RESUMO
Many actinobacterial species contain structural genes for iron-dependent enzymes that consume ergothioneine by way of O2-dependent dioxygenation. The resulting product ergothioneine sulfinic acid is stable under physiological conditions unless cleavage to sulfur dioxide and trimethyl histidine is catalyzed by a dedicated desulfinase. This report documents that two types of ergothioneine sulfinic desulfinases have evolved by convergent evolution. One type is related to metal-dependent decarboxylases while the other belongs to the superfamily of rhodanese-like enzymes. Pairs of ergothioneine dioxygenases (ETDO) and ergothioneine sulfinic acid desulfinase (ETSD) occur in thousands of sequenced actinobacteria, suggesting that oxidative ergothioneine degradation is a common activity in this phylum.
Assuntos
Ergotioneína , Ergotioneína/metabolismo , Ergotioneína/química , Actinobacteria/enzimologia , Biocatálise , Ácidos Sulfínicos/química , Ácidos Sulfínicos/metabolismo , Dioxigenases/metabolismo , Dioxigenases/químicaRESUMO
Biocatalysis has become a major driver in the innovation of preparative chemistry. Enzyme discovery, engineering and computational design have matured to reliable strategies in the development of biocatalytic processes. By comparison, substrate engineering has received much less attention. In this Minireview, we highlight the idea that the design of synthetic reagents may be an equally fruitful and complementary approach to develop novel enzyme-catalysed group transfer chemistry. This Minireview discusses key examples from the literature that illustrate how synthetic substrates can be devised to improve the efficiency, scalability and sustainability, as well as the scope of such reactions. We also provide an opinion as to how this concept might be further developed in the future, aspiring to replicate the evolutionary success story of natural group transfer reagents, such as adenosine triphosphate (ATP) and S-adenosyl methionine (SAM).
Assuntos
BiocatáliseRESUMO
Ergothioneine is a sulfur-containing metabolite that is produced by bacteria and fungi, and is absorbed by plants and animals as a micronutrient. Ergothioneine reacts with harmful oxidants, including singlet oxygen and hydrogen peroxide, and may therefore protect cells against oxidative stress. Herein we describe two enzymes from actinobacteria that cooperate in the specific oxidative degradation of ergothioneine. The first enzyme is an iron-dependent thiol dioxygenase that produces ergothioneine sulfinic acid. A crystal structure of ergothioneine dioxygenase from Thermocatellispora tengchongensis reveals many similarities with cysteine dioxygenases, suggesting that the two enzymes share a common mechanism. The second enzyme is a metal-dependent ergothioneine sulfinic acid desulfinase that produces Nα-trimethylhistidine and SO2 . The discovery that certain actinobacteria contain the enzymatic machinery for O2 -dependent biosynthesis and O2 -dependent degradation of ergothioneine indicates that these organisms may actively manage their ergothioneine content.
Assuntos
Dioxigenases , Ergotioneína , Animais , Ergotioneína/metabolismo , Dioxigenases/metabolismo , Cisteína Dioxigenase/metabolismo , Oxirredução , CatáliseRESUMO
S-adenosylmethionine-dependent methyltransferases are involved in countless biological processes, including signal transduction, epigenetics, natural product biosynthesis, and detoxification. Only a handful of carboxylate methyltransferases have evolved to participate in amide bond formation. In this report we show that enzyme-catalyzed F-methylation of carboxylate substrates produces F-methyl esters that readily react with N- or S-nucleophiles under physiological conditions. We demonstrate the applicability of this approach to the synthesis of small amides, hydroxamates, and thioesters, as well as to site-specific protein modification and native chemical ligation.
