RESUMO
The reaction of benzylsuccinate synthase, the radical-based addition of toluene to a fumarate cosubstrate, is initiated by hydrogen transfer from a conserved cysteine to the nearby glycyl radical in the active center of the enzyme. In this study, we analyze this step by comprehensive computer modeling, predicting (i) the influence of bound substrates or products, (ii) the energy profiles of forward- and backward hydrogen-transfer reactions, (iii) their kinetic constants and potential mechanisms, (iv) enantiospecificity differences, and (v) kinetic isotope effects. Moreover, we support several of the computational predictions experimentally, providing evidence for the predicted H/D-exchange reactions into the product and at the glycyl radical site. Our data indicate that the hydrogen transfer reactions between the active site glycyl and cysteine are principally reversible, but their rates differ strongly depending on their stereochemical orientation, transfer of protium or deuterium, and the presence or absence of substrates or products in the active site. This is particularly evident for the isotope exchange of the remaining protium atom of the glycyl radical to deuterium, which appears dependent on substrate or product binding, explaining why the exchange is observed in some, but not all, glycyl-radical enzymes.
Assuntos
Biocatálise , Cinética , Liases de Carbono-Enxofre/química , Liases de Carbono-Enxofre/metabolismo , Domínio Catalítico , Modelos Moleculares , Cisteína/química , Cisteína/metabolismo , Hidrogênio/química , Radicais Livres/química , Radicais Livres/metabolismo , Carbono-Carbono LiasesRESUMO
The Escherichia coli cysteine desulfurase SufS (EcSufS) is a dimeric, PLP-dependent enzyme responsible for sulfur mobilization in the SUF Fe-S cluster bioassembly pathway. The enzyme uses cysteine as a sulfur source and generates alanine and a covalent persulfide located on an active site of cysteine. Optimal in vitro activity of EcSufS requires the presence of the transpersulfurase protein, EcSufE, and a strong reductant. Here, presteady-state and single-turnover kinetics are used to investigate the mechanism of EcSufS activation by EcSufE. In the absence of EcSufE, EcSufS exhibits a presteady-state burst of product production with an amplitude of â¼0.4 active site equivalents, consistent with a half-sites reactivity. KinTek Explorer was used to isolate the first turnover of alanine formation and fit the data with a simplified kinetic mechanism with steps for alanine formation (k3) and a net rate constant for the downstream steps (k5). Using this treatment, microscopic rate constants of 2.3 ± 0.5 s-1 and 0.10 ± 0.01 s-1 were determined for k3 and k5, respectively. The inclusion of EcSufE in the reaction results in a similar rate constant for k3 but induces a 10-fold enhancement of k5 to 1.1 ± 0.2 s-1, such that both steps are partially rate-determining. The most likely downstream step where EcSufE could exert influence on EcSufS activity is the removal of the persulfide intermediate. Importantly, this step appears to serve as a limiting feature in the half-sites activity such that activating persulfide transfer allows for rapid shifting between active sites. Single-turnover assays show that the presence of EcSufE slightly slowed the rates of alanine-forming steps, suggesting it does not activate steps in the desulfurase half reaction.
Assuntos
Liases de Carbono-Enxofre , Proteínas de Escherichia coli , Escherichia coli , Sulfetos , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Sulfetos/metabolismo , Sulfetos/química , Escherichia coli/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Cinética , Liases de Carbono-Enxofre/metabolismo , Liases de Carbono-Enxofre/química , Alanina/metabolismo , Alanina/química , Domínio Catalítico , Cisteína/metabolismo , Cisteína/química , Proteínas Ferro-Enxofre/metabolismo , Proteínas Ferro-Enxofre/químicaRESUMO
The sulfite-reducing bacterium Bilophila wadsworthia, a common human intestinal pathobiont, is unique in its ability to metabolize a wide variety of sulfonates to generate sulfite as a terminal electron acceptor (TEA). The resulting formation of H2S is implicated in inflammation and colon cancer. l-cysteate, an oxidation product of l-cysteine, is among the sulfonates metabolized by B. wadsworthia, although the enzymes involved remain unknown. Here we report a pathway for l-cysteate dissimilation in B. wadsworthia RZATAU, involving isomerization of l-cysteate to d-cysteate by a cysteate racemase (BwCuyB), followed by cleavage into pyruvate, ammonia and sulfite by a d-cysteate sulfo-lyase (BwCuyA). The strong selectivity of BwCuyA for d-cysteate over l-cysteate was rationalized by protein structural modeling. A homolog of BwCuyA in the marine bacterium Silicibacter pomeroyi (SpCuyA) was previously reported to be a l-cysteate sulfo-lyase, but our experiments confirm that SpCuyA too displays a strong selectivity for d-cysteate. Growth of B. wadsworthia with cysteate as the electron acceptor is accompanied by production of H2S and induction of BwCuyA. Close homologs of BwCuyA and BwCuyB are present in diverse bacteria, including many sulfate- and sulfite-reducing bacteria, suggesting their involvement in cysteate degradation in different biological environments.
