly depend on the chosen linker strategy. Herein, the influence of a novel nucleosidic linker moiety on the photoregulation efficiency of azobenzene is investigated. To this purpose, two azobenzene C-nucleosides were stereoselectively synthesized, characterized, and incorporated into RNA oligonucleotides. Spectroscopic characterization revealed a reversible and fast switching process, even at 20 °C, and a high thermal stability of the respective cis isomers. The photoregulation efficiency of RNA duplexes upon trans-to-cis isomerization was investigated by using melting point studies and compared with the known D-threoninol-based azobenzene system, revealing a photoswitching amplitude of the new residues exceeding 90 % even at room temperature. Structural changes in the duplexes upon photoisomerization were investigated by using MM/MD calculations. The excellent photoswitching performance at room temperature and the high thermal stability make these new azobenzene residues promising candidates for in-vivo and nanoarchitecture photoregulation applications of RNA.


Assuntos
Compostos Azo/química , Nucleosídeos/química , RNA/química , Hibridização Genética , Estrutura Molecular , Oligonucleotídeos
20.
Chembiochem ; 15(18): 2719-28, 2014 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-25358800

RESUMO

Bacterial cytochrome P450s form a remarkable clade of the P450 superfamily of oxidative hemoproteins, and are often involved in the biosynthesis of complex natural products. Those in a subgroup known as "Oxy enzymes" play a crucial role in the biosynthesis of glycopeptide antibiotics, including vancomycin and teicoplanin. The Oxy enzymes catalyze crosslinking of aromatic residues in the non-ribosomal antibiotic precursor peptide while it remains bound to the non-ribosomal peptide synthetase (NRPS); this crosslinking secures the three-dimensional structure of the glycopeptide, crucial for antibiotic activity. We have characterized OxyBtei , the first of the Oxy enzymes in teicoplanin biosynthesis. Our results reveal that OxyBtei possesses a structure similar to those of other Oxy proteins and is active in crosslinking NRPS-bound peptide substrates. However, OxyBtei displays a significantly altered activity spectrum against peptide substrates compared to its well-studied vancomycin homologue.


Assuntos
Antibacterianos/metabolismo , Proteínas de Bactérias/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Micromonosporaceae/metabolismo , Fenóis/metabolismo , Teicoplanina/metabolismo , Antibacterianos/química , Proteínas de Bactérias/química , Biocatálise , Sistema Enzimático do Citocromo P-450/química , Micromonosporaceae/química , Modelos Moleculares , Fenóis/química , Teicoplanina/química
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