RESUMEN
Heme trafficking is essential for cellular function, yet mechanisms of transport and/or heme interaction are not well defined. The System I and System II bacterial cytochrome c biogenesis pathways are developing into model systems for heme trafficking due to their functions in heme transport, heme stereospecific positioning, and mediation of heme attachment to apocytochrome c. Here we focus on the System II pathway, CcsBA, that is proposed to be a bi-functional heme transporter and holocytochrome c synthase. An extensive structure-function analysis of recombinantly expressed Helicobacter pylori and Campylobacter jejuni CcsBAs revealed key residues required for heme interaction and holocytochrome c synthase activity. Homologous residues were previously identified to be required for heme interaction in Helicobacter hepaticus CcsBA. This study provides direct, biochemical evidence that mechanisms of heme interaction are conserved, leading to the proposal that the CcsBA WWD heme-handling domain represents a novel target for therapeutics.
Asunto(s)
Proteínas Bacterianas , Campylobacter jejuni , Helicobacter pylori , Hemo , Hemo/metabolismo , Helicobacter pylori/enzimología , Helicobacter pylori/genética , Helicobacter pylori/metabolismo , Campylobacter jejuni/enzimología , Campylobacter jejuni/genética , Campylobacter jejuni/metabolismo , Relación Estructura-Actividad , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Unión Proteica , Modelos Moleculares , LiasasRESUMEN
Metal-tetrapyrrole cofactors are involved in multiple cellular functions, and chelatases are key enzymes for the biosynthesis of these cofactors. CfbA is an ancestral, homodimeric-type class II chelatase which is able to use not only Ni2+ as a physiological metal substrate, but also Co2+ as a nonphysiological substrate with higher activity than for Ni2+. The Ni/Co-chelatase function found in CfbA is also observed in SirB, a descendant, monomeric-type class II chelatase. This is despite the distinct active site structure of CfbA and SirB; specifically, CfbA shows a unique four His residue arrangement, unlike other monomeric class II chelatases such as SirB. Herein, we studied the Ni-chelatase activity of SirB variants R134H, L200H, and R134H/L200H, the latter of which mimics the His alignment of CfbA. Our results showed that the SirB R134H variant exhibited the highest Ni-chelatase activity among the SirB enzymes, which in turn suggests that the position of His134 could be more important for the Ni-chelatase activity than that of His200. The SirB R134H/L200H variant showed lower activity than R134H, despite the four His residues found in SirB R134H/L200H. CD spectroscopy showed secondary structure denaturation and a slight difficulty in Ni-binding of SirB R134H/L200H, which may be related to its lower activity. Finally, a docking simulation suggested that the His134 of the SirB R134H variant could function as a base catalyst for the Ni-chelatase reaction in a class II chelatase architecture.
Asunto(s)
Níquel , Níquel/metabolismo , Níquel/química , Dominio Catalítico/genética , Histidina/metabolismo , Histidina/química , Histidina/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Modelos Moleculares , Liasas/metabolismo , Liasas/química , Liasas/genéticaRESUMEN
The thiamine diphosphate (ThDP)-binding motif, characterized by the canonical GDG(X)24-27N sequence, is highly conserved among ThDP-dependent enzymes. We investigated a ThDP-dependent lyase (JanthE from Janthinobacterium sp. HH01) with an unusual cysteine (C458) replacing the first glycine of this motif. JanthE exhibits a high substrate promiscuity and accepts long aliphatic α-keto acids as donors. Sterically hindered aromatic aldehydes or non-activated ketones are acceptor substrates, giving access to a variety of secondary and tertiary alcohols as carboligation products. The crystal structure solved at a resolution of 1.9â Å reveals that C458 is not primarily involved in cofactor binding as previously thought for the canonical glycine. Instead, it coordinates methionine 406, thus ensuring the integrity of the active site and the enzyme activity. In addition, we have determined the long-sought genuine tetrahedral intermediates formed with pyruvate and 2-oxobutyrate in the pre-decarboxylation states and deciphered the atomic details for their stabilization in the active site. Collectively, we unravel an unexpected role for the first residue of the ThDP-binding motif and unlock a family of lyases that can perform valuable carboligation reactions.