Assuntos
Cisteína , Cisteína/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Bilophila/metabolismo , Bilophila/enzimologia , Racemases e Epimerases/metabolismo , Oxirredução , Liases de Carbono-Enxofre/metabolismo , Liases de Carbono-Enxofre/química , Sulfitos/metabolismo , HumanosRESUMO
Limited alliinase resources cause difficulties in the biosynthesis of thiosulfinates (e.g., allicin), restricting their applications in the agricultural and food industries. To effectively biosynthesize thiosulfinates, this study aimed to excavate bacterial alliinase resources and elucidate their catalytic properties. Two bacterial cystathionine ß-lyases (MetCs) possessing high alliinase activity (>60 U mg -1) toward L-(-)-alliin were identified from Allium sativum rhizosphere isolates. Metagenomic exploration revealed that cystathionine ß-lyase from Bacillus cereus (BcPatB) possessed high activity toward both L-(±)-alliin and L-(+)-alliin (208.6 and 225.1 U mg -1), respectively. Although these enzymes all preferred l-cysteine S-conjugate sulfoxides as substrates, BcPatB had a closer phylogenetic relationship with Allium alliinases and shared several similar features with A. sativum alliinase. Interestingly, the Trp30Ile31Ala32Asp33 Met34 motif in a cuspate loop of BcPatB, especially sites 31 and 32 at the top of the motif, was modeled to locate near the sulfoxide of L-(+)-alliin and is important for substrate stereospecificity. Moreover, the stereoselectivity and activity of mutants I31V and A32G were higher toward L-(+)-alliin than those of mutant I31L/D33E toward L-(-)-alliin. Using bacterial alliinases and chemically synthesized substrates, we obtained thiosulfinates with high antimicrobial and antinematode activities that could provide insights into the protection of crops and food.
Assuntos
Proteínas de Bactérias , Alho , Especificidade por Substrato , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Alho/química , Alho/enzimologia , Alho/genética , Ácidos Sulfínicos/química , Ácidos Sulfínicos/metabolismo , Bacillus cereus/enzimologia , Bacillus cereus/genética , Bacillus cereus/metabolismo , Dissulfetos/química , Dissulfetos/metabolismo , Filogenia , Estereoisomerismo , Sequência de Aminoácidos , Bactérias/enzimologia , Bactérias/genética , Bactérias/classificação , Bactérias/metabolismo , Cinética , Liases de Carbono-Enxofre/metabolismo , Liases de Carbono-Enxofre/genética , Liases de Carbono-Enxofre/química , Cisteína/análogos & derivadosRESUMO
The Dph1â¢Dph2 heterodimer from yeast is a radical SAM (RS) enzyme that generates the 3-amino-3-carboxy-propyl (ACP) precursor for diphthamide, a clinically relevant modification on eukaryotic elongation factor 2 (eEF2). ACP formation requires SAM cleavage and atypical Cys-bound Fe-S clusters in each Dph1 and Dph2 subunit. Intriguingly, the first Cys residue in each motif is found next to another ill-defined cysteine that we show is conserved across eukaryotes. As judged from structural modeling, the orientation of these tandem cysteine motifs (TCMs) suggests a candidate Fe-S cluster ligand role. Hence, we generated, by site-directed DPH1 and DPH2 mutagenesis, Dph1â¢Dph2 variants with cysteines from each TCM replaced individually or in combination by serines. Assays diagnostic for diphthamide formation in vivo reveal that while single substitutions in the TCM of Dph2 cause mild defects, double mutations almost entirely inactivate the RS enzyme. Based on enhanced Dph1 and Dph2 subunit instability in response to cycloheximide chases, the variants with Cys substitutions in their cofactor motifs are particularly prone to protein degradation. In sum, we identify a fourth functionally cooperative Cys residue within the Fe-S motif of Dph2 and show that the Cys-based cofactor binding motifs in Dph1 and Dph2 are critical for the structural integrity of the dimeric RS enzyme in vivo.