Assuntos
Amidas , Metiltransferases , Metiltransferases/metabolismo , Metilação , Amidas/química , S-Adenosilmetionina/química , Ácidos Carboxílicos , Trifosfato de Adenosina/metabolismo , BiocatáliseRESUMO
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
Late-stage methylation is a key technology in the development of pharmaceutical compounds. Methyltransferase biocatalysis may provide powerful options to insert methyl groups into complex molecules with high regio- and chemoselectivity. The challenge of a large-scale application of methyltransferases is their dependence on S-adenosylmethionine (SAM) as a stoichiometric, and thus exceedingly expensive co-substrate. As a solution to this problem, we and others have explored the use of methyl halides as reagents for the in situ regeneration of SAM. However, the need to handle volatile electrophiles, such as methyl iodide (MeI), may also hamper applications at scale. As a more practical solution, we have now developed an enzyme-catalyzed process for the regeneration of SAM with methyl toluene sulfonate. Herein, we describe enzymes from the thiopurine methyltransferase family that accept sulfate- and sulfonate-based methyl donors to convert S-adenosylhomocysteine into SAM with efficiencies that rival MeI-based reactions.
Assuntos
S-Adenosil-Homocisteína , S-Adenosilmetionina , Biocatálise , Indicadores e Reagentes , Metilação , Metiltransferases/metabolismo , Preparações Farmacêuticas , S-Adenosilmetionina/química , Sulfatos , ToluenoRESUMO
We introduce the UV-vis spectra-activated droplet sorter (UVADS) for high-throughput label-free chemical identification and enzyme screening. In contrast to previous absorbance-based droplet sorters that relied on single-wavelength absorbance in the visible range, our platform collects full UV-vis spectra from 200 to 1050 nm at up to 2100 spectra per second. Our custom-built open-source software application, "SpectraSorter," enables real-time data processing, analysis, visualization, and selection of droplets for sorting with any set of UV-vis spectral features. An optimized UV-vis detection region extended the absorbance path length for droplets and allowed for the direct protein quantification down to 10 µM of bovine serum albumin at 280 nm. UV-vis spectral data can distinguish a variety of different chemicals or spurious events (such as air bubbles) that are inaccessible at a single wavelength. The platform is used to measure ergothionase enzyme activity from monoclonal microcolonies isolated in droplets. In a label-free manner, we directly measure the ergothioneine substrate to thiourocanic acid product conversion while tracking the microcolony formation. UVADS represents an important new tool for high-throughput label-free in-droplet chemical analysis.
Assuntos
SoftwareRESUMO
In multicellular organisms, cell type diversity and fate depend on specific sets of transcript isoforms generated by post-transcriptional RNA processing. Here, we used Schmidtea mediterranea, a flatworm with extraordinary regenerative abilities and a large pool of adult stem cells, as an in vivo model to study the role of Uridyl-rich small nuclear RNAs (UsnRNAs), which participate in multiple RNA processing reactions including splicing, in stem cell regulation. We characterized the planarian UsnRNA repertoire, identified stem cell-enriched variants and obtained strong evidence for an increased rate of UsnRNA 3'-processing in stem cells compared to their differentiated counterparts. Consistently, components of the Integrator complex showed stem cell-enriched expression and their depletion by RNAi disrupted UsnRNA processing resulting in global changes of splicing patterns and reduced processing of histone mRNAs. Interestingly, loss of Integrator complex function disrupted both stem cell maintenance and regeneration of tissues. Our data show that the function of the Integrator complex in UsnRNA 3'-processing is conserved in planarians and essential for maintaining their stem cell pool. We propose that cell type-specific modulation of UsnRNA composition and maturation contributes to in vivo cell fate choices, such as stem cell self-renewal in planarians.
