RESUMEN
Self-cleaving ribozymes are RNA molecules that catalyze a site-specific self-scission reaction. Analysis of self-cleavage is a crucial aspect of the biochemical study and understanding of these molecules. Here we describe a co-transcriptional assay that allows the analysis of self-cleaving ribozymes in different reaction conditions and in the presence of desired ligands and/or cofactors. Utilizing a standard T7 RNA polymerase in vitro transcription system under limiting Mg2+ concentration, followed by a 25-fold dilution of the reaction in desired conditions of self-cleavage (buffer, ions, ligands, pH, temperature, etc.) to halt the synthesis of new RNA molecules, allows the study of self-scission of these molecules without the need for purification or additional preparation steps, such as refolding procedures. Furthermore, because the transcripts are not denatured, this assay likely yields RNAs in conformations relevant to co-transcriptionally folded species in vivo.
Asunto(s)
Proteínas Bacterianas/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Pruebas de Enzimas/métodos , Faecalibacterium prausnitzii/metabolismo , Magnesio/metabolismo , ARN Catalítico/metabolismo , Transcripción Genética , Proteínas Virales/metabolismo , Proteínas Bacterianas/genética , Catálisis , Electroforesis en Gel de Poliacrilamida , Faecalibacterium prausnitzii/enzimología , Faecalibacterium prausnitzii/genética , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Iones/química , Cinética , Ligandos , Magnesio/química , Fosfoglucomutasa/metabolismo , ARN Catalítico/genéticaRESUMEN
Inorganic arsenic is one of the most toxic and carcinogenic substances in the environment, but many organisms, including humans, methylate inorganic arsenic to mono-, di-, and trimethylated arsenic metabolites, which the organism can excrete. In humans and other eukaryotic organisms, the arsenite methyltransferase (AS3MT) protein methylates arsenite. AS3MT sequences from eukaryotic organisms group phylogenetically with predicted eubacterial AS3MT sequences, which has led to the suggestion that AS3MT was acquired from eubacteria by multiple events of horizontal gene transfer. In this study, we evaluated whether 55 (out of which 47 were predicted based on protein sequence similarity) sequences encoding putative AS3MT orthologues in 47 species from different kingdoms can indeed methylate arsenic. Fifty-three of the proteins showed arsenic methylating capacity. For example, the predicted AS3MT of the human gut bacterium Faecalibacterium prausnitzii methylated arsenic efficiently. We performed a kinetic analysis of 14 AS3MT proteins representing two phylogenetically distinct clades (Group 1 and 2) that each contain both eubacterial and eukaryotic sequences. We found that animal and bacterial AS3MTs in Group 1 rarely produce trimethylated arsenic, whereas Hydra vulgaris and the bacterium Rhodopseudomonas palustris in Group 2 produce trimethylated arsenic metabolites. These findings suggest that animals during evolution have acquired different arsenic methylating phenotypes from different bacteria. Further, it shows that humans carry two bacterial systems for arsenic methylation: one bacterium-derived AS3MT from Group 1 incorporated in the human genome and one from Group 2 in F. prausnitzii present in the gut microbiome.
Asunto(s)
Arsénico/metabolismo , Metiltransferasas/metabolismo , Animales , Faecalibacterium prausnitzii/enzimología , Microbioma Gastrointestinal , Humanos , Hydra/enzimología , Metilación , Metiltransferasas/genética , Filogenia , Rhodopseudomonas/enzimologíaRESUMEN
Bacterial ß-glucuronidase (GUS) enzymes cause drug toxicity by reversing Phase II glucuronidation in the gastrointestinal tract. While many human gut microbial GUS enzymes have been examined with model glucuronide substrates like p-nitrophenol-ß-D-glucuronide (pNPG), the GUS orthologs that are most efficient at processing drug-glucuronides remain unclear. Here we present the crystal structures of GUS enzymes from human gut commensals Lactobacillus rhamnosus, Ruminococcus gnavus, and Faecalibacterium prausnitzii that possess an active site loop (Loop 1; L1) analogous to that found in E. coli GUS, which processes drug substrates. We also resolve the structure of the No Loop GUS from Bacteroides dorei. We then compare the pNPG and diclofenac glucuronide processing abilities of a panel of twelve structurally diverse GUS proteins, and find that the new L1 GUS enzymes presented here process small glucuronide substrates inefficiently compared to previously characterized L1 GUS enzymes like E. coli GUS. We further demonstrate that our GUS inhibitors, which are effective against some L1 enzymes, are not potent towards all. Our findings pinpoint active site structural features necessary for the processing of drug-glucuronide substrates and the inhibition of such processing.
Asunto(s)
Microbioma Gastrointestinal/fisiología , Tracto Gastrointestinal/microbiología , Glucuronidasa/antagonistas & inhibidores , Glucuronidasa/metabolismo , Glucurónidos/metabolismo , Bacteroides/enzimología , Dominio Catalítico , Clostridiales/enzimología , Cristalografía por Rayos X , Inhibidores Enzimáticos/farmacología , Faecalibacterium prausnitzii/enzimología , Tracto Gastrointestinal/metabolismo , Humanos , Lacticaseibacillus rhamnosus/enzimología , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Relación Estructura-ActividadRESUMEN
Self-cleaving ribozymes were discovered 30 years ago and have been found throughout nature, from bacteria to animals, but little is known about their biological functions and regulation, particularly how cofactors and metabolites alter their activity. A hepatitis delta virus-like self-cleaving ribozyme maps upstream of a phosphoglucosamine mutase (glmM) open reading frame in the genome of the human gut bacterium Faecalibacterium prausnitzii. The presence of a ribozyme in the untranslated region of glmM suggests a regulation mechanism of gene expression. In the bacterial hexosamine biosynthesis pathway, the enzyme glmM catalyzes the isomerization of glucosamine 6-phosphate into glucosamine 1-phosphate. In this study, we investigated the effect of these metabolites on the co-transcriptional self-cleavage rate of the ribozyme. Our results suggest that glucosamine 6-phosphate, but not glucosamine 1-phosphate, is an allosteric ligand that increases the self-cleavage rate of drz-Fpra-1, providing the first known example of allosteric modulation of a self-cleaving ribozyme by the substrate of the adjacent gene product. Given that the ribozyme is activated by the glmM substrate, but not the product, this allosteric modulation may represent a potential feed-forward mechanism of gene expression regulation in bacteria.