Assuntos
Motivos de Aminoácidos , Cisteína , Histidina/análogos & derivados , Proteínas Repressoras , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Cisteína/metabolismo , Cisteína/genética , Cisteína/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Multimerização Proteica , Liases de Carbono-Enxofre/metabolismo , Liases de Carbono-Enxofre/química , Liases de Carbono-Enxofre/genética , Mutagênese Sítio-DirigidaRESUMO
Iron-sulfur (Fe-S) clusters are inorganic prosthetic groups in proteins composed exclusively of iron and inorganic sulfide. These cofactors are required in a wide range of critical cellular pathways. Iron-sulfur clusters do not form spontaneously in vivo; several proteins are required to mobilize sulfur and iron, assemble and traffic-nascent clusters. Bacteria have developed several Fe-S assembly systems, such as the ISC, NIF, and SUF systems. Interestingly, in Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), the SUF machinery is the primary Fe-S biogenesis system. This operon is essential for the viability of Mtb under normal growth conditions, and the genes it contains are known to be vulnerable, revealing the Mtb SUF system as an interesting target in the fight against tuberculosis. In the present study, two proteins of the Mtb SUF system were characterized for the first time: Rv1464(sufS) and Rv1465(sufU). The results presented reveal how these two proteins work together and thus provide insights into Fe-S biogenesis/metabolism by this pathogen. Combining biochemistry and structural approaches, we showed that Rv1464 is a type II cysteine-desulfurase enzyme and that Rv1465 is a zinc-dependent protein interacting with Rv1464. Endowed with a sulfurtransferase activity, Rv1465 significantly enhances the cysteine-desulfurase activity of Rv1464 by transferring the sulfur atom from persulfide on Rv1464 to its conserved Cys40 residue. The zinc ion is important for the sulfur transfer reaction between SufS and SufU, and His354 in SufS plays an essential role in this reaction. Finally, we showed that Mtb SufS-SufU is more resistant to oxidative stress than E. coli SufS-SufE and that the presence of zinc in SufU is likely responsible for this improved resistance. This study on Rv1464 and Rv1465 will help guide the design of future anti-tuberculosis agents.
Assuntos
Escherichia coli , Mycobacterium tuberculosis , Escherichia coli/metabolismo , Mycobacterium tuberculosis/metabolismo , Cisteína/metabolismo , Zinco/metabolismo , Liases de Carbono-Enxofre/química , Liases de Carbono-Enxofre/genética , Liases de Carbono-Enxofre/metabolismo , Enxofre/metabolismo , Ferro/metabolismoRESUMO
Purification of L-methionine γ-lyase (MGL) from A. fumigatus was sequentially conducted using heat treatment and gel filtration, resulting in 3.04 of purification fold and 73.9% of enzymatic recovery. The molecular mass of the purified MGL was approximately apparent at 46 KDa based on SDS-PAGE analysis. The enzymatic biochemical properties showed a maximum activity at pH 7 and exhibited plausible stability within pH range 5.0-7.5; meanwhile the highest catalytic activity of MGL was observed at 30-40 °C and the enzymatic stability was noted up to 40 °C. The enzyme molecule was significantly inhibited in the presence of Cu2+, Cd2+, Li2+, Mn2+, Hg2+, sodium azide, iodoacetate, and mercaptoethanol. Moreover, MGL displayed a maximum activity toward the following substrates, L-methionine < DL-methionine < Ethionine < Cysteine. Kinetic studies of MGL for L-methioninase showed catalytic activity at 20.608 mM and 12.34568 µM.min-1. Furthermore, MGL exhibited anticancer activity against cancerous cell lines, where IC50 were 243 ± 4.87 µg/ml (0.486 U/ml), and 726 ± 29.31 µg/ml (1.452 U/ml) against Hep-G2, and HCT116 respectively. In conclusion, A. fumigatus MGL had good catalytic properties along with significantly anticancer activity at low concentration which makes it a probably candidate to apply in the enzymotherapy field.
