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This review article aims to highlight the role of methyltransferases within the context of ribosomally synthesised and post-translationally modified peptide (RiPP) natural products. Methyltransferases play a pivotal role in the biosynthesis of diverse natural products with unique chemical structures and bioactivities. They are highly chemo-, regio-, and stereoselective allowing methylation at various positions. The different possible acceptor regions in ribosomally synthesised peptides are described in this article. Furthermore, we will discuss the potential application of these methyltransferases as powerful biocatalytic tools in the synthesis of modified peptides and other bioactive compounds. By providing an overview of the various methylation options available, this review is intended to emphasise the biocatalytic potential of RiPP methyltransferases and their impact on the field of natural product chemistry.
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Covering: up to 2024Ribosomally synthesised and post-translationally modified peptides (RiPPs) comprise a substantial group of peptide natural products exhibiting noteworthy bioactivities ranging from antiinfective to anticancer and analgesic effects. Furthermore, RiPP biosynthetic pathways represent promising production routes for complex peptide drugs, and the RiPP technology is well-suited for peptide engineering to produce derivatives with specific functions. Thus, RiPP natural products possess features that render them potentially ideal candidates for drug discovery and development. Nonetheless, only a small number of RiPP-derived compounds have successfully reached the market thus far. This review initially outlines the therapeutic opportunities that RiPP-based compounds can offer, whilst subsequently discussing the limitations that require resolution in order to fully exploit the potential of RiPPs towards the development of innovative drugs.
Assuntos
Produtos Biológicos , Peptídeos , Processamento de Proteína Pós-Traducional , Produtos Biológicos/química , Produtos Biológicos/farmacologia , Humanos , Peptídeos/química , Peptídeos/farmacologia , Peptídeos/uso terapêutico , Descoberta de Drogas , Antineoplásicos/farmacologia , Antineoplásicos/química , Animais , Anti-Infecciosos/farmacologia , Anti-Infecciosos/química , Anti-Infecciosos/uso terapêuticoRESUMO
S-Adenosylmethionine (SAM) is an enzyme cofactor involved in methylation, aminopropyl transfer, and radical reactions. This versatility renders SAM-dependent enzymes of great interest in biocatalysis. The usage of SAM analogues adds to this diversity. However, high cost and instability of the cofactor impedes the investigation and usage of these enzymes. While SAM regeneration protocols from the methyltransferase (MT) byproduct S-adenosylhomocysteine are available, aminopropyl transferases and radical SAM enzymes are not covered. Here, we report a set of efficient one-pot systems to supply or regenerate SAM and SAM analogues for all three enzyme classes. The systems' flexibility is showcased by the transfer of an ethyl group with a cobalamin-dependent radical SAM MT using S-adenosylethionine as a cofactor. This shows the potential of SAM (analogue) supply and regeneration for the application of diverse chemistry, as well as for mechanistic studies using cofactor analogues.
Assuntos
Biomimética , S-Adenosilmetionina , S-Adenosilmetionina/metabolismo , Biocatálise , Alquilação , Metilação , Metiltransferases/metabolismoRESUMO
Landornamide A is a ribosomally synthesized and post-translationally modified peptide (RiPP) natural product with antiviral activity. Its biosynthetic gene cluster encodesâamong other maturasesâthe peptide arginase OspR, which converts arginine to ornithine units in an unusual post-translational modification. Peptide arginases are a recently discovered RiPP maturase family with few characterized representatives. They show little sequence similarity to conventional arginases, a well-characterized enzyme family catalyzing the hydrolysis of free arginine to ornithine and urea. Peptide arginases are highly promiscuous and accept a variety of substrate sequences. The molecular basis for binding the large peptide substrate and for the high promiscuity of peptide arginases remains unclear. Here, we report the first crystal structure of a peptide arginase at a resolution of 2.6 Å. The three-dimensional structure reveals common features and differences between conventional arginases and the peptide arginase: the binuclear metal cluster and the active-site environment strongly resemble each other, while the quaternary structures diverge. Kinetic analyses of OspR with various substrates provide new insights into the order of biosynthetic reactions during the post-translational maturation of landornamide A. These results provide the basis for pathway engineering to generate derivatives of landornamide A and for the general application of peptide arginases as biosynthetic tools for peptide engineering.
