RESUMEN
A heterodisulfide reductase-like complex (sHdr) and novel lipoate-binding proteins (LbpAs) are central players of a wide-spread pathway of dissimilatory sulfur oxidation. Bioinformatic analysis demonstrate that the cytoplasmic sHdr-LbpA systems are always accompanied by sets of sulfur transferases (DsrE proteins, TusA, and rhodaneses). The exact composition of these sets may vary depending on the organism and sHdr system type. To enable generalizations, we studied model sulfur oxidizers from distant bacterial phyla, that is, Aquificota and Pseudomonadota. DsrE3C of the chemoorganotrophic Alphaproteobacterium Hyphomicrobium denitrificans and DsrE3B from the Gammaproteobacteria Thioalkalivibrio sp. K90mix, an obligate chemolithotroph, and Thiorhodospira sibirica, an obligate photolithotroph, are homotrimers that donate sulfur to TusA. Additionally, the hyphomicrobial rhodanese-like protein Rhd442 exchanges sulfur with both TusA and DsrE3C. The latter is essential for sulfur oxidation in Hm. denitrificans. TusA from Aquifex aeolicus (AqTusA) interacts physiologically with AqDsrE, AqLbpA, and AqsHdr proteins. This is particularly significant as it establishes a direct link between sulfur transferases and the sHdr-LbpA complex that oxidizes sulfane sulfur to sulfite. In vivo, it is unlikely that there is a strict unidirectional transfer between the sulfur-binding enzymes studied. Rather, the sulfur transferases form a network, each with a pool of bound sulfur. Sulfur flux can then be shifted in one direction or the other depending on metabolic requirements. A single pair of sulfur-binding proteins with a preferred transfer direction, such as a DsrE3-type protein towards TusA, may be sufficient to push sulfur into the sink where it is further metabolized or needed.
Asunto(s)
Proteínas Bacterianas , Oxidación-Reducción , Oxidorreductasas , Azufre , Sulfurtransferasas , Azufre/metabolismo , Sulfurtransferasas/metabolismo , Sulfurtransferasas/química , Sulfurtransferasas/genética , Oxidorreductasas/metabolismo , Oxidorreductasas/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genéticaRESUMEN
Sulfurtransferases transfer of sulfur atoms from thiols to acceptors like cyanide. They are categorized as thiosulfate sulfurtransferases (TSTs) and 3-mercaptopyruvate sulfurtransferases (MSTs). TSTs transfer sulfur from thiosulfate to cyanide, producing thiocyanate. MSTs transfer sulfur from 3-mercaptopyruvate to cyanide, yielding pyruvate and thiocyanate. The present study aimed to isolate and characterize the sulfurtransferase FrST from Frondihabitans sp. PAMC28461 using biochemical and structural analyses. FrST exists as a dimer and can be classified as a TST rather than an MST according to sequence-based clustering and enzyme activity. Furthermore, the discovery of activity over a wide temperature range and the broad substrate specificity exhibited by FrST suggest promising prospects for its utilization in industrial applications, such as the detoxification of cyanide.
Asunto(s)
Cisteína/análogos & derivados , Tiocianatos , Tiosulfatos , Sulfurtransferasas/química , Tiosulfato Azufretransferasa , Ácido Pirúvico , Cianuros , AzufreRESUMEN
Enzymes carrying Iron-Sulfur (Fe-S) clusters perform many important cellular functions and their biogenesis require complex protein machinery. In mitochondria, the IBA57 protein is essential and promotes assembly of [4Fe-4S] clusters and their insertion into acceptor proteins. YgfZ is the bacterial homologue of IBA57 but its precise role in Fe-S cluster metabolism is uncharacterized. YgfZ is needed for activity of the radical S-adenosyl methionine [4Fe-4S] cluster enzyme MiaB which thiomethylates some tRNAs. The growth of cells lacking YgfZ is compromised especially at low temperature. The RimO enzyme is homologous to MiaB and thiomethylates a conserved aspartic acid in ribosomal protein S12. To quantitate thiomethylation by RimO, we developed a bottom-up LC-MS2 analysis of total cell extracts. We show here that the in vivo activity of RimO is very low in the absence of YgfZ and independent of growth temperature. We discuss these results in relation to the hypotheses relating to the role of the auxiliary 4Fe-4S cluster in the Radical SAM enzymes that make Carbon-Sulfur bonds.
