Gotowa bibliografia na temat „Desulfidase”

AbstractIn all domains of life, transfer RNAs (tRNAs) contain post-transcriptionally sulfur-modified nucleosides such as 2- and 4-thiouridine. We have previously reported that a recombinant [4Fe-4S] cluster-containing bacterial desulfidase (TudS) from an uncultured bacterium catalyzes the desulfuration of 2- and 4-thiouracil via a [4Fe-5S] cluster intermediate. However, the in vivo function of TudS enzymes has remained unclear and direct evidence for substrate binding to the [4Fe-4S] cluster during catalysis was lacking. Here, we provide kinetic evidence that 4-thiouridine-5’-monophosphate rather than sulfurated tRNA, thiouracil, thiouridine or 4-thiouridine-5’-triphosphate is the preferred substrate of TudS. The occurrence of sulfur- and substrate-bound catalytic intermediates was uncovered from the observed switch of the S = 3/2 spin state of the catalytic [4Fe-4S] cluster to a S = 1/2 spin state upon substrate addition. We show that a putative gene product from Pseudomonas putida KT2440 acts as a TudS desulfidase in vivo and conclude that TudS-like enzymes are widespread desulfidases involved in recycling and detoxifying tRNA-derived 4-thiouridine monophosphate nucleosides for RNA synthesis.

Cysteine is a singularly reactive amino acid; in high concentrations, it can disrupt cytoplasmic metabolism. This phenomenon prompted the view that the cyuPA operon of Escherichia coli serves to detoxify cysteine by degrading it.

ABSTRACT Salmonella enterica has two CyuR-activated enzymes that degrade cysteine, i.e., the aerobic CdsH and an unidentified anaerobic enzyme; Escherichia coli has only the latter. To identify the anaerobic enzyme, transcript profiling was performed for E. coli without cyuR and with overexpressed cyuR. Thirty-seven genes showed at least 5-fold changes in expression, and the cyuPA (formerly yhaOM) operon showed the greatest difference. Homology suggested that CyuP and CyuA represent a cysteine transporter and an iron-sulfur-containing cysteine desulfidase, respectively. E. coli and S. enterica ΔcyuA mutants grown with cysteine generated substantially less sulfide and had lower growth yields. Oxygen affected the CyuR-dependent genes reciprocally; cyuP-lacZ expression was greater anaerobically, whereas cdsH-lacZ expression was greater aerobically. In E. coli and S. enterica, anaerobic cyuP expression required cyuR and cysteine and was induced by l-cysteine, d-cysteine, and a few sulfur-containing compounds. Loss of either CyuA or RidA, both of which contribute to cysteine degradation to pyruvate, increased cyuP-lacZ expression, which suggests that CyuA modulates intracellular cysteine concentrations. Phylogenetic analysis showed that CyuA homologs are present in obligate and facultative anaerobes, confirming an anaerobic function, and in archaeal methanogens and bacterial acetogens, suggesting an ancient origin. Our results show that CyuA is the major anaerobic cysteine-catabolizing enzyme in both E. coli and S. enterica, and it is proposed that anaerobic cysteine catabolism can contribute to coordination of sulfur assimilation and amino acid synthesis. IMPORTANCE Sulfur-containing compounds such as cysteine and sulfide are essential and reactive metabolites. Exogenous sulfur-containing compounds can alter the thiol landscape and intracellular redox reactions and are known to affect several cellular processes, including swarming motility, antibiotic sensitivity, and biofilm formation. Cysteine inhibits several enzymes of amino acid synthesis; therefore, increasing cysteine concentrations could increase the levels of the inhibited enzymes. This inhibition implies that control of intracellular cysteine levels, which is the immediate product of sulfide assimilation, can affect several pathways and coordinate metabolism. For these and other reasons, cysteine and sulfide concentrations must be controlled, and this work shows that cysteine catabolism contributes to this control.