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Arginase , Arginina , Arginase/metabolismo , Arginina/metabolismo , Ornitina/metabolismo , Peptídeos/metabolismo , Processamento de Proteína Pós-TraducionalRESUMO
Peptide natural products are important lead structures for human drugs and many nonribosomal peptides possess antibiotic activity. This makes them interesting targets for engineering approaches to generate peptide analogues with, for example, increased bioactivities. Nonribosomal peptides are produced by huge mega-enzyme complexes in an assembly-line like manner, and hence, these biosynthetic pathways are challenging to engineer. In the past decade, more and more structural features thought to be unique to nonribosomal peptides were found in ribosomally synthesised and posttranslationally modified peptides as well. These streamlined ribosomal pathways with modifying enzymes that are often promiscuous and with gene-encoded precursor proteins that can be modified easily, offer several advantages to produce designer peptides. This review aims to provide an overview of recent progress in this emerging research area by comparing structural features common to both nonribosomal and ribosomally synthesised and posttranslationally modified peptides in the first part and highlighting synthetic biology strategies for emulating nonribosomal peptides by ribosomal pathway engineering in the second part.
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The ubiquitous cofactor S-adenosyl-l-methionine (SAM) is part of numerous biochemical reactions in metabolism, epigenetics, and cancer development. As methylation usually improves physiochemical properties of compounds relevant for pharmaceutical use, the sustainable use of SAM as a methyl donor in biotechnological applications is an important goal. SAM-dependent methyltransferases are consequently an emerging biocatalytic tool for environmentally friendly and selective alkylations. However, SAM shows undesirable characteristics such as degradation under mild conditions and its stoichiometric use is economically not reasonable. Here, we report an optimised biomimetic system for the regeneration of SAM and SAM analogues consisting of effective nucleoside triphosphate formation and an additional l-methionine regeneration cycle without by-product accumulation. The bicyclic system uses seven enzymes, S-methylmethionine as methyl donor and a surplus of inorganic polyphosphate, along with catalytic amounts of l-methionine and cofactor building block reaching conversions of up to 99% (up to 200 turnovers). We also show that the cycle can be run with cofactor building blocks containing different purine and pyrimidine nucleobases, which can be fed in at the nucleoside or nucleotide stage. These alternative cofactors are in turn converted to the corresponding SAM analogues, which are considered to be a key for the development of bioorthogonal systems. In addition to purified enzymes, the bicyclic system can also be used with crude lysates highlighting its broad biocatalytic applicability.
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Ornithine is a component of many bioactive nonribosomal peptides but is challenging to incorporate into ribosomal products. We recently identified OspR, a cyanobacterial arginase-like enzyme that installs ornithines in the antiviral ribosomally synthesised and posttranslationally modified peptide (RiPP) landornamideâ A. Here we report that OspR belongs to a larger family of peptide arginases from diverse organisms and RiPP types. In E. coli, seven selected enzymes converted arginine into ornithine with little preference for the leader type. A broad range of peptide sequences was modified, including polyarginine repeats. We also generated analogues of ornithine-containing nonribosomal peptides using RiPP technology. Five pseudo-nonribosomal products with ornithines at the correct positions were obtained, including a brevicidine analogue containing ornithine and a d-amino acid installed by the peptide epimerase OspD. These results suggest new opportunities for peptide bioengineering.
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Arginase/metabolismo , Cianobactérias/enzimologia , Ornitina/metabolismo , Peptídeos/metabolismo , Ribossomos/metabolismo , Sequência de Aminoácidos , Arginase/química , Conformação Molecular , Ornitina/química , Peptídeos/química , Processamento de Proteína Pós-Traducional , Ribossomos/químicaRESUMO
Covering: up to the beginning of 2020Enzymes depending on cofactors are essential in many biosynthetic pathways of natural products. They are often involved in key steps: catalytic conversions that are difficult to achieve purely with synthetic organic chemistry. Hence, cofactor-dependent enzymes have great potential for biocatalysis, on the condition that a corresponding cofactor regeneration system is available. For some cofactors, these regeneration systems require multiple steps; such complex enzyme cascades/multi-enzyme systems are (still) challenging for in vitro biocatalysis. Further, artificial cofactor analogues have been synthesised that are more stable, show an altered reaction range, or act as inhibitors. The development of bio-orthogonal systems that can be used for the production of modified natural products in vivo is an ongoing challenge. In light of the recent progress in this field, this review aims to provide an overview of general strategies involving enzyme cofactors, cofactor analogues, and regeneration systems; highlighting the current possibilities for application of enzymes using some of the most common cofactors.