Asunto(s)
Tiamina Pirofosfato , Tiamina Pirofosfato/metabolismo , Tiamina Pirofosfato/química , Liasas/metabolismo , Liasas/química , Secuencias de Aminoácidos , Dominio Catalítico , Cristalografía por Rayos X , Especificidad por Sustrato , Modelos MolecularesRESUMEN
BACKGROUND: γ-Hexachlorocyclohexane (γ-HCH), an organochlorine insecticide of anthropogenic origin, is a persistent organic pollutant (POP) that causes environmental pollution concerns worldwide. Although many γ-HCH-degrading bacterial strains are available, inoculating them directly into γ-HCH-contaminated soil is ineffective because of the low survival rate of the exogenous bacteria. Another strategy for the bioremediation of γ-HCH involves the use of transgenic plants expressing bacterial enzyme for γ-HCH degradation through phytoremediation. RESULTS: We generated transgenic Arabidopsis thaliana expressing γ-HCH dehydrochlroninase LinA from bacterium Sphingobium japonicum strain UT26. Among the transgenic Arabidopsis T2 lines, we obtained one line (A5) that expressed and accumulated LinA well. The A5-derived T3 plants showed higher tolerance to γ-HCH than the non-transformant control plants, indicating that γ-HCH is toxic for Arabidopsis thaliana and that this effect is relieved by LinA expression. The crude extract of the A5 plants showed γ-HCH degradation activity, and metabolites of γ-HCH produced by the LinA reaction were detected in the assay solution, indicating that the A5 plants accumulated the active LinA protein. In some A5 lines, the whole plant absorbed and degraded more than 99% of γ-HCH (10 ppm) in the liquid medium within 36 h. CONCLUSION: The transgenic Arabidopsis expressing active LinA absorbed and degraded γ-HCH in the liquid medium, indicating the high potential of LinA-expressing transgenic plants for the phytoremediation of environmental γ-HCH. This study marks a crucial step toward the practical use of transgenic plants for the phytoremediation of POPs.
Asunto(s)
Arabidopsis , Biodegradación Ambiental , Hexaclorociclohexano , Plantas Modificadas Genéticamente , Sphingomonadaceae , Arabidopsis/genética , Arabidopsis/metabolismo , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Hexaclorociclohexano/metabolismo , Sphingomonadaceae/genética , Sphingomonadaceae/metabolismo , Sphingomonadaceae/enzimología , Contaminantes del Suelo/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Liasas/genética , Liasas/metabolismoRESUMEN
The WRKY transcription factor (TF) genes form a large family in higher plants, with 72 members in Arabidopsis (Arabidopsis thaliana). The gaseous phytohormone ethylene (ET) regulates multiple physiological processes in plants. It is known that 1-aminocyclopropane-1-carboxylic acid (ACC) synthases (ACSs, EC 4.4.1.14) limit the enzymatic reaction rate of ethylene synthesis. However, whether WRKY TFs regulate the expression of ACSs and/or ACC oxidases (ACOs, EC 1.14.17.4) remains largely elusive. Here, we demonstrated that Arabidopsis WRKY22 positively regulated the expression of a few ACS and ACO genes, thus promoting ethylene production. Inducible overexpression of WRKY22 caused shorter hypocotyls without ACC treatment. A qRT-PCR screening demonstrated that overexpression of WRKY22 activates the expression of several ACS and ACO genes. The promoter regions of ACS5, ACS11, and ACO5 were also activated by WRKY22, which was revealed by a dual luciferase reporter assay. A follow-up chromatin immunoprecipitation coupled with quantitative PCR (ChIP-qPCR) and electrophoretic mobility shift assay (EMSA) showed that the promoter regions of ACS5 and ACO5 could be bound by WRKY22 directly. Moreover, wrky22 mutants had longer primary roots and more lateral roots than wild type, while WRKY22-overexpressing lines showed the opposite phenotype. In conclusion, this study revealed that WRKY22 acts as a novel TF activating, at least, the expression of ACS5 and ACO5 to increase ethylene synthesis and modulate root development.