Abstract Background To investigate the efficacy of a PLGA-based nanobody complex in photodynamic therapy (PDT) and NIR-II imaging in A549 tumor hypoxic model. Method IR1048-MZ was firstly synthesized by conjugating a nitro imidazole group to IR1048. IR1048-MZ and Cat were then encapsulated in PLGA-SH solution. Anti-EGFR-Nanobody was also expressed and purified, and finally Anti-EGFR-Nanobody@PLGA-IR1048MZ-Cat (Nb@IC-NPs) nanobody complex was obtained based on the formation of desulfide bond between PLGA-SH and Anti-EGFR-Nanobody. Size distribution and morphology were characterized by TEM and DLS. Spectrum of Nb@IC-NPs towards NTR was measured by UV and fluorescence, while the particle’s selective response was studied using fluorescence. The uptake of Nb@IC-NPs in A549 cells was observed by flow cytometry and CLSM. In the meantime, its’ catalytic ability that decomposes H2O2 both extra-and intra-cellular was observed by fluorescence and CLSM. In vitro photodynamic toxicity of Nb@IC-NPs was examined by MTT, Live/Dead Cell Staining, Flow Cytometry and Apoptosis Assay. Tumor-bearing model was constructed to observe a semi-quantitative fluorescent distribution and the possibility of NIR-II fluorescence/photoacoustic (PA) imaging. Effect of Nb@IC-NPs on enhancing A549 tumor hypoxia and expression profile of HIF-1α was investigated in the presence of NIR. An A549 tumor metastasis model was also constructed to confirm the complex’ potential to destroy primary tumor, inhibit lung metastasis, and prolong mice’ survival. Lastly, impact of Nb@IC-NPs on mice’ main organs and blood indices was observed. Results Nb@IC-NPs was successfully fabricated with good homogeneity. The fluorescent absorbance of Nb@IC-NPs showed a linear relationship with the concentration of NTR, and a higher concentration of NTR corresponded to a stronger photoacoustic signal. In addition, Nb@IC-NPs showed a stable selectivity toward NTR. Our results also suggested a high efficient uptake of Nb@IC-NPs in A549 cells, which was more efficient than IC-NPs and IR1048-MZ alone. In vitro assays confirmed the effects of Nb@IC-NPs on catalytic O2 generation even in hypoxic cells. The cell viability was upregulated with the nanocomplex at the absence of the laser, whereas it was dramatically declined with laser treatment that excited at 980 nm. Nb@IC-NPs achieved tumor hypoxia NIR-II/PA imaging through assisting A549 gathering. When NIR was applied, Nb@IC-NPs can significantly relieve A549 cellular/tumor hypoxia by generating more reactive oxygen species (ROS), which in turn helps lower the expression level of HIF-1α. In summary, Nb@IC-NPs based PDT can efficiently decimate A549 primary tumor, inhibit metastatic lung cancer, and prolong the lifespan of the mice under tolerable dosage. At last, in vivo toxicity tests of the nanocomplex showed its biosafety to the main organs and normal blood indices values. Conclusion Nb@IC-NPs improves tumor hypoxia through catalytic reaction and lowers the expression level of HIF-1α. It achieves tumor PA imaging through intensified NIR-II fluorescence signal that caused by response of the complex to the lesion’s nitroreductase (NTR). Nb@IC-NPs based PDT can efficiently kill A549 primary tumor, inhibit a lung metastasis, as well as prolong mice’ survival cycle.