Assuntos
Células-Tronco Adultas/fisiologia , Planárias/genética , Planárias/fisiologia , RNA de Helmintos/genética , RNA de Helmintos/metabolismo , RNA Nuclear Pequeno/genética , RNA Nuclear Pequeno/metabolismo , Regeneração/genética , Regeneração/fisiologia , Células-Tronco Adultas/citologia , Animais , Sequência de Bases , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Autorrenovação Celular/genética , Autorrenovação Celular/fisiologia , Modelos Biológicos , Conformação de Ácido Nucleico , Planárias/citologia , Interferência de RNA , Processamento Pós-Transcricional do RNA , Splicing de RNA , RNA de Helmintos/química , Homologia de Sequência do Ácido NucleicoRESUMO
Molecular oxygen (O2 ) is a sustainable oxidation reagent. O2 is strongly oxidizing but kinetically stable and its final reaction product is water. For these reasons learning how to activate O2 and how to steer its reactivity along desired reaction pathways is a longstanding challenge in chemical research.[1] Activation of ground-state diradical O2 can occur either via conversion to singlet oxygen or by one-electron reduction to superoxide. Many enzymes facilitate activation of O2 by direct fomation of a metal-oxygen coordination complex concomitant with inner sphere electron transfer. The formylglycine generating enzyme (FGE) is an unusual mononuclear copper enzyme that appears to follow a different strategy. Atomic-resolution crystal structures of the precatalytic complex of FGE demonstrate that this enzyme binds O2 juxtaposed, but not coordinated to the catalytic CuI . Isostructural complexes that contain AgI instead of CuI or nitric oxide instead of O2 confirm that formation of the initial oxygenated complex of FGE does not depend on redox activity. A stepwise mechanism that decouples binding and activation of O2 is unprecedented for metal-dependent oxidases, but is reminiscent of flavin-dependent enzymes.
RESUMO
Strategic replacement of protons with fluorine atoms or functional groups with fluorine-containing fragments has proven a powerful strategy to optimize the activity of therapeutic compounds. For this reason, the synthetic chemistry of organofluorides has been the subject of intense development and innovation for many years. By comparison, the literature on fluorine biocatalysis still makes for a slim chapter. Herein we introduce S-adenosylmethionine (SAM) dependent methyltransferases as a new tool for the production of fluorinated compounds. We demonstrate the ability of halide methyltransferases to form fluorinated SAM (S-adenosyl-S-(fluoromethyl)-L-homocysteine) from S-adenosylhomocysteine and fluoromethyliodide. Fluorinated SAM (F-SAM) is too unstable for isolation, but is accepted as a substrate by C-, N- and O-specific methyltransferases for enzyme-catalyzed fluoromethylation of small molecules.
Assuntos
Hidrocarbonetos Fluorados/síntese química , Indicadores e Reagentes/química , Metiltransferases/química , S-Adenosilmetionina/análogos & derivados , Bactérias/enzimologia , Proteínas de Bactérias/química , Halogenação , MetilaçãoRESUMO
Ergothioneine is an emerging component of the redox homeostasis system in human cells and in microbial pathogens, such as Mycobacterium tuberculosis and Burkholderia pseudomallei. The synthesis of stable isotope-labeled ergothioneine derivatives may provide important tools for deciphering the distribution, function, and metabolism of this compound in vivo. We describe a general protocol for the production of ergothioneine isotopologues with programmable 2 H, 15 N, 13 C, 34 S, and 33 S isotope labeling patterns. This enzyme-based approach makes efficient use of commercial isotope reagents and is also directly applicable to the synthesis of radio-isotopologues.
Assuntos
Ergotioneína/síntese química , Proteínas de Bactérias/química , Biocatálise , Marcação por Isótopo , Metiltransferases/química , Mycobacterium smegmatis/enzimologia , Radioisótopos/química , Sulfurtransferases/químicaRESUMO
Enantioselective synthesis of bioactive compounds bearing a pyrroloindole framework is often laborious. In contrast, there are several S-adenosyl methionine (SAM)-dependent methyl transferases known for stereo- and regioselective methylation at the C3 position of various indoles, directly leading to the formation of the desired pyrroloindole moiety. Herein, the SAM-dependent methyl transferase PsmD from Streptomyces griseofuscus, a key enzyme in the biosynthesis of physostigmine, is characterized in detail. The biochemical properties of PsmD and its substrate scope were demonstrated. Preparative scale enzymatic methylation including SAM regeneration was achieved for three selected substrates after a design-of-experiment optimization.