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Etilenos , Regulación de la Expresión Génica de las Plantas , Liasas , Raíces de Plantas , Factores de Transcripción , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Etilenos/metabolismo , Etilenos/biosíntesis , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Raíces de Plantas/genética , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/metabolismo , Liasas/genética , Liasas/metabolismo , Aminoácido Oxidorreductasas/genética , Aminoácido Oxidorreductasas/metabolismo , Regiones Promotoras Genéticas/genética , Liasas de Carbono-Carbono/metabolismo , Liasas de Carbono-Carbono/genética , Activación Transcripcional/genéticaRESUMEN
When subjected to γ-irradiation at cryogenic temperatures the oxygenated complexes of Cytochrome P450 CYP17A1 (CYP17A1) bound with either of the lyase substrates, 17α-Hydroxypregnenolone (17-OH PREG) or 17α-Hydroxyprogesterone (17-OH PROG) are shown to generate the corresponding lyase products, dehydroepiandrosterone (DHEA) and androstenedione (AD) respectively. The current study uses gas chromatography-mass spectrometry (GC/MS) to document the presence of the initial substrates and products in extracts of the processed samples. A rapid and efficient method for the simultaneous determination of residual substrate and products by GC/MS is described without derivatization of the products. It is also shown that no lyase products were detected for similarly treated control samples containing no nanodisc associated CYP17 enzyme, demonstrating that the product is formed during the enzymatic reaction and not by GC/MS conditions, nor the conditions produced by the cryoradiolysis process.
Asunto(s)
Cromatografía de Gases y Espectrometría de Masas , Esteroide 17-alfa-Hidroxilasa , Esteroide 17-alfa-Hidroxilasa/metabolismo , Deshidroepiandrosterona/química , Deshidroepiandrosterona/metabolismo , 17-alfa-Hidroxiprogesterona/química , 17-alfa-Hidroxiprogesterona/metabolismo , 17-alfa-Hidroxipregnenolona/química , 17-alfa-Hidroxipregnenolona/metabolismo , Androstenodiona/química , Androstenodiona/metabolismo , Humanos , Liasas/metabolismo , Liasas/química , Rayos gamma , Especificidad por Sustrato , Oxígeno/químicaRESUMEN
KEY MESSAGE: Identification of selenium stress-responsive expression and molecular docking of serine acetyltransferase (SAT) and O-acetyl serine (thiol) lyase (OASTL) in Cardamine hupingshanensis. A complex coupled with serine acetyltransferase (SAT) and O-acetyl serine (thiol) lyase (OASTL) is the key enzyme that catalyzes selenocysteine (Sec) synthesis in plants. The functions of SAT and OASTL genes were identified in some plants, but it is still unclear whether SAT and OASTL are involved in the selenium metabolic pathway in Cardamine hupingshanensis. In this study, genome-wide identification and comparative analysis of ChSATs and ChOASTLs were performed. The eight ChSAT genes were divided into three branches, and the thirteen ChOASTL genes were divided into four branches by phylogenetic analysis and sequence alignment, indicating the evolutionary conservation of the gene structure and its association with other plant species. qRT-PCR analysis showed that the ChSAT and ChOASTL genes were differentially expressed in different tissues under various selenium levels, suggesting their important roles in Sec synthesis. The ChSAT1;2 and ChOASTLA1;2 were silenced by the VIGS system to investigate their involvement in selenium metabolites in C. hupingshanensis. The findings contribute to understanding the gene functions of ChSATs and ChOASTLs in the selenium stress and provide a reference for further exploration of the selenium metabolic pathway in plants.