L’atome de soufre est un élément abondant et indispensable à la vie. Il est présent dans une grande diversité de biomolécules contenant du soufre, tels que certains acides aminés essentiels - comme la cystéine et la méthionine, qui sont à la base de diverses voies métaboliques -, des thionucléosides des ARNs de transfert, et de certains cofacteurs indispensables à de nombreux processus biologiques, comme les centres fer-soufre [Fe-S]. Les centres [Fe-S] sont connus pour leur activité rédox et leur rôle dans des réactions de transferts d’électrons. Ils ont des fonctions cellulaires importantes dans la photosynthèse, la respiration et la régulation de la traduction génétique dans des conditions de stress. Ma thèse a consisté en l’étude de deux familles d’enzymes à centres [4Fe-4S] impliquées dans le métabolisme du soufre : plusieurs thiouridylases d'ARN de transfert (MnmA d’E. coli, ThiI de l’archée Methanococcus maripaludis), catalysant l’insertion d’un soufre dans les uridines d’ARNt, et une ThioUracile DéSulfidase (TudS) catalysant l’abstraction du soufre du thiouracile. En combinant diverses méthodes de caractérisation biochimique (tests d’activité in vitro, mutagénèse dirigée) et biophysiques (spectroscopies UV-visible, RPE, Mössbauer, cristallographie aux rayons X), nous avons pu démontrer la nature chimique et le rôle de cofacteur du centre [4Fe-4S] dans les réactions non rédox catalysées par ces métalloenzymes. L’identification d’un intermédiaire réactionnel [4Fe-5S] in crystallo, dans la structure de l’enzyme TudS, a permis de confirmer une nouvelle fonction des centres [4Fe-4S] dans la catalyse de réactions non-redox, précédemment proposée pour les thiouridylases d’ARN de transfert (TtuA) : le centre [4Fe-4S] étant ligandé par trois acides aminés seulement, le quatrième fer non coordonné jouerait le rôle d’acide Lewis en liant et activant l’atome de soufre du substrat (sulfure exogène ou thiouracile, respectivement) pour catalyser la réaction de thiolation (thiouridylases d’ARNt) ou déthiolation (TudS)
The sulfur atom is an abundant and essential element for life. It is present in a wide variety of sulfur-containing biomolecules, such as certain essential amino acids - the cysteine and the methionine, which are at the center of various metabolic pathways -, transfer RNA thionucleosides, and certain essential cofactors participating in many biological processes, such as iron-sulfur centers [Fe-S]. The [Fe-S] centers are known for their redox activity and their role in electron transfer reactions. They have important cellular functions in photosynthesis, respiration, and regulation of gene translation under stress conditions. My thesis consisted of the study of two families of enzymes with [4Fe-4S] centers involved in sulfur metabolism: several transfer RNA thiouridylases (MnmA from E. coli, ThiI from archaea Methanococcus maripaludis), catalyzing sulfur insertion into tRNA uridines, as well as a ThioUracil DeSulfidase (TudS) catalyzing sulfur abstraction from thiouracil. By combining various biochemical (in vitro activity tests, site-directed mutagenesis) and biophysical (UV-visible spectroscopy, EPR, Mössbauer, X-ray crystallography) characterization methods, we were able to demonstrate the chemical nature and the role of the [4Fe-4S] cluster in the non-redox reactions catalyzed by these metalloenzymes. Identifying a reaction intermediate [4Fe-5S] in crystal, in the structure of the enzyme TudS, has confirmed a new function of the [4Fe-4S] clusters in the catalysis of non-redox reactions, previously proposed for transfer RNA thiouridylases (TtuA): the [4Fe-4S] cluster being liganded by only three amino acids, the fourth uncoordinated iron would play the role of Lewis acid by binding and activating the sulfur atom of the substrate (exogenous sulfide or thiouracil, respectively) to catalyze the reaction of thiolation (tRNA thiouridylases) or dethiolation (TudS)