Asunto(s)
Proteínas de Escherichia coli , Proteínas Hierro-Azufre , Escherichia coli/metabolismo , Sulfurtransferasas/química , Proteínas Ribosómicas/metabolismo , Azufre/metabolismo , Proteínas Hierro-Azufre/metabolismo , Proteínas de Escherichia coli/metabolismoRESUMEN
Numerous post-transcriptional modifications of transfer RNAs have vital roles in translation. The 2-methylthio-N6-isopentenyladenosine (ms2i6A) modification occurs at position 37 (A37) in transfer RNAs that contain adenine in position 36 of the anticodon, and serves to promote efficient A:U codon-anticodon base-pairing and to prevent unintended base pairing by near cognates, thus enhancing translational fidelity1-4. The ms2i6A modification is installed onto isopentenyladenosine (i6A) by MiaB, a radical S-adenosylmethionine (SAM) methylthiotransferase. As a radical SAM protein, MiaB contains one [Fe4S4]RS cluster used in the reductive cleavage of SAM to form a 5'-deoxyadenosyl 5'-radical, which is responsible for removing the C2 hydrogen of the substrate5. MiaB also contains an auxiliary [Fe4S4]aux cluster, which has been implicated6-9 in sulfur transfer to C2 of i6A37. How this transfer takes place is largely unknown. Here we present several structures of MiaB from Bacteroides uniformis. These structures are consistent with a two-step mechanism, in which one molecule of SAM is first used to methylate a bridging µ-sulfido ion of the auxiliary cluster. In the second step, a second SAM molecule is cleaved to a 5'-deoxyadenosyl 5'-radical, which abstracts the C2 hydrogen of the substrate but only after C2 has undergone rehybridization from sp2 to sp3. This work advances our understanding of how enzymes functionalize inert C-H bonds with sulfur.
Asunto(s)
Bacteroides/enzimología , Metiltransferasas/química , ARN de Transferencia/química , ARN de Transferencia/metabolismo , S-Adenosilmetionina/metabolismo , Compuestos de Sulfhidrilo/metabolismo , Sulfurtransferasas/química , Adenosina/análogos & derivados , Adenosina/metabolismo , Sitios de Unión , Biocatálisis , Isopenteniladenosina/metabolismo , Metiltransferasas/metabolismo , Modelos Moleculares , Dominios Proteicos , ARN/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Especificidad por Sustrato , Sulfurtransferasas/metabolismoRESUMEN
The knowledge on sulfur incorporation mechanism involved in sulfur-containing molecule biosynthesis remains limited. Chuangxinmycin is a sulfur-containing antibiotic with a unique thiopyrano[4,3,2-cd]indole (TPI) skeleton and selective inhibitory activity against bacterial tryptophanyl-tRNA synthetase. Despite the previously reported biosynthetic gene clusters and the recent functional characterization of a P450 enzyme responsible for C-S bond formation, the enzymatic mechanism for sulfur incorporation remains unknown. Here, we resolve this central biosynthetic problem by in vitro biochemical characterization of the key enzymes and reconstitute the TPI skeleton in a one-pot enzymatic reaction. We reveal that the JAMM/MPN+ protein Cxm3 functions as a deubiquitinase-like sulfurtransferase to catalyze a non-classical sulfur-transfer reaction by interacting with the ubiquitin-like sulfur carrier protein Cxm4GG. This finding adds a new mechanism for sulfurtransferase in nature.