Asunto(s)
Cardamine , Regulación de la Expresión Génica de las Plantas , Simulación del Acoplamiento Molecular , Filogenia , Proteínas de Plantas , Selenio , Selenio/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Cardamine/genética , Cardamine/metabolismo , Redes y Vías Metabólicas/genética , Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Liasas/metabolismo , Liasas/genéticaRESUMEN
The ethylene-forming enzyme (EFE) is an Fe(II), 2-oxoglutarate (2OG), and l-arginine (l-Arg)-dependent oxygenase that either forms ethylene and three CO2/bicarbonate from 2OG or couples the decarboxylation of 2OG to C5 hydroxylation of l-Arg. l-Arg binds with C5 toward the metal center, causing 2OG to change from monodentate to chelate metal interaction and OD1 to OD2 switch of D191 metal coordination. We applied anaerobic UV-visible spectroscopy, X-ray crystallography, and computational approaches to three EFE systems with high-resolution structures. The ineffective l-Arg analogue l-canavanine binds to the EFE with O5 pointing away from the metal center while promoting chelate formation by 2OG but fails to switch the D191 metal coordination from OD1 to OD2. Substituting alanine for R171 that interacts with 2OG and l-Arg inactivates the protein, prevents metal chelation by 2OG, and weakens l-Arg binding. The R171A EFE had electron density at the 2OG binding site that was identified by mass spectrometry as benzoic acid. The substitution by alanine of Y306 in the EFE, a residue 12 Å away from the catalytic metal center, generates an interior cavity that leads to multiple local and distal structural changes that reduce l-Arg binding and significantly reduce the enzyme activity. Flexibility analyses revealed correlated and anticorrelated motions in each system, with important distinctions from the wild-type enzyme. In combination, the results are congruent with the currently proposed enzyme mechanism, reinforce the importance of metal coordination by OD2 of D191, and highlight the importance of the second coordination sphere and longer range interactions in promoting EFE activity.
Asunto(s)
Canavanina , Compuestos Ferrosos , Liasas , Compuestos Ferrosos/metabolismo , Sitios de Unión , Alanina , Ácidos Cetoglutáricos/metabolismoRESUMEN
Mitochondrial DNA (mtDNA) plays a key role in mitochondrial and cellular functions. mtDNA is maintained by active DNA turnover and base excision repair (BER). In BER, one of the toxic repair intermediates is 5'-deoxyribose phosphate (5'dRp). Human mitochondrial DNA polymerase γ has weak dRp lyase activities, and another known dRp lyase in the nucleus, human DNA polymerase ß, can also localize to mitochondria in certain cell and tissue types. Nonetheless, whether additional proteins have the ability to remove 5'dRp in mitochondria remains unknown. Our prior work on the AP lyase activity of mitochondrial transcription factor A (TFAM) has prompted us to examine its ability to remove 5'dRp residues in vitro. TFAM is the primary DNA-packaging factor in human mitochondria and interacts with mitochondrial DNA extensively. Our data demonstrate that TFAM has the dRp lyase activity with different DNA substrates. Under single-turnover conditions, TFAM removes 5'dRp residues at a rate comparable to that of DNA polymerase (pol) ß, albeit slower than that of pol λ. Among the three proteins examined, pol λ shows the highest single-turnover rates in dRp lyase reactions. The catalytic effect of TFAM is facilitated by lysine residues of TFAM via Schiff base chemistry, as evidenced by the observation of dRp-lysine adducts in mass spectrometry experiments. The catalytic effect of TFAM observed here is analogous to the AP lyase activity of TFAM reported previously. Together, these results suggest a potential role of TFAM in preventing the accumulation of toxic DNA repair intermediates.