Assuntos
Aspergillus fumigatus , Liases de Carbono-Enxofre , Aspergillus fumigatus/metabolismo , Cinética , Liases de Carbono-Enxofre/química , Liases de Carbono-Enxofre/metabolismo , MetioninaRESUMO
Frataxin is a kinetic activator of the mitochondrial supercomplex for iron-sulfur cluster assembly. Low frataxin expression or a decrease in its functionality results in Friedreich's Ataxia (FRDA). With the aim of creating new molecular tools to study this metabolic pathway, and ultimately, to explore new therapeutic strategies, we have investigated the possibility of obtaining small proteins exhibiting a high affinity for frataxin. In this study, we applied the ribosome display approach, using human frataxin as the target. We focused on Affi_224, one of the proteins that we were able to select after five rounds of selection. We have studied the interaction between both proteins and discussed some applications of this specific molecular tutor, concerning the modulation of the supercomplex activity. Affi_224 and frataxin showed a KD value in the nanomolar range, as judged by surface plasmon resonance analysis. Most likely, it binds to the frataxin acidic ridge, as suggested by the analysis of chemical shift perturbations (nuclear magnetic resonance) and computational simulations. Affi_224 was able to increase Cys NFS1 desulfurase activation exerted by the FRDA frataxin variant G130V. Importantly, Affi_224 interacts with frataxin in a human cellular model. Our results suggest quaternary addition may be a new tool to modulate frataxin function in vivo. Nevertheless, more functional experiments under physiological conditions should be carried out to evaluate Affi_224 effectiveness in FRDA cell models.
Assuntos
Liases de Carbono-Enxofre , Proteínas de Ligação ao Ferro , Humanos , Proteínas de Ligação ao Ferro/genética , Proteínas de Ligação ao Ferro/química , Proteínas de Ligação ao Ferro/metabolismo , Liases de Carbono-Enxofre/química , Liases de Carbono-Enxofre/metabolismo , FrataxinaRESUMO
The microbial cleavage of dimethylsulfoniopropionate (DMSP) produces volatile dimethyl sulfide (DMS) via the lyase pathway, playing a crucial role in the global sulfur cycle. Herein, the DMSP decomposition catalyzed by PuDddK (a DMSP lyase) devised with various transition metal ion cofactors are investigated using density functional calculations. The PuDddK reaction has been demonstrated to employ a concerted ß-elimination mechanism, where the substrate α-proton abstraction by the deprotonated Tyr64 occurs simultaneously with the Cß-S bond cleavage and Cα = Cß double bond formation. The PuDddK enzymes with diverse divalent metal ions (Ni2+, Mn2+, Fe2+, Co2+, Zn2+, and Cu2+) incorporated prefer DMSP as a monodentate ligand. The cases of Ni2+, Mn2+, Fe2+, Co2+, and Zn2+ with the same 3His-1Glu ligands have close reaction energy barriers, indicating that the lyase activity may be hardly affected by the divalent transition metal type with the same ligand type and number. The coordination loss of one histidine in Cu2+, forming a 2His-1Glu architecture, leads to a lower activity, revealing that the 3His-1Glu ligand set used by DddK appears to be a scaffold capable of more efficiently catalyzing the DMSP decomposition. Further analysis reveals that the inactivation of Fe3+-dependent PuDddK is derived from an electron transfer from the Tyr64 phenolate to Fe3+, with the implication that the PuDddK activity may be primarily affected by the redox effects induced by a strongly oxidizing transition metal ion (like Fe3+).
Assuntos
Histidina , Elementos de Transição , Liases de Carbono-Enxofre/química , Liases de Carbono-Enxofre/metabolismo , Ligantes , Prótons , Compostos de Sulfônio , EnxofreRESUMO
Crohn's disease (CD) is characterized by the chronic inflammation of the gastrointestinal tract. A dysbiotic microbiome and a defective immune system are linked to CD, where hydrogen sulfide (H2 S) microbial producers positively correlate with the severity of the disease. Atopobium parvulum is a key H2 S producer from the microbiome of CD patients. In this study, the biochemical characterization of two Atopobium parvulum cysteine desulfurases, ApSufS and ApCsdB, shows that the enzymes are allosterically regulated. Structural analyses reveal that ApSufS forms a dimer with conserved characteristics observed in type II cysteine desulfurases. Four residues surrounding the active site are essential to catalyse cysteine desulfurylation, and a segment of short-chain residues grant access for substrate binding. A better understanding of ApSufS will help future avenues for CD treatment.