Asunto(s)
Antibacterianos/biosíntesis , Proteínas Bacterianas/metabolismo , Sulfurtransferasas/metabolismo , Actinoplanes/genética , Actinoplanes/metabolismo , Antibacterianos/química , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Indoles/análisis , Indoles/química , Indoles/metabolismo , Familia de Multigenes , Pyrococcus/enzimología , Pyrococcus/genética , Azufre/metabolismo , Sulfurtransferasas/química , Sulfurtransferasas/genética , Ubiquitinación , Ubiquitinas/genética , Ubiquitinas/metabolismoRESUMEN
The enzyme 3-mercaptopyruvate sulfurtransferase (3-MST) is one of the more recently identified mammalian sources of H2S. A recent study identified several novel 3-MST inhibitors with micromolar potency. Among those, (2-[(4-hydroxy-6-methylpyrimidin-2-yl)sulfanyl]-1-(naphthalen-1-yl)ethan-1-one) or HMPSNE was found to be the most potent and selective. We now took the central core of this compound and modified the pyrimidone and the arylketone sides independently. A 63-compound library was synthesized; compounds were tested for H2S generation from recombinant 3-MST in vitro. Active compounds were subsequently tested to elucidate their potency and selectivity. Computer modeling studies have delineated some of the key structural features necessary for binding to the 3-MST's active site. Six novel 3-MST inhibitors were tested in cell-based assays: they exerted inhibitory effects in murine MC38 and CT26 colon cancer cell proliferation; the antiproliferative effect of the compound with the highest potency and best cell-based activity (1b) was also confirmed on the growth of MC38 tumors in mice.
Asunto(s)
Neoplasias del Colon/patología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Pirimidinonas/química , Pirimidinonas/farmacología , Sulfurtransferasas/antagonistas & inhibidores , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Dominio Catalítico , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ratones , Sulfurtransferasas/química , Sulfurtransferasas/metabolismoRESUMEN
Lipoyl synthase (LIAS) is an iron-sulfur cluster protein and a member of the radical S-adenosylmethionine (SAM) superfamily that catalyzes the final step of lipoic acid biosynthesis. The enzyme contains two [4Fe-4S] centers (reducing and auxiliary clusters) that promote radical formation and sulfur transfer, respectively. Most information concerning LIAS and its mechanism has been determined from prokaryotic enzymes. Herein, we detail the expression, isolation, and characterization of human LIAS, its reactivity, and evaluation of natural iron-sulfur (Fe-S) cluster reconstitution mechanisms. Cluster donation by a number of possible cluster donor proteins and heterodimeric complexes has been evaluated. [2Fe-2S]-cluster-bound forms of human ISCU and ISCA2 were found capable of reconstituting human LIAS, such that complete product turnover was enabled for LIAS, as monitored via a liquid chromatography-mass spectrometry (LC-MS) assay. Electron paramagnetic resonance (EPR) studies of native LIAS and substituted derivatives that lacked the ability to bind one or the other of LIAS's two [4Fe-4S] clusters revealed a likely order of cluster addition, with the auxiliary cluster preceding the reducing [4Fe-4S] center. These results detail the trafficking of Fe-S clusters in human cells and highlight differences with respect to bacterial LIAS analogs. Likely in vivo Fe-S cluster donors to LIAS are identified, with possible connections to human disease states, and a mechanistic ordering of [4Fe-4S] cluster reconstitution is evident.
Asunto(s)
Proteínas Hierro-Azufre/metabolismo , Sulfurtransferasas/metabolismo , Sustitución de Aminoácidos , Biocatálisis , Espectroscopía de Resonancia por Spin del Electrón , Humanos , Técnicas In Vitro , Hierro/metabolismo , Proteínas Hierro-Azufre/química , Proteínas Hierro-Azufre/genética , Cinética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análisis Espectral , Azufre/metabolismo , Sulfurtransferasas/química , Sulfurtransferasas/genética , Ácido Tióctico/biosíntesisRESUMEN
3-Mercaptopyruvate sulfurtransferase (MPST) catalyzes the desulfuration of 3-mercaptopyruvate to generate an enzyme-bound hydropersulfide. Subsequently, MPST transfers the persulfide's outer sulfur atom to proteins or small molecule acceptors. MPST activity is known to be involved in hydrogen sulfide generation, tRNA thiolation, protein urmylation and cyanide detoxification. Tissue-specific changes in MPST expression correlate with ageing and the development of metabolic disease. Deletion and overexpression experiments suggest that MPST contributes to oxidative stress resistance, mitochondrial respiratory function and the regulation of fatty acid metabolism. However, the role and regulation of MPST in the larger physiological context remain to be understood.