Asunto(s)
ADN Polimerasa beta , Liasas , Liasas de Fósforo-Oxígeno , Humanos , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Liasas/metabolismo , Lisina , ADN Polimerasa beta/metabolismo , Reparación del ADN , ADN Polimerasa gamma/metabolismo , ADN Mitocondrial/metabolismo , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción , Proteínas Mitocondriales/metabolismoRESUMEN
The gaseous hormone ethylene plays pivotal roles in plant growth and development. The rate-limiting enzyme of ethylene biosynthesis in seed plants is 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS). ACS proteins are encoded by a multigene family and the expression of ACS genes is highly regulated, especially at a post-translational level. AtACS7, the only type III ACS in Arabidopsis, is degraded in a 26S proteasome-dependent pathway. Here, by using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis, two lysine residues of AtACS7, lys285 (K285) and lys366 (K366), were revealed to be ubiquitin-modified in young, light-grown Arabidopsis seedlings but not in etiolated seedlings. Deubiquitylation-mimicking mutations of these residues significantly increased the stability of the AtACS7K285RK366R mutant protein in cell-free degradation assays. All results suggest that K285 and K366 are the major ubiquitination sites on AtACS7, providing deeper insights into the post-translational regulation of AtACS7 in Arabidopsis.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Liasas , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Cromatografía Liquida , Etilenos/metabolismo , Regulación de la Expresión Génica de las Plantas , Liasas/metabolismo , Espectrometría de Masas en Tándem , UbiquitinaciónRESUMEN
Many human health problems and property losses caused by pathogenic contamination cannot be underestimated. Bactericidal techniques have been extensively studied to address this issue of public health and economy. Bacterial resistance develops as a result of the extensive use of single or multiple but persistent usage of sterilizing drugs, and the emergence of super-resistant bacteria brings new challenges. Therefore, it is crucial to control pathogen contamination by applying innovative and effective sterilization techniques. As organisms that exist in nature and can specifically kill bacteria, phages have become the focus as an alternative to antibacterial agents. Furthermore, phage-encoded lyases are proteins that play important roles in phage sterilization. The in vitro sterilization of phage lyase has been developed as a novel biosterilization technique to reduce bacterial resistance and is more environmentally friendly than conventional sterilization treatments. For the shortcomings of enzyme applications, this review discusses the enzyme immobilization methods and the application potential of immobilized lyases for sterilization. Although some techniques provide effective solutions, immobilized lyase sterilization technology has been proven to be a more effective innovation for efficient pathogen killing and reducing bacterial resistance. We hope that this review can provide new insights for the development of sterilization techniques.
Asunto(s)
Infecciones Bacterianas , Bacteriófagos , Liasas , Humanos , Liasas/farmacología , Bacterias , Infecciones Bacterianas/microbiología , Esterilización , Antibacterianos/farmacologíaRESUMEN
AIMS: Sphingolipids are involved in the regulation of insulin signaling, which is linked to the development of insulin resistance, leading to diabetes mellitus. We aimed to study whether modulation of sphingolipid levels by GT-11 may regulate insulin signaling in C2C12 myotubes. MAIN METHODS: We investigated the effects of sphingolipid metabolism on Akt phosphorylation and glucose uptake using C2C12 myotubes. Either GT-11, an inhibitor of dihydroceramide desaturase 1 and S1P lyase, or siRNA targeting Sgpl1, the gene encoding the enzyme, was employed to determine the effect of sphingolipid metabolism modulation on insulin signaling. Western blotting and glucose uptake assays were used to evaluate the effect of treatments on insulin signaling. Sphingolipid metabolites were analyzed by high performance liquid chromatography (HPLC). KEY FINDINGS: Treatment with GT-11 resulted in decreased Akt phosphorylation and reduced glucose uptake. Silencing the Sgpl1 gene, which encodes S1P lyase, mimicked these findings, suggesting the potential for regulating insulin signaling through S1P lyase modulation. GT-11 modulated sphingolipid metabolism, inducing the accumulation of sphingolipids. Using PF-543 and ARN14974 to inhibit sphingosine kinases and acid ceramidase, respectively, we identified a significant interplay between sphingosine, S1P lyase, and insulin signaling. Treatment with either exogenous sphingosine or palmitic acid inhibited Akt phosphorylation, and reduced S1P lyase activity. SIGNIFICANCE: Our findings highlight the importance of close relationship between sphingolipid metabolism and insulin signaling in C2C12 myotubes, pointing to its potential therapeutic relevance for diabetes mellitus.