Assuntos
Doença de Crohn , Cisteína , Actinobacteria , Liases de Carbono-Enxofre/química , Cisteína/metabolismo , HumanosRESUMO
l-Methionine γ-lyse (MGL), a pyridoxal 5'-phosphate-dependent enzyme, catalyzes the α,γ-elimination of l-methionine (l-Met) and l-homocysteine (l-Hcy) to produce α-keto acids, thiols, and ammonia. Previously, various mutant enzymes of Pseudomonas putida MGL (PpMGL) were prepared to identify a homocysteine (Hcy)-specific enzyme that would assist the diagnosis of homocystinuria. Among the mutat enzymes the Q349S mutant exhibited high degradation activity toward l-Hcy. In the present study, PpMGL Q349S was characterized; the results suggested that it could be applied to determine the amount of l-Hcy. Compared to the wild-type PpMGL, specific activities of the Q349S mutant with l-Hcy and l-Met were 1.5 and 0.7 times, respectively. Additionally, we confirmed that l-Hcy in plasma samples could be accurately detected using the Q349S mutant by preincubating it with cysteine desulfurase (CsdA). Furthermore, we determined the X-ray crystal structure of PpMGL Q349S l-Met or l-Hcy complexes Michaelis complex, germinal diamine, and external aldimine at 2.25-2.40 Å. These 3D structures showed that the interaction partner of the ß-hydroxyl group of Thr355 in the wild-type PpMGL was changed to the carboxyl group of the Hcy-PLP external aldimine in the Q349S mutant. The interaction of Ser349 and Arg375 was different between l-Met and l-Hcy recognition, indicating that it was important for the recognition of the carboxyl group of the substrate.
Assuntos
Liases de Carbono-Enxofre , Pseudomonas putida , Liases de Carbono-Enxofre/química , Liases de Carbono-Enxofre/genética , Liases de Carbono-Enxofre/metabolismo , Homocisteína , Metionina/metabolismo , Pseudomonas putida/metabolismo , Fosfato de PiridoxalRESUMO
BACKGROUND: L-Methioninase (EC 4.4.1.11; MGL) is a pyridoxal phosphate (PLP)-dependent enzyme that is produced by a variety of bacteria, fungi, and plants. L-methioninase, especially from Pseudomonas and Citrobacter sp., is considered as the efficient therapeutic enzyme, particularly in cancers such as glioblastomas, medulloblastoma, and neuroblastoma that are more sensitive to methionine starvation. OBJECTIVE: The low stability is one of the main drawbacks of the enzyme; in this regard, in the current study, different features of the enzyme, including phylogenetic, functional, and structural from Pseudomonas, Escherichia, Clostridium, and Citrobacter strains were evaluated to find the best bacterial L-Methioninase. METHODS: After the initial screening of L-Methioninase sequences from the above-mentioned bacterial strains, the three-dimensional structures of enzymes from Escherichia fergusonii, Pseudomonas fluorescens, and Clostridium homopropionicum were determined through homology modeling via GalaxyTBM server and refined by GalaxyRefine server. RESULTS AND CONCLUSION: Afterwards, PROCHECK, verify 3D, and ERRAT servers were used for verification of the obtained models. Moreover, antigenicity, allergenicity, and physico-chemical analysis of enzymes were also carried out. In order to get insight into the interaction of the enzyme with other proteins, the STRING server was used. The secondary structure of the enzyme is mainly composed of random coils and alpha-helices. However, these outcomes should further be validated by wet-lab investigations.
Assuntos
Proteínas de Bactérias/genética , Liases de Carbono-Enxofre/genética , Proteínas de Bactérias/química , Liases de Carbono-Enxofre/química , Citrobacter/enzimologia , Citrobacter/genética , Clostridium/enzimologia , Clostridium/genética , Escherichia/enzimologia , Escherichia/genética , Patentes como Assunto , Filogenia , Pseudomonas/enzimologia , Pseudomonas/genéticaRESUMO
Dimethylsulfoniopropionate (DMSP) is an abundant and ubiquitous organosulfur molecule in marine environments with important roles in global sulfur and nutrient cycling. Diverse DMSP lyases in some algae, bacteria, and fungi cleave DMSP to yield gaseous dimethyl sulfide (DMS), an infochemical with important roles in atmospheric chemistry. Here, we identified a novel ATP-dependent DMSP lyase, DddX. DddX belongs to the acyl-CoA synthetase superfamily and is distinct from the eight other known DMSP lyases. DddX catalyses the conversion of DMSP to DMS via a two-step reaction: the ligation of DMSP with CoA to form the intermediate DMSP-CoA, which is then cleaved to DMS and acryloyl-CoA. The novel catalytic mechanism was elucidated by structural and biochemical analyses. DddX is found in several Alphaproteobacteria, Gammaproteobacteria, and Firmicutes, suggesting that this new DMSP lyase may play an overlooked role in DMSP/DMS cycles.