Asunto(s)
Azufre/metabolismo , Sulfurtransferasas/metabolismo , Animales , Cisteína/análogos & derivados , Cisteína/química , Cisteína/metabolismo , Humanos , Estructura Molecular , Azufre/química , Sulfurtransferasas/químicaRESUMEN
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.
Asunto(s)
Ergotioneína/síntesis química , Proteínas Bacterianas/química , Biocatálisis , Marcaje Isotópico , Metiltransferasas/química , Mycobacterium smegmatis/enzimología , Radioisótopos/química , Sulfurtransferasas/químicaRESUMEN
It is estimated that over 1.5 billion people suffer from various forms of chronic liver disease worldwide. The emerging prevalence of metabolic syndromes and alcohol misuse, along with the lack of disease-modifying agents for the therapy of many severe liver conditions predicts that chronic liver disease will continue to be a major problem in the future. Better understanding of the underlying pathogenetic mechanisms and identification of potential therapeutic targets remains a priority. Herein, we explored the potential role of the 3-mercaptopyruvate sulfurtransferase/hydrogen sulfide (H2S) system in the regulation of the endoplasmic reticulum (ER) stress and of its downstream processes in the immortalized hepatic cell line HepG2 in vitro. ER stress suppressed endogenous H2S levels and pharmacological supplementation of H2S with sodium hydrogen sulfide (NaHS) mitigated many aspects of ER stress, culminating in improved cellular bioenergetics and prevention of autophagic arrest, thereby switching cells' fate towards survival. Genetic silencing of 3-MST or pharmacological inhibition of the key enzymes involved in hepatocyte H2S biosynthesis exacerbated many readouts related to ER-stress or its downstream functional responses. Our findings implicate the 3-MST/H2S system in the intracellular network that governs proteostasis and ER-stress adaptability in hepatocytes and reinforce the therapeutic potential of pharmacological H2S supplementation.
Asunto(s)
Estrés del Retículo Endoplásmico , Regulación de la Expresión Génica , Hepatocitos/metabolismo , Sulfuro de Hidrógeno/química , Sulfurtransferasas/química , Autofagia , Linaje de la Célula , Supervivencia Celular , Células Hep G2 , Humanos , Hígado/efectos de los fármacos , Hígado/metabolismo , Mitocondrias/metabolismo , Superóxidos/metabolismo , Tapsigargina/química , Transfección , Respuesta de Proteína DesplegadaRESUMEN
The chemical modification of tRNA bases by sulfur is crucial to tune translation and to optimize protein synthesis. In eukaryotes, the ubiquitin-related modifier 1 (Urm1) pathway is responsible for the synthesis of 2-thiolated wobble uridine (U34 ). During the key step of the modification cascade, the E1-like activating enzyme ubiquitin-like protein activator 4 (Uba4) first adenylates and thiocarboxylates the C-terminus of its substrate Urm1. Subsequently, activated thiocarboxylated Urm1 (Urm1-COSH) can serve as a sulfur donor for specific tRNA thiolases or participate in ubiquitin-like conjugation reactions. Structural and mechanistic details of Uba4 and Urm1 have remained elusive but are key to understand the evolutionary branch point between ubiquitin-like proteins (UBL) and sulfur-relay systems. Here, we report the crystal structures of full-length Uba4 and its heterodimeric complex with its substrate Urm1. We show how the two domains of Uba4 orchestrate recognition, binding, and thiocarboxylation of the C-terminus of Urm1. Finally, we uncover how the catalytic domains of Uba4 communicate efficiently during the reaction cycle and identify a mechanism that enables Uba4 to protect itself against self-conjugation with its own product, namely activated Urm1-COSH.