Asunto(s)
Diabetes Mellitus , Liasas , Humanos , Insulina/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Esfingosina/metabolismo , Esfingolípidos/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Glucosa/metabolismo , Liasas/metabolismo , Liasas/farmacología , Diabetes Mellitus/metabolismo , Lisofosfolípidos/metabolismoRESUMEN
Gum arabic (GA) is widely used as an emulsion stabilizer and edible coating and consists of a complex carbohydrate moiety with a rhamnosyl-glucuronate group capping the non-reducing ends. Enzymes that can specifically cleave the glycosidic chains of GA and modify their properties are valuable for structural analysis and industrial application. Cryogenic X-ray crystal structure of GA-specific L-rhamnose-α-1,4-D-glucuronate lyase from Fusarium oxysporum (FoRham1), belonging to the polysaccharide lyase (PL) family 42, has been previously reported. To determine the specific reaction mechanism based on its hydrogen-containing enzyme structure, we performed joint X-ray/neutron crystallography of FoRham1. Large crystals were grown in the presence of L-rhamnose (a reaction product), and neutron and X-ray diffraction datasets were collected at room temperature at 1.80 and 1.25 Å resolutions, respectively. The active site contained L-rhamnose and acetate, the latter being a partial analog of glucuronate. Incomplete H/D exchange between Arg166 and acetate suggested that a strong salt-bridge interaction was maintained. Doubly deuterated His105 and deuterated Tyr150 supported the interaction between Arg166 and the acetate. The unique hydrogen-rich environment functions as a charge neutralizer for glucuronate and stabilizes the oxyanion intermediate. The NE2 atom of His85 was deprotonated and formed a hydrogen bond with the deuterated O1 hydroxy of L-rhamnose, indicating the function of His85 as the base/acid catalyst for bond cleavage via ß-elimination. Asp83 functions as a pivot between the two catalytic histidine residues by bridging them. This His-His-Asp structural motif is conserved in the PL 24, 25, and 42 families.
Asunto(s)
Fusarium , Polisacárido Liasas , Humanos , Acetatos , Cristalografía por Rayos X , Ácido Glucurónico/química , Hidrógeno , Liasas , Polisacárido Liasas/química , Ramnosa/química , Fusarium/enzimologíaRESUMEN
Aromatic ammonia lyases (AALs) and tyrosine/phenylalanine ammonia mutases (TAM/PAM) are 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO)-dependent enzymes. Usually, the MIO moiety is autocatalytically formed from the tripeptide Ala-Ser-Gly (ASG) and acts as an electrophile during the enzymatic reaction. However, the MIO-forming residues (ASG) have some diversity in this enzyme class. In this work, a systematic investigation on the variety of MIO-forming residues was carried out using in-depth sequence analyses. Several protein clusters of AAL-like enzymes with unusual MIO-forming residues such as ACG, TSG, SSG, and CSG were identified, including two novel histidine ammonia lyases and one PAM with CSG and TSG residues, respectively, as well as three novel ergothioneine trimethylammonia lyases without MIO motif. The mutagenesis of common MIO-groups confirmed the function of these MIO variants, which provides good starting points for future functional prediction and mutagenesis research of AALs.
Asunto(s)
Amoníaco-Liasas , Liasas , Amoníaco-Liasas/química , Amoníaco , Histidina Amoníaco-Liasa/químicaRESUMEN
Ethylene-forming enzyme (EFE) is an iron(II)-dependent dioxygenase that fragments 2-oxoglutarate (2OG) to ethylene (from C3 and C4) and 3 equivs of carbon dioxide (from C1, C2, and C5). This major ethylene-forming pathway requires l-arginine as the effector and competes with a minor pathway that merely decarboxylates 2OG to succinate as it oxidatively fragments l-arginine. We previously proposed that ethylene forms in a polar-concerted (Grob-like) fragmentation of a (2-carboxyethyl)carbonatoiron(II) intermediate, formed by the coupling of a C3-C5-derived propion-3-yl radical to a C1-derived carbonate coordinated to the Fe(III) cofactor. Replacement of one or both C4 hydrogens of 2OG by fluorine, methyl, or hydroxyl favored the elimination products 2-(F1-2/Me/OH)-3-hydroxypropionate and CO2 over the expected olefin or carbonyl products, implying strict stereoelectronic requirements in the final step, as is known for Grob fragmentations. Here, we substituted active-site residues expected to interact sterically with the proposed Grob intermediate, aiming to disrupt or enable the antiperiplanar disposition of the carboxylate electrofuge and carbonate nucleofuge required for concerted fragmentation. The bulk-increasing A198L substitution barely affects the first partition between the major and minor pathways but then, as intended, markedly diminishes ethylene production in favor of 3-hydroxypropionate. Conversely, the bulk-diminishing L206V substitution enables propylene formation from (4R)-methyl-2OG, presumably by allowing the otherwise sterically disfavored antiperiplanar conformation of the Grob intermediate bearing the extra methyl group. The results provide additional evidence for a polar-concerted ethylene-yielding step and thus for the proposed radical-polar crossover via substrate-radical coupling to the Fe(III)-coordinated carbonate.