The global sulfur cycle is a collection of geological and biological processes that circulate sulfur-containing compounds through the oceans, rocks and atmosphere. Sulfur itself is essential for life and important for plant growth, hence its widespread use in fertilizers. Marine organisms such as bacteria, algae and phytoplankton produce one particular sulfur compound, called dimethylsulfoniopropionate, or DMSP, in massive amounts. DMSP made in the oceans gets readily converted into a gas called dimethyl sulfide (DMS), which is the largest natural source of sulfur entering the atmosphere. In the air, DMS is converted to sulfate and other by-products that can act as cloud condensation nuclei, which, as the name suggests, are involved in cloud formation. In this way, DMS can influence weather and climate, so it is often referred to as 'climate-active' gas. At least eight enzymes are known to cleave DMSP into DMS gas with a few by-products. These enzymes are found in algae, bacteria and fungi, and are referred to as lyases, for the way they breakdown their target compounds (DMSP, in this case). Recently, researchers have identified some bacteria that produce DMS from DMSP without using known DMSP lyases. This suggests there are other, unidentified enzymes that act on DMSP in nature, and likely contribute to global sulfur cycling. Li, Wang et al. set out to uncover new enzymes responsible for converting the DMSP that marine bacteria produce into gaseous DMS. One new enzyme called DddX was identified and found to belong to a superfamily of enzymes quite separate to other known DMSP lyases. Li, Wang et al. also showed how DddX drives the conversion of DMSP to DMS in a two-step reaction, and that the enzyme is found across several classes of bacteria. Further experiments to characterise the protein structure of DddX also revealed the molecular mechanism for its catalytic action. This study offers important insights into how marine bacteria generate the climatically important gas DMS from DMSP, leading to a better understanding of the global sulfur cycle. It gives microbial ecologists a more comprehensive perspective of these environmental processes, and provides biochemists with data on a family of enzymes not previously known to act on sulfur-containing compounds.
Assuntos
Liases de Carbono-Enxofre/química , Psychrobacter/enzimologia , Compostos de Sulfônio/metabolismo , Acil Coenzima A/metabolismo , Trifosfato de Adenosina , Bactérias/crescimento & desenvolvimento , Bactérias/isolamento & purificação , Proteínas de Bactérias/química , Liases de Carbono-Enxofre/genética , Psychrobacter/genética , Psychrobacter/crescimento & desenvolvimento , Sulfetos/metabolismoRESUMO
The methionine dependence is a well known phenomenon in metabolism of cancer cells. Methionine γ-lyase (EC 4.4.1.11, MGL) catalyzes the γ-elimination reaction of L-methionine and thus could effectively inhibit the growth of malignant cells. Recently we have demonstrated that the mutant form of the enzyme C115H MGL can be used as a component of the pharmacological pair enzyme/S-(allyl/alkyl)-L-cysteine sulfoxides to yield thiosulfinates in situ. Thiosulfinates were shown to be toxic to various cancer cell lines. Therefore the application of the enzyme in enzyme pro-drug therapy may be promising. The conjugates of MGL and C115H MGL with polysialic acid were obtained and their kinetic and pharmacokinetic parameters were determined. The formation of polysialic shell around the enzyme was confirmed by atomic force microscopy. The half-life of conjugated enzymes increased 3-6 times compared to the native enzyme. The cytotoxic effect of conjugated MGL against methionine dependent cancer cell lines was increased two times compared to the values for the native enzymes. The anticancer efficiency of thiosulfinates produced by pharmacological pair C115H MGL/S-(allyl/alkyl)-L-cysteine sulfoxides was demonstrated in vitro. The results indicate that the conjugates of MGL with polysialic acid could be new antitumor drugs.
Assuntos
Antineoplásicos/farmacologia , Liases de Carbono-Enxofre/química , Neoplasias/tratamento farmacológico , Ácidos Siálicos/química , Animais , Antineoplásicos/uso terapêutico , Liases de Carbono-Enxofre/metabolismo , Liases de Carbono-Enxofre/farmacocinética , Liases de Carbono-Enxofre/farmacologia , Linhagem Celular Tumoral , Feminino , Humanos , Cinética , Células MCF-7 , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias/terapia , Ácidos Siálicos/farmacologia , Ácidos Siálicos/uso terapêuticoRESUMO
Methionine-γ-lyase (MGL) is a pyridoxal-5'-phosphate dependent enzyme found in bacteria and protozoa that catalyzes a variety of reactions, including the γ-elimination of L-methionine (L-Met). Here we report experimental kinetic data and density functional theory (DFT) computational data for the γ-elimination reaction of L-Met and several other substrate analogues by a recombinant MGL from P. gingivalis (MGL_Pg). UV-Visible spectrophotometry experiments revealed a heavily populated species with maximum absorbance at 478 nm during steady-state catalysis of L-Met, L-ethionine, L-methionine sulfone and L-homoserine, which we assign to a late crotonate intermediate formed after the γ-cleavage step in the reaction and thus common to all substrates. A more red-shifted (498 nm) species was observed during the reaction of L-homoserine lactone, which we assign to an early quinonoid intermediate with the aid of time-dependent self-consistent field calculations. Significant differences in both binding and the rate of turnover were observed for the substrates. MGL_Pg's highest catalytic efficiency was recorded for L-vinylglycine (kcat/Km = 6455 s-1 M-1), exceeding that of L-Met (kcat/Km = 4211 s-1 M-1), while L-Met sulfone displayed the largest turnover number (kcat = 1638 min-1). A direct correlation between experimental kcat values and DFT-calculated γ-cleavage Gibbs activation energies was identified for the various substrates. In light of these data, we propose that the γ-cleavage step in the catalytic reaction pathway is rate-limiting. This conclusion has direct implications for the rational design of substrates or inhibitors aimed at regulating MGL activity.