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Coenzimas/química , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Catálise , Coenzima A/química , Coenzima A/metabolismo , Coenzimas/síntese química , NADP/química , NADP/metabolismo , Nucleosídeos/metabolismo , Fosfoadenosina Fosfossulfato/química , Fosfoadenosina Fosfossulfato/metabolismo , FosforilaçãoRESUMO
Chorismatases catalyse the cleavage of chorismate, yielding (dihydroxy-)benzoate derivatives, which often constitute starter units for pharmaceutically relevant secondary metabolites. Depending on their products, chorismatases have been classified into three different subfamilies. These can be assigned using a set of amino acid residues in the active site. Here, we describe five new chorismatases, two of them members of a new subfamily, which has been discovered through correlation analysis of homologous protein sequences. The enzymes from the new subfamily produce exclusively 4-hydroxybenzoate, the same compound as produced by the structurally unrelated chorismate lyases. This showcase of convergent evolution is an example of the existence of more than one pathway to central building blocks. In contrast to chorismate lyases, however, chorismatases do not suffer from product inhibition (up to 2 mM 4-HBA), while the remaining kinetic parameters are in the same range; this makes them an interesting alternative for biocatalytic applications.
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Bactérias/enzimologia , Proteínas de Bactérias/metabolismo , Ácido Corísmico/metabolismo , Oxo-Ácido-Liases/metabolismo , Parabenos/metabolismo , Bactérias/química , Bactérias/metabolismo , Proteínas de Bactérias/química , Ácido Corísmico/química , Modelos Moleculares , Oxo-Ácido-Liases/química , Parabenos/química , Streptomyces/química , Streptomyces/enzimologia , Streptomyces/metabolismo , Especificidade por SubstratoRESUMO
Polyphosphate kinases (PPKs) are involved in many metabolic processes; enzymes of the second family (PPK2) are responsible for nucleotide synthesis fuelled by the consumption of inorganic polyphosphate. They catalyse the phosphorylation of nucleotides with various numbers of phosphate residues, such as monophosphates or diphosphates. Hence, these enzymes are promising candidates for cofactor regeneration systems. Besides adenosine 5'-triphosphate, PPK2s also catalyse the synthesis of highly phosphorylated nucleotides in vitro, as shown here for adenosine 5'-tetraphosphate and adenosine 5'-pentaphosphate. These unusually phosphorylated adenosine 5'-polyphosphates add up to 50 % of the whole adenosine nucleotides in the assay. The two new products were chemically synthesised to serve as standards and compared with the two enzymatically produced compounds by high-performance ion chromatography and 31 Pâ NMR analysis. This study shows that PPK2s are highly suitable for biocatalytic synthesis of different phosphorylated nucleotides.
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Monofosfato de Adenosina/metabolismo , Fosfotransferases (Aceptor do Grupo Fosfato)/metabolismo , Polifosfatos/metabolismo , Monofosfato de Adenosina/química , Catálise , Especificidade por SubstratoRESUMO
Inorganic polyphosphate is a ubiquitous, linear biopolymer built of up to thousands of phosphate residues that are linked by energy-rich phosphoanhydride bonds. Polyphosphate kinases of the family 2 (PPK2) use polyphosphate to catalyze the reversible phosphorylation of nucleotide phosphates and are highly relevant as targets for new pharmaceutical compounds and as biocatalysts for cofactor regeneration. PPK2s can be classified based on their preference for nucleoside mono- or diphosphates or both. The detailed mechanism of PPK2s and the molecular basis for their substrate preference is unclear, which is mainly due to the lack of high-resolution structures with substrates or substrate analogs. Here, we report the structural analysis and comparison of a class I PPK2 (ADP-phosphorylating) and a class III PPK2 (AMP- and ADP-phosphorylating), both complexed with polyphosphate and/or nucleotide substrates. Together with complementary biochemical analyses, these define the molecular basis of nucleotide specificity and are consistent with a Mg2+ catalyzed in-line phosphoryl transfer mechanism. This mechanistic insight will guide the development of PPK2 inhibitors as potential antibacterials or genetically modified PPK2s that phosphorylate alternative substrates.
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Deinococcus/enzimologia , Fosfotransferases (Aceptor do Grupo Fosfato)/química , Fosfotransferases (Aceptor do Grupo Fosfato)/metabolismo , Polifosfatos/metabolismo , Cristalografia por Raios X , Cinética , Ligantes , Fosforilação , Conformação Proteica , Especificidade por SubstratoRESUMO
S-Adenosylmethionine-dependent methyltransferases (MTs) play a decisive role in the biosynthesis of natural products and in epigenetic processes. MTs catalyze the methylation of heteroatoms and even of carbon atoms, which, in many cases, is a challenging reaction in conventional synthesis. However, C-MTs are often highly substrate-specific. Herein, we show that SgvM from Streptomyces griseoviridis features an extended substrate scope with respect to the nucleophile as well as the electrophile. Aside from its physiological substrate 4-methyl-2-oxovalerate, SgvM catalyzes the (di)methylation of pyruvate, 2-oxobutyrate, 2-oxovalerate, and phenylpyruvate at the ß-carbon atom. Chiral-phase HPLC analysis revealed that the methylation of 2-oxovalerate occurs with Râ selectivity while the ethylation of 2-oxobutyrate with S-adenosylethionine results in the Sâ enantiomer of 3-methyl-2-oxovalerate. Thus SgvM could be a valuable tool for asymmetric biocatalytic C-alkylation reactions.