Asunto(s)
Alquenos , Etilenos , Compuestos Férricos , Ácido Láctico/análogos & derivados , Liasas , Etilenos/química , Arginina/metabolismo , Dominio Catalítico , CarbonatosRESUMEN
OBJECTIVES: How gaseous signalling molecules affect ion transport processes contributing to the physiological functions of the gastrointestinal tract under hypoxic conditions still needs to be clarified. The objective of the present study was to characterize the impact of gaseous signalling molecules on parameters of colonic ion transport during a hypoxia/reoxygenation cycle and the remaining secretory capacity of the epithelium after such a cycle. METHODS: Short-circuit current (Isc) and tissue conductance (Gt) recordings in Ussing chamber experiments were performed on rat colon samples using CORM-2 (putative CO donor; 35 and 350⯵M), sodium nitroprusside (NO donor; 100⯵M), NaHS (fast H2S donor; 10 - 1,000⯵M), GYY 4137 (slow H2S donor; 50⯵M) and Angeli's salt (HNO donor; 100⯵M) as donors for gasotransmitters. Inhibition of endogenous synthesis of H2S was operated by inhibitors of cystathionin-γ-lyase, i.e. dl-propargylglycine (1â¯mM) or ß-cyano-l-alanine (5â¯mM), and the inhibitor of cystathionine-ß-synthase, amino-oxyacetate (5â¯mM). RESULTS: The fast gasotransmitter donors NaHS, sodium nitroprusside and Angeli's salt, administered 5â¯min before the onset of hypoxia, induced an increase in Isc. The response to the subsequently applied hypoxia was characterized by a decrease in Isc, which tended to be reduced only in the presence of the lowest concentration of NaHS (10⯵M) tested. Reoxygenation resulted in a slow increase in Isc, which was unaffected by all donors or inhibitors tested. The stable acetylcholine derivative carbachol (50⯵M) was administered at the end of each hypoxia/reoxygenation cycle to test the secretory capacity of the epithelium. Pretreatment of the tissue with the putative CO donor CORM-2 suppressed the secretory response induced by carbachol. The same was observed when cystathionin-γ-lyase and cystathionin-γ-synthase were inhibited simultaneously. Under both conditions, Gt drastically increased suggesting an impaired tissue integrity. CONCLUSIONS: The present results demonstrate that none of the exogenous gasotransmitter releasing drugs significantly ameliorated the changes in epithelial ion transport during the hypoxia/reoxygenation cycle ex vivo. In contrast, the putative CO donor CORM-2 exerted a toxic effect on the epithelium. The endogenous production of H2S, however, seems to have a protective effect on the mucosal integrity and the epithelial transport functions, which - when inhibited - leads to a loss of the secretory ability of the mucosa. This observation together with the trend for improvement observed with a low concentration of the H2S donor NaHS suggests a moderate protective role of low concentrations of H2S under hypoxic conditions.