Asunto(s)
Nucleotidiltransferasas/química , ARN de Transferencia/química , Azufre/química , Sulfurtransferasas/química , Ubiquitinas/química , Humanos , Nucleotidiltransferasas/metabolismo , ARN de Transferencia/metabolismo , Azufre/metabolismo , Sulfurtransferasas/metabolismo , Ubiquitinas/metabolismoRESUMEN
TtuA and TtuB are the sulfurtransferase and sulfur donor proteins, respectively, for biosynthesis of 2-thioribothymidine (s2T) at position 54 of transfer RNA (tRNA), which is responsible for adaptation to high temperature environments in Thermus thermophilus. The enzymatic activity of TtuA requires an iron-sulfur (Fe-S) cluster, by which a sulfur atom supplied by TtuB is transferred to the tRNA substrate. Here, we demonstrate that the Fe-S cluster directly receives sulfur from TtuB through its inherent coordination ability. TtuB forms a [4Fe-4S]-TtuB intermediate, but that sulfur is not immediately released from TtuB. Further desulfurization assays and mutation studies demonstrated that the release of sulfur from the thiocarboxylated C-terminus of TtuB is dependent on adenylation of the substrate tRNA, and the essential residue for TtuB desulfurization was identified. Based on these findings, the molecular mechanism of sulfur transfer from TtuB to Fe-S cluster is proposed.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas Hierro-Azufre/metabolismo , ARN de Transferencia/metabolismo , Sulfurtransferasas/metabolismo , Thermus thermophilus/enzimología , Tiouridina/análogos & derivados , Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Cristalografía por Rayos X , Espectroscopía de Resonancia por Spin del Electrón , Proteínas Hierro-Azufre/química , Proteínas Hierro-Azufre/genética , Modelos Moleculares , Familia de Multigenes , Mutación , Unión Proteica , Conformación Proteica , ARN de Transferencia/genética , Relación Estructura-Actividad , Especificidad por Sustrato , Sulfurtransferasas/química , Sulfurtransferasas/genética , Thermus thermophilus/genética , Tiouridina/metabolismoRESUMEN
3-Mercaptopyruvate sulfur transferase (MPST) catalyzes the desulfuration of 3-mercaptopyruvate (3-MP) and transfers sulfane sulfur from an enzyme-bound persulfide intermediate to thiophilic acceptors such as thioredoxin and cysteine. Hydrogen sulfide (H2S), a signaling molecule implicated in many physiological processes, can be released from the persulfide product of the MPST reaction. Two splice variants of MPST, differing by 20 amino acids at the N terminus, give rise to the cytosolic MPST1 and mitochondrial MPST2 isoforms. Here, we characterized the poorly-studied MPST1 variant and demonstrated that substitutions in its Ser-His-Asp triad, proposed to serve a general acid-base role, minimally affect catalytic activity. We estimated the 3-MP concentration in murine liver, kidney, and brain tissues, finding that it ranges from 0.4 µmol·kg-1 in brain to 1.4 µmol·kg-1 in kidney. We also show that N-acetylcysteine, a widely-used antioxidant, is a poor substrate for MPST and is unlikely to function as a thiophilic acceptor. Thioredoxin exhibits substrate inhibition, increasing the KM for 3-MP â¼15-fold compared with other sulfur acceptors. Kinetic simulations at physiologically-relevant substrate concentrations predicted that the proportion of sulfur transfer to thioredoxin increases â¼3.5-fold as its concentration decreases from 10 to 1 µm, whereas the total MPST reaction rate increases â¼7-fold. The simulations also predicted that cysteine is a quantitatively-significant sulfane sulfur acceptor, revealing MPST's potential to generate low-molecular-weight persulfides. We conclude that the MPST1 and MPST2 isoforms are kinetically indistinguishable and that thioredoxin modulates the MPST-catalyzed reaction in a physiologically-relevant concentration range.