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S-Adenosylmethionine-dependent methyltransferases are versatile tools for the specific alkylation of many compounds, such as pharmaceuticals, but their biocatalytic application is severely limited owing to the lack of a cofactor regeneration system. We report a biomimetic, polyphosphate-based, cyclic cascade for methyltransferases. In addition to the substrate to be methylated, only methionine and polyphosphate have to be added in stoichiometric amounts. The system acts catalytically with respect to the cofactor precursor adenosine in methylation and ethylation reactions of selected substrates, as shown by HPLC analysis. Furthermore, 1 H and 13 Câ NMR measurements were performed to unequivocally identify methionine as the methyl donor and to gain insight into the selectivity of the reactions. This system constitutes a vital stage in the development of economical and environmentally friendly applications of methyltransferases.
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Mg2+ -dependent catechol-O-methyltransferases occur in animals as well as in bacteria, fungi and plants, often with a pronounced selectivity towards one of the substrate's hydroxyl groups. Here, we show that the bacterial MxSafC exhibits excellent regioselectivity for para as well as for meta methylation, depending on the substrate's characteristics. The crystal structure of MxSafC was solved in apo and in holo form. The structure complexed with a full set of substrates clearly illustrates the plasticity of the active site region. The awareness that a wide range of factors influences the regioselectivity will aid the further development of catechol-O-methyltransferases as well as other methyltransferases as selective and efficient biocatalysts for chemical synthesis.
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Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Catecol O-Metiltransferase/química , Catecol O-Metiltransferase/metabolismo , Myxococcus/enzimologia , Domínio Catalítico , Cromatografia Líquida de Alta Pressão , Modelos Moleculares , Estereoisomerismo , Especificidade por SubstratoRESUMO
S-Adenosylmethionine (SAM)-dependent enzymes have great potential for selective alkylation processes. In this study we investigated the regiocomplementary O-methylation of catechols. Enzymatic methylation is often hampered by the need for a stoichiometric supply of SAM and the inhibitory effect of the SAM-derived byproduct on most methyltransferases. To counteract these issues we set up an enzyme cascade. Firstly, SAM was generated from l-methionine and ATP by use of an archaeal methionine adenosyltransferase. Secondly, 4-O-methylation of the substrates dopamine and dihydrocaffeic acid was achieved by use of SafC from the saframycin biosynthesis pathway in 40-70 % yield and high selectivity. The regiocomplementary 3-O-methylation was catalysed by catechol O-methyltransferase from rat. Thirdly, the beneficial influence of a nucleosidase on the overall conversion was demonstrated. The results of this study are important milestones on the pathway to catalytic SAM-dependent alkylation processes.
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Catecol O-Metiltransferase/metabolismo , Catecóis/metabolismo , Metionina Adenosiltransferase/metabolismo , N-Glicosil Hidrolases/metabolismo , Animais , Archaea/enzimologia , Archaea/metabolismo , Proteínas Arqueais/metabolismo , Catecóis/química , Cromatografia Líquida de Alta Pressão , Metionina/metabolismo , Metilação , Oxigênio/química , Oxigênio/metabolismo , Ratos , S-Adenosilmetionina/metabolismo , Espectrofotometria , EstereoisomerismoRESUMO
Chorismatases and isochorismatases catalyse the hydrolysis of chorismate or isochorismate leading to unsaturated cyclohexenoic acid derivatives. Based on simplification of the physiological substrates, two cinnamic acid-derived compounds, differing in the saturation of the side chain, were developed. In contrast to earlier inhibitor studies, the compounds described here do not have an ether bond and therefore can be synthesised very easily in one or two steps without the need for protective groups. Both substances demonstrate inhibition of the isochorismatase EntB from Escherichia coli and the chorismatases FkbO and Hyg5 from Streptomyces. For chorismatases, the unsaturated compound shows IC(50) values in the millimolar range, while the saturated compound is the better inhibitor with IC(50) values in the micromolar/low millimolar range; for the isochorismatase tested both compounds inhibit in the micromolar range. Further, an analysis of the apparent K(m) values for FkbO and EntB was performed, showing that both inhibitors act in a competitive manner. Due to the ease of modifying these new inhibitors they are a suitable starting point for exploring further functionalised derivatives.