Assuntos
Liases de Carbono-Enxofre/metabolismo , Metionina/metabolismo , Liases de Carbono-Enxofre/química , Catálise , Cisteína/metabolismo , Cinética , Metionina/análogos & derivados , Metionina/química , Porphyromonas gingivalis/metabolismo , Espectrofotometria/métodos , Especificidade por SubstratoRESUMO
While two-dimensional infrared (2D-IR) spectroscopy is uniquely suitable for monitoring femtosecond (fs) to picosecond (ps) water dynamics around static protein structures, its utility for probing enzyme active-site dynamics is limited due to the lack of site-specific 2D-IR probes. We demonstrate the genetic incorporation of a novel 2D-IR probe, m-azido-L-tyrosine (N3Y) in the active-site of DddK, an iron-dependent enzyme that catalyzes the conversion of dimethylsulfoniopropionate to dimethylsulphide. Our results show that both the oxidation of active-site iron to FeIII , and the addition of denaturation reagents, result in significant decrease in enzyme activity and active-site water motion confinement. As tyrosine residues play important roles, including as general acids and bases, and electron transfer agents in many key enzymes, the genetically encoded 2D-IR probe N3Y should be broadly applicable to investigate how the enzyme active-site motions at the fs-ps time scale direct reaction pathways to facilitating specific chemical reactions.
Assuntos
Azidas/metabolismo , Liases de Carbono-Enxofre/metabolismo , Compostos Férricos/metabolismo , Tirosina/análogos & derivados , Azidas/química , Liases de Carbono-Enxofre/química , Domínio Catalítico , Compostos Férricos/química , Estrutura Molecular , Espectrofotometria Infravermelho , Tirosina/química , Tirosina/metabolismoRESUMO
Ergothioneine is a histidine-derived sulfur metabolite that is biosynthesized by bacteria and fungi. Plants and animals absorb ergothioneine as a micronutrient from their environment or nutrition. Several different mechanisms of microbial ergothioneine production have been described in the past ten years. Much less is known about the genetic and structural basis for ergothioneine catabolism. In this report, we describe the in vitro reconstitution of a five-step pathway that degrades ergothioneine to l-glutamate, trimethylamine, hydrogen sulfide, carbon dioxide, and ammonia. The first two steps are catalyzed by the two enzymes ergothionase and thiourocanate hydratase. These enzymes are closely related to the first two enzymes in histidine catabolism. However, the crystal structure of thiourocanate hydratase from the firmicute Paenibacillus sp. reveals specific structural features that strictly differentiate the activity of this enzyme from that of urocanate hydratases. The final two steps are catalyzed by metal-dependent hydrolases that share most homology with the last two enzymes in uracil catabolism. The early and late part of this pathway are connected by an entirely new enzyme type that catalyzes desulfurization of a thiohydantoin intermediate. Homologous enzymes are encoded in many soil-dwelling firmicutes and proteobacteria, suggesting that bacterial activity may have a significant impact on the environmental availability of ergothioneine.