Assuntos
Indóis/síntese química , Metiltransferases/química , Pirróis/síntese química , Biocatálise , Cinética , Metilação , S-Adenosilmetionina/química , Estereoisomerismo , Streptomyces/enzimologiaRESUMO
Tissue regeneration depends on proliferative cells and on cues that regulate cell division, differentiation, patterning and the restriction of these processes once regeneration is complete. In planarians, flatworms with high regenerative potential, muscle cells express some of these instructive cues. Here, we show that members of the integrin family of adhesion molecules are required for the integrity of regenerating tissues, including the musculature. Remarkably, in regenerating ß1-integrin RNAi planarians, we detected increased numbers of mitotic cells and progenitor cell types, as well as a reduced ability of stem cells and lineage-restricted progenitor cells to accumulate at wound sites. These animals also formed ectopic spheroid structures of neural identity in regenerating heads. Interestingly, those polarized assemblies comprised a variety of neural cells and underwent continuous growth. Our study indicates that integrin-mediated cell adhesion is required for the regenerative formation of organized tissues and for restricting neurogenesis during planarian regeneration.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Integrina beta1/fisiologia , Neurogênese/fisiologia , Planárias/fisiologia , Regeneração/fisiologia , Animais , Padronização Corporal , Adesão Celular , Diferenciação Celular , Proliferação de Células , Hibridização In Situ , Neurônios/citologia , Filogenia , Interferência de RNA , Transdução de Sinais , Células-Tronco/citologiaRESUMO
Ergothioneine has emerged as a crucial cytoprotectant in the pathogenic lifestyle of Mycobacterium tuberculosis. Production of this antioxidant from primary metabolites may be regulated by phosphorylation of Thr213 in the active site of the methyltransferase EgtD. The structure of mycobacterial EgtD suggests that this post-translational modification would require a large-scale change in conformation to make the active-site residue accessible to a protein kinase. In this report, we show that, under inâ vitro conditions, EgtD is not a substrate of protein kinase PknD.
Assuntos
Ergotioneína/biossíntese , Metiltransferases/metabolismo , Mycobacterium tuberculosis/enzimologia , Ergotioneína/química , Metiltransferases/química , Modelos Moleculares , Conformação Molecular , Proteínas Quinases/metabolismo , Especificidade por SubstratoRESUMO
Se-benzyl selenoimidazolium salts are characterized by remarkable alkyl-transfer potential under physiological conditions. Structure-activity relationship studies show that selective monoalkylation of primary amines depends on supramolecular interactions between the selenoimidazole leaving group and the target nucleophile. We demonstrate that these reagents can be used for site-selective and nearly quantitative modification of the model protein lysozyme on Lys13, bypassing the higher intrinsic reactivities of Lys1 and Lys33. These observations introduce selenoimidazolium salts as novel class of electrophiles for selective N-alkylation of native proteins.
Assuntos
Imidazóis/química , Indicadores e Reagentes/química , Muramidase/síntese química , Compostos Organosselênicos/química , Alquilação , Substâncias Macromoleculares/química , Modelos Moleculares , Estrutura Molecular , Muramidase/química , Muramidase/metabolismo , SaisRESUMO
Cupin-type cysteine dioxygenases (CDOs) are non-heme iron enzymes that occur in animals, plants, bacteria and in filamentous fungi. In this report, we show that agaricomycetes contain an entirely unrelated type of CDO that emerged by convergent evolution from enzymes involved in the biosynthesis of ergothioneine. The activity of this CDO type is dependent on the ergothioneine precursor N-α-trimethylhistidine. The metabolic link between ergothioneine production and cysteine oxidation suggests that the two processes might be part of the same chemical response in fungi, for example against oxidative stress.
Assuntos
Basidiomycota/enzimologia , Cisteína Dioxigenase/metabolismo , Evolução Molecular , Basidiomycota/classificação , Biocatálise , Estrutura MolecularRESUMO
Sulfoxide synthases are non-heme iron enzymes that participate in the biosynthesis of thiohistidines, such as ergothioneine and ovothiolâ A. The sulfoxide synthase EgtB from Chloracidobacterium thermophilum (CthEgtB) catalyzes oxidative coupling between the side chains of N-α-trimethyl histidine (TMH) and cysteine (Cys) in a reaction that entails complete reduction of molecular oxygen, carbon-sulfur (C-S) and sulfur-oxygen (S-O) bond formation as well as carbon-hydrogen (C-H) bond cleavage. In this report, we show that CthEgtB and other bacterial sulfoxide synthases cannot efficiently accept selenocysteine (SeCys) as a substrate in place of cysteine. In contrast, the sulfoxide synthase from the filamentous fungus Chaetomium thermophilum (CthEgt1) catalyzes C-S and C-Se bond formation at almost equal efficiency. We discuss evidence suggesting that this functional difference between bacterial and fungal sulfoxide synthases emerges from different modes of oxygen activation.