Les composés soufrés, tels que la cystéine et certains cofacteurs, jouent un rôle essentiel dans les processus cellulaires. Cette thèse se concentre sur deux enzymes dépendantes d’un centre [4Fe-4S], impliquées dans le métabolisme du soufre chez l'archée anaérobie Methanococcus maripaludis : MmCyuA, une L-cystéine désulfidase, et MmLarE, une sulfurtransférase dépendante de l'ATP. La première partie porte sur MmCyuA, qui catalyse la conversion de la L-cystéine en sulfure d’hydrogène et 2-aminoacrylate, ultérieurement transformé en pyruvate et ammoniac. Les structures cristallographiques de MmCyuA, que nous avons obtenues, seule et en présence de l'inhibiteur sérine ou du produit pyruvate, sont les premières structures d'une cystéine désulfidase. Ces structures ainsi que nos résultats biochimiques et analyses spectroscopiques révèlent l’aptitude de MmCyuA à lier un cluster [4Fe-4S], indispensable à l’activité catalytique, via trois ou quatre cystéines. La structure de l'enzyme en complexe avec la sérine mime l'étape initiale de la réaction et suggère un mécanisme de désulfuration de la cystéine impliquant la formation d'un intermédiaire [4Fe-5S]. Des expériences comparatives de croissance de la souche sauvage de M. maripaludis et de la souche mutante dépourvue de l'enzyme MmCyuA soulignent l’importance de MmCyuA pour une croissance cellulaire optimale et pour permettre une croissance où la cystéine est utilisée comme unique source de soufre. Nous proposons que MmCyuA puisse transférer le sulfure lié au cluster à des accepteurs en aval des voies de biosynthèse des composés soufrés, tels que les enzymes de thiolation dépendantes d’un centre [4Fe-4S]. La deuxième partie concerne la structure et le mécanisme de MmLarE. Cette enzyme catalyse la conversion séquentielle des deux groupes carboxylates du précurseur du cofacteur de la lactate racémase en thiocarboxylates. Deux classes d’enzymes LarE existent, qui utilisent un mécanisme sacrificiel où une cystéine sert de source de soufre ou un mécanisme dépendant d’un cluster [4Fe-4S]. Nous rapportons la première structure cristallographique d'une enzyme LarE [4Fe-4S]-dépendante, sous ses formes apo (sans cluster) et holo (avec cluster). La structure de holo-MmLarE montre un cluster [4Fe- 4S], coordonné par trois cystéines seulement, avec le quatrième atome de fer lié à un ligand anionique (chlorure ou groupement phosphate). Ces structures, appuyées par nos études spectroscopiques, nous permettent de proposer un mécanisme dans lequel le cluster [4Fe-4S] lie un hydrogénosulfure, formant un intermédiaire [4Fe-5S]. Ce processus est similaire à celui des enzymes de thiolation de l'ARNt dépendantes d’un cluster [4Fe-4S]
Sulfur-containing compounds, such as cysteine and certain cofactors, play crucial roles in cellular processes. This thesis explores the sulfur metabolism in the anaerobic archaeum Methanococcus maripaludis, focusing on two [4Fe-4S]-dependent enzymes: L-cysteine desulfidase MmCyuA and ATP-dependent sulfur insertase MmLarE. The first part focuses on MmCyuA, which catalyzes the decomposition of L-cysteine into hydrogenosulfide and 2-aminoacrylate, subsequently converted into pyruvate and ammonia. The crystal structures of MmCyuA that we obtained, alone and in the presence of the serine inhibitor or the pyruvate product, are the first structures of a cysteine desulfidase. These structures, together with our biochemical results and spectroscopic analysis, reveal the capacity of MmCyuA to bind a [4Fe-4S] cluster, required for activity, using three or four cysteines. The structure of the enzyme in complex with serine mimics the initial step of the reaction and suggest a desulfuration mechanism for cysteine that involves the formation of a [4Fe-5S] intermediate. Comparative growth experiments between wild-type and CyuAdeficient M. maripaludis strains highlight the important role of MmCyuA for optimal growth and to enables growth using cysteine as the sole sulfur source. We propose that MmCyuA could transfer the cluster-bound sulfide to downstream acceptors, along the biosynthetic pathways of sulfurated compounds, such as [4Fe-4S]-dependent thiolation enzymes. The second part details the structure and mechanism of MmLarE. This enzyme catalyzes the sequential conversion of the two carboxylate groups of the precursor of the lactate racemase cofactor into thiocarboxylates. Two classes of LarE enzymes exist, using a sacrificial mechanism, in which a cysteine serves as the sulfur source, or a [4Fe-4S] cluster-dependent mechanism. We present the first crystal structure of a [4Fe-4S]-dependent LarE enzyme, in both its apo (without cluster) and holo (with cluster) forms. The crystal structure of holo-MmLarE reveals a [4Fe-4S] cluster coordinated by three cysteines only, with the fourth iron atom bound to an anionic ligand (chloride or phosphate group). These structures, along with our spectroscopic studies, support a mechanism in which the [4Fe-4S] cluster binds a hydrogenosulfide ligand, forming a [4Fe-5S]