Asunto(s)
Sulfurtransferasas , Tiorredoxinas , Animales , Catálisis , Células HEK293 , Células Hep G2 , Humanos , Isoenzimas/química , Isoenzimas/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Especificidad de Órganos , Sulfurtransferasas/química , Sulfurtransferasas/metabolismo , Tiorredoxinas/química , Tiorredoxinas/metabolismoRESUMEN
Transfer RNA (tRNA) is an adaptor molecule indispensable for assigning amino acids to codons on mRNA during protein synthesis. 2-thiouridine (s2U) derivatives in the anticodons (position 34) of tRNAs for glutamate, glutamine, and lysine are post-transcriptional modifications essential for precise and efficient codon recognition in all organisms. s2U34 is introduced either by (i) bacterial MnmA/eukaryote mitochondrial Mtu1 or (ii) eukaryote cytosolic Ncs6/archaeal NcsA, and the latter enzymes possess iron-sulfur (Fe-S) cluster. Here, we report the identification of novel-type MnmA homologs containing three conserved Cys residues, which could support Fe-S cluster binding and catalysis, in a broad range of bacteria, including thermophiles, Cyanobacteria, Mycobacteria, Actinomyces, Clostridium, and Helicobacter Using EPR spectroscopy, we revealed that Thermus thermophilus MnmA (TtMnmA) contains an oxygen-sensitive [4Fe-4S]-type cluster. Efficient in vitro formation of s2U34 in tRNALys and tRNAGln by holo-TtMnmA occurred only under anaerobic conditions. Mutational analysis of TtMnmA suggested that the Fe-S cluster is coordinated by the three conserved Cys residues (Cys105, Cys108, and Cys200), and is essential for its activity. Evolutionary scenarios for the sulfurtransferases, including the Fe-S cluster containing Ncs6/NcsA s2U thiouridylases and several distantly related sulfurtransferases, are proposed.
Asunto(s)
Anticodón/genética , Proteínas de Escherichia coli/genética , ARN de Transferencia/genética , Sulfurtransferasas/genética , Codón/genética , Cianobacterias/genética , Escherichia coli/genética , Ácido Glutámico/genética , Glutamina/genética , Hierro/metabolismo , Lisina/genética , Mycobacterium/genética , Azufre/metabolismo , Sulfurtransferasas/química , Tiouridina/análogos & derivados , Tiouridina/metabolismoRESUMEN
Cyanide is a toxic compound that is converted to the non-toxic thiocyanate by a rhodanese enzyme. Rhodaneses belong to the family of transferases (sulfurtransferases), which are largely studied. The sulfur donor defines the subfamily of these enzymes as thiosulfate:cyanide sulfurtransferases or rhodaneses (TSTs) or 3-mercaptopyruvate sulfurtransfeases (MSTs). In Mycobacterium tuberculosis, the causative agent of tuberculosis, the gene Rv0815c encodes the protein CysA2, a putative uncharacterized thiosulfate:cyanide sulfurtransferase that belongs to the essential sulfur assimilation pathway in the bacillus and is secreted during infection. In this work, we characterized the functional and structural properties of CysA2 and its kinetic parameters. The recombinant CysA2 is a α/ß protein with two rhodanese-like domains that maintains the functional motifs and a catalytic cysteine. Sulfurtransferase activity was determined using thiosulfate and 3-mercaptopyruvate as sulfur donors. The assays showed Km values of 2.89 mM and 7.02 mM for thiosulfate and 3-mercaptopyruvate, respectively, indicating the protein has dual activity as TST and MST. Immunological assays revealed that CysA2 interacted with pulmonary cells, and it was capable to activate macrophages and dendritic cells, indicating the stimulation of the immune response, which is important for its use as an antigen for vaccine development and immunodiagnostic.