Assuntos
Proteínas de Bactérias/metabolismo , Carbono-Nitrogênio Liases/metabolismo , Carbono-Oxigênio Liases/metabolismo , Liases de Carbono-Enxofre/metabolismo , Ergotioneína/metabolismo , Hidrolases/metabolismo , Proteínas de Bactérias/química , Biocatálise , Carbono-Nitrogênio Liases/química , Carbono-Oxigênio Liases/química , Liases de Carbono-Enxofre/química , Hidrolases/química , Paenibacillus/metabolismo , Metabolismo Secundário , Especificidade por SubstratoRESUMO
Body odour is a characteristic trait of Homo sapiens, however its role in human behaviour and evolution is poorly understood. Remarkably, body odour is linked to the presence of a few species of commensal microbes. Herein we discover a bacterial enzyme, limited to odour-forming staphylococci that are able to cleave odourless precursors of thioalcohols, the most pungent components of body odour. We demonstrated using phylogenetics, biochemistry and structural biology that this cysteine-thiol lyase (C-T lyase) is a PLP-dependent enzyme that moved horizontally into a unique monophyletic group of odour-forming staphylococci about 60 million years ago, and has subsequently tailored its enzymatic function to human-derived thioalcohol precursors. Significantly, transfer of this enzyme alone to non-odour producing staphylococci confers odour production, demonstrating that this C-T lyase is both necessary and sufficient for thioalcohol formation. The structure of the C-T lyase compared to that of other related enzymes reveals how the adaptation to thioalcohol precursors has evolved through changes in the binding site to create a constrained hydrophobic pocket that is selective for branched aliphatic thioalcohol ligands. The ancestral acquisition of this enzyme, and the subsequent evolution of the specificity for thioalcohol precursors implies that body odour production in humans is an ancient process.
Assuntos
Álcoois/metabolismo , Corpo Humano , Odorantes/análise , Compostos de Sulfidrila/metabolismo , Álcoois/química , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Teorema de Bayes , Sítios de Ligação , Liases de Carbono-Enxofre/química , Liases de Carbono-Enxofre/metabolismo , Cisteína/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Modelos Moleculares , Filogenia , Staphylococcus/metabolismo , Compostos de Sulfidrila/química , Fatores de TempoRESUMO
The relationships between conformational dynamics, stability and protein function are not obvious. Frataxin (FXN) is an essential protein that forms part of a supercomplex dedicated to the iron-sulfur (Fe-S) cluster assembly within the mitochondrial matrix. In humans, the loss of FXN expression or a decrease in its functionality results in Friedreich's Ataxia, a cardio-neurodegenerative disease. Recently, the way in which FXN interacts with the rest of the subunits of the supercomplex was uncovered. This opens a window to explore relationships between structural dynamics and function. In this study, we prepared a set of FXN variants spanning a broad range of conformational stabilities. Variants S160I, S160M and A204R were more stable than the wild-type and showed similar biological activity. Additionally, we prepared SILCAR, a variant that combines S160I, L203C and A204R mutations. SILCAR was 2.4 kcal mol-1 more stable and equally active. Some of the variants were significantly more resistant to proteolysis than the wild-type FXN. SILCAR showed the highest resistance, suggesting a more rigid structure. It was corroborated by means of molecular dynamics simulations. Relaxation dispersion NMR experiments comparing SILCAR and wild-type variants suggested similar internal motions in the microsecond to millisecond timescale. Instead, variant S157I showed higher denaturation resistance but a significant lower function, similarly to that observed for the FRDA variant N146K. We concluded that the contribution of particular side chains to the conformational stability of FXN might be highly subordinated to their impact on both the protein function and the stability of the functional supercomplex.
Assuntos
Proteínas de Ligação ao Ferro/química , Liases de Carbono-Enxofre/química , Biologia Computacional , Humanos , Proteínas de Ligação ao Ferro/genética , Simulação de Dinâmica Molecular , Mutação Puntual , Conformação Proteica , Engenharia de Proteínas , Estabilidade Proteica , Proteólise , FrataxinaRESUMO
Auxin is a crucial plant growth regulator. Forward genetic screens for auxin-related mutants have led to the identification of key genes involved in auxin biosynthesis, transport, and signaling. Loss-of-function mutations in genes involved in glucosinolate biosynthesis, a metabolically related route that produces defense compounds, result in auxin overproduction. We identified an allelic series of fertile, hypomorphic Arabidopsis (Arabidopsis thaliana) mutants for the essential glucosinolate biosynthetic gene ROOTY (RTY) that exhibit a range of phenotypic defects characteristic of enhanced auxin production. Genetic characterization of these lines uncovered phenotypic suppression by cyp79b2 cyp79b3, wei2, and wei7 mutations and revealed the phenomenon of interallelic complementation in several RTY transheterozygotes. Structural modeling of RTY elucidated the relationships between structure and function in the RTY homo- and heterodimers, and unveiled the likely structural basis of interallelic complementation. This work underscores the importance of employing true null mutants in genetic complementation studies.