Assuntos
Acidobacteria/enzimologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas Fúngicas/antagonistas & inibidores , Selenocisteína/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Ligação Competitiva , Biocatálise , Domínio Catalítico , Cisteína Dioxigenase/antagonistas & inibidores , Cisteína Dioxigenase/metabolismo , Ergotioneína/química , Ergotioneína/metabolismo , Proteínas Fúngicas/metabolismo , Cinética , Simulação de Dinâmica Molecular , Mycobacteriaceae/enzimologia , Selenocisteína/metabolismoRESUMO
This report describes a modular enzyme-catalyzed cascade reaction that transforms l- or d-α-amino acids to ß-methyl-α-amino acids. In this process an α-amino acid transaminase, an α-keto acid methyltransferase, and a halide methyltransferase cooperate in two orthogonal reaction cycles that mediate product formation and regeneration of the cofactor pyridoxal-5'-phosphate and the co-substrate S-adenosylmethionine. The only stoichiometric reagents consumed in this process are the unprotected l- or d-α-amino acid and methyl iodide.
Assuntos
Aminoácidos/metabolismo , Metiltransferases/metabolismo , Transaminases/metabolismo , Aminoácidos/química , Metilação , Estrutura Molecular , EstereoisomerismoRESUMO
Ergothioneine is an emergent factor in cellular redox biochemistry in humans and pathogenic bacteria. Broad consensus has formed around the idea that ergothioneine protects cells against reactive oxygen species. The recent discovery that anaerobic microorganisms make the same metabolite using oxygen-independent chemistry indicates that ergothioneine also plays physiological roles under anoxic conditions. In this report, we describe the crystal structure of the anaerobic ergothioneine biosynthetic enzyme EanB from green sulfur bacterium Chlorobium limicola. This enzyme catalyzes the oxidative sulfurization of N-α-trimethyl histidine. On the basis of structural and kinetic evidence, we describe the catalytic mechanism of this unusual C-S bond-forming reaction. Significant active-site conservation among distant EanB homologues suggests that the oxidative sulfurization of heterocyclic substrates may occur in a broad range of bacteria.
Assuntos
Biocatálise , Ergotioneína/biossíntese , Sulfurtransferases/química , Domínio Catalítico/genética , Chlorobium/enzimologia , Cristalografia por Raios X , Cinética , Modelos Químicos , Mutagênese Sítio-Dirigida , Sulfurtransferases/genética , Sulfurtransferases/metabolismoRESUMO
Sulfoxide synthases are nonheme iron enzymes that catalyze oxidative carbon-sulfur bond formation between cysteine derivatives and N-α-trimethylhistidine as a key step in the biosynthesis of thiohistidines. The complex catalytic mechanism of this enzyme reaction has emerged as the controversial subject of several biochemical and computational studies. These studies all used the structure of the γ-glutamyl cysteine utilizing sulfoxide synthase, MthEgtB from Mycobacterium thermophilum (EC 1.14.99.50), as a structural basis. To provide an alternative model system, we have solved the crystal structure of CthEgtB from Chloracidobacterium thermophilum (EC 1.14.99.51) that utilizes cysteine as a sulfur donor. This structure reveals a completely different configuration of active site residues that are involved in oxygen binding and activation. Furthermore, comparison of the two EgtB structures enables a classification of all ergothioneine biosynthetic EgtBs into five subtypes, each characterized by unique active-site features. This active site diversity provides an excellent platform to examine the catalytic mechanism of sulfoxide synthases by comparative enzymology, but also raises the question as to why so many different solutions to the same biosynthetic problem have emerged.