Asunto(s)
Cisteína/análogos & derivados , Mycobacterium tuberculosis/enzimología , Sulfurtransferasas/química , Sulfurtransferasas/metabolismo , Tiosulfatos/metabolismo , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/metabolismo , Sitios de Unión , Línea Celular , Cisteína/química , Cisteína/metabolismo , Células Dendríticas/citología , Células Dendríticas/inmunología , Cinética , Macrófagos/citología , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Mycobacterium tuberculosis/genética , Unión Proteica , Conformación Proteica , Especificidad por Sustrato , Sulfurtransferasas/genética , Sulfurtransferasas/inmunologíaRESUMEN
Sulfur is an essential element for the growth and development of plants, which synthesize cysteine and methionine from the reductive assimilation of sulfate. Besides its incorporation into proteins, cysteine is the building block for the biosynthesis of numerous sulfur-containing molecules and cofactors. The required sulfur atoms are extracted either directly from cysteine by cysteine desulfurases or indirectly after its catabolic transformation to 3-mercaptopyruvate, a substrate for sulfurtransferases (STRs). Both enzymes are transiently persulfidated in their reaction cycle, i.e. the abstracted sulfur atom is bound to a reactive cysteine residue in the form of a persulfide group. Trans-persulfidation reactions occur when sulfur atoms are transferred to nucleophilic acceptors such as glutathione, proteins, or small metabolites. STRs form a ubiquitous, multigenic protein family. They are characterized by the presence of at least one rhodanese homology domain (Rhd), which usually contains the catalytic, persulfidated cysteine. In this review, we focus on Arabidopsis STRs, presenting the sequence characteristics of all family members as well as their biochemical and structural features. The physiological functions of particular STRs in the biosynthesis of molybdenum cofactor, thio-modification of cytosolic tRNAs, arsenate tolerance, cysteine catabolism, and hydrogen sulfide formation are also discussed.
Asunto(s)
Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Plantas/enzimología , Azufre/metabolismo , Sulfurtransferasas/química , Sulfurtransferasas/metabolismo , Proteínas de Plantas/genética , Plantas/química , Plantas/genética , Plantas/metabolismo , Dominios Proteicos , Transporte de Proteínas , Sulfurtransferasas/genéticaRESUMEN
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.
Asunto(s)
Biocatálisis , Ergotioneína/biosíntesis , Sulfurtransferasas/química , Dominio Catalítico/genética , Chlorobium/enzimología , Cristalografía por Rayos X , Cinética , Modelos Químicos , Mutagénesis Sitio-Dirigida , Sulfurtransferasas/genética , Sulfurtransferasas/metabolismoRESUMEN
The molybdenum cofactor, composed of molybdopterin and molybdenum, is a necessary compound for the catalytic activity of molybdenum enzymes. Molybdenum cofactor biosynthesis is a conserved multi-step process involving several enzymes. Molybdopterin synthase, a hetero-tetrameric enzyme composed of a pair of MoaE-MoaD subunits, catalyzes the generation of the cis-dithiolene group of molybdopterin in the second step of the process. The cis-dithiolene group can covalently bind molybdenum. Most mycobacterial species possess several genes encoding the full pathway of molybdenum cofactor biosynthesis. In M. smegmatis, the moaD2 and moaE2 genes encode the functional molybdopterin synthase. However, M. tuberculosis has genes encoding several molybdopterin synthase subunit homologs, including moaD1, moaD2, moaE1, moaE2, and moaX, which encodes a MoaD-MoaE fusion protein. Previous studies have shown that moaD2 and moaE2 encode functional molybdopterin synthase. Here, we report the crystal structures of two substrate-free molybdopterin synthases from two different mycobacterial pathogens, M. tuberculosis and M. smegmatis, at 2.1â¯Å and 2.6â¯Å resolutions, respectively. The overall structure of both molybdopterin synthases was hetero-tetrameric, consisting of a MoaE2 dimer flanked on either side by single MoaD2 subunits. The carboxyl-terminal domain of MoaD2 inserted into MoaE2, forming the active pocket. A comparison with previously reported molybdopterin synthase structures showed that substrate-binding and catalytic residues were conserved, despite low sequence similarity among these enzymes. The low sequence identity at the MoaE-MoaD heterodimer interface may provide the structural basis to explore mycobacterial inhibitors.