Asunto(s)
Gasotransmisores , Sulfuro de Hidrógeno , Liasas , Nitritos , Compuestos Organometálicos , Sulfuros , Ratas , Animales , Gasotransmisores/farmacología , Sulfuro de Hidrógeno/farmacología , Nitroprusiato , Carbacol , Hipoxia , Transporte IónicoRESUMEN
Flowering is an essential process in fruit trees. Flower number and timing have a substantial impact on the yield and maturity of fruit. Ethylene and gibberellin (GA) play vital roles in flowering, but the mechanism of coordinated regulation of flowering in woody plants by GA and ethylene is still unclear. In this study, a lemon (Citrus limon L. Burm) 1-aminocyclopropane-1-carboxylic acid synthase gene (CiACS4) was overexpressed in Nicotiana tabacum and resulted in late flowering and increased flower number. Further transformation of citrus revealed that ethylene and starch content increased, and soluble sugar content decreased in 35S:CiACS4 lemon. Inhibition of CiACS4 in lemon resulted in effects opposite to that of 35S:CiACS4 in transgenic plants. Overexpression of the CiACS4-interacting protein ETHYLENE RESPONSE FACTOR3 (CiERF3) in N. tabacum resulted in delayed flowering and more flowers. Further experiments revealed that the CiACS4-CiERF3 complex can bind the promoters of FLOWERING LOCUS T (CiFT) and GOLDEN2-LIKE (CiFE) and suppress their expression. Moreover, overexpression of CiFE in N. tabacum led to early flowering and decreased flowers, and ethylene, starch, and soluble sugar contents were opposite to those in 35S:CiACS4 transgenic plants. Interestingly, CiFE also bound the promoter of CiFT. Additionally, GA3 and 1-aminocyclopropanecarboxylic acid (ACC) treatments delayed flowering in adult citrus, and treatment with GA and ethylene inhibitors increased flower number. ACC treatment also inhibited the expression of CiFT and CiFE. This study provides a theoretical basis for the application of ethylene to regulate flower number and mitigate the impacts of extreme weather on citrus yield due to delayed flowering.
Asunto(s)
Citrus , Etilenos , Flores , Regulación de la Expresión Génica de las Plantas , Giberelinas , Proteínas de Plantas , Plantas Modificadas Genéticamente , Giberelinas/metabolismo , Citrus/genética , Citrus/fisiología , Citrus/crecimiento & desarrollo , Flores/genética , Flores/fisiología , Flores/crecimiento & desarrollo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Etilenos/metabolismo , Nicotiana/genética , Nicotiana/fisiología , Nicotiana/crecimiento & desarrollo , Liasas/metabolismo , Liasas/genéticaRESUMEN
Ethylene is an essential plant hormone, critical in various physiological processes. These processes include seed germination, leaf senescence, fruit ripening, and the plant's response to environmental stressors. Ethylene biosynthesis is tightly regulated by two key enzymes, namely 1-aminocyclopropane-1-carboxylate synthase (ACS) and 1-aminocyclopropane-1-carboxylate oxidase (ACO). Initially, the prevailing hypothesis suggested that ACS is the limiting factor in the ethylene biosynthesis pathway. Nevertheless, accumulating evidence from various studies has demonstrated that ACO, under specific circumstances, acts as the rate-limiting enzyme in ethylene production. Under normal developmental processes, ACS and ACO collaborate to maintain balanced ethylene production, ensuring proper plant growth and physiology. However, under abiotic stress conditions, such as drought, salinity, extreme temperatures, or pathogen attack, the regulation of ethylene biosynthesis becomes critical for plants' survival. This review highlights the structural characteristics and examines the transcriptional, post-transcriptional, and post-translational regulation of ACS and ACO and their role under abiotic stress conditions. Reviews on the role of ethylene signaling in abiotic stress adaptation are available. However, a review delineating the role of ACS and ACO in abiotic stress acclimation is unavailable. Exploring how particular ACS and ACO isoforms contribute to a specific plant's response to various abiotic stresses and understanding how they are regulated can guide the development of focused strategies. These strategies aim to enhance a plant's ability to cope with environmental challenges more effectively.
Asunto(s)
Aminoácido Oxidorreductasas , Liasas , Óxido Nítrico Sintasa , Aminoácido Oxidorreductasas/genética , Ácidos Carboxílicos , Etilenos , Estrés Fisiológico , Fenómenos Fisiológicos de las Plantas/genéticaRESUMEN
Tea (Camellia sinensis) is a highly important beverage crop renowned for its unique flavour and health benefits. Chlorotic mutants of tea, known worldwide for their umami taste and economic value, have gained global popularity. However, the genetic basis of this chlorosis trait remains unclear. In this study, we identified a major-effect quantitative trait locus (QTL), qChl-3, responsible for the chlorosis trait in tea