Asunto(s)
Mycobacterium smegmatis/enzimología , Mycobacterium tuberculosis/enzimología , Sulfurtransferasas/química , Secuencia de Aminoácidos , Dominio Catalítico , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Infecciones por Mycobacterium no Tuberculosas/microbiología , Mycobacterium smegmatis/química , Mycobacterium tuberculosis/química , Conformación Proteica , Alineación de Secuencia , Tuberculosis/microbiologíaRESUMEN
The synthesis, absolute stereochemical configuration, complete biological characterization, mechanism of action and resistance, and pharmacokinetic properties of ( S)-(-)-acidomycin are described. Acidomycin possesses promising antitubercular activity against a series of contemporary drug susceptible and drug-resistant M. tuberculosis strains (minimum inhibitory concentrations (MICs) = 0.096-6.2 µM) but is inactive against nontuberculosis mycobacteria and Gram-positive and Gram-negative pathogens (MICs > 1000 µM). Complementation studies with biotin biosynthetic pathway intermediates and subsequent biochemical studies confirmed acidomycin inhibits biotin synthesis with a Ki of approximately 1 µM through the competitive inhibition of biotin synthase (BioB) and also stimulates unproductive cleavage of S-adenosyl-l-methionine (SAM) to generate the toxic metabolite 5'-deoxyadenosine. Cell studies demonstrate acidomycin selectively accumulates in M. tuberculosis providing a mechanistic basis for the observed antibacterial activity. The development of spontaneous resistance by M. tuberculosis to acidomycin was difficult, and only low-level resistance to acidomycin was observed by overexpression of BioB. Collectively, the results provide a foundation to advance acidomycin and highlight BioB as a promising target.
Asunto(s)
Antituberculosos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/enzimología , Sulfurtransferasas/antagonistas & inhibidores , Tiazolidinas/farmacología , Tuberculosis/microbiología , Animales , Antituberculosos/síntesis química , Antituberculosos/química , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Productos Biológicos/síntesis química , Productos Biológicos/química , Productos Biológicos/farmacología , Biotina/biosíntesis , Caproatos/síntesis química , Caproatos/química , Caproatos/farmacología , Farmacorresistencia Bacteriana , Humanos , Cinética , Ratones , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/genética , Sulfurtransferasas/química , Sulfurtransferasas/genética , Sulfurtransferasas/metabolismo , Tiazolidinas/síntesis química , Tiazolidinas/química , Tuberculosis/tratamiento farmacológicoRESUMEN
Biotin (vitamin B7) is an enzyme cofactor required by organisms from all branches of life but synthesized only in microbes and plants. In the final step of biotin biosynthesis, a radical S-adenosyl-l-methionine (SAM) enzyme, biotin synthase (BioB), converts the substrate dethiobiotin to biotin through the stepwise formation of two C-S bonds. Previous electron paramagnetic resonance (EPR) spectroscopic studies identified a semistable intermediate in the formation of the first C-S bond as 9-mercaptodethiobiotin linked to a paramagnetic [2Fe-2S] cluster through one of its bridging sulfides. Herein, we report orientation-selected pulse EPR spectroscopic results that reveal hyperfine interactions between the [2Fe-2S] cluster and a number of magnetic nuclei (e.g., 57Fe, 15N, 13C, and 2H) introduced in a site-specific manner via biosynthetic methods. Combining these results with quantum chemical modeling gives a structural model of the intermediate showing that C6, the target of the second hydrogen-atom abstraction, is now in close proximity to the nascent thioether sulfur and is ideally positioned for the second C-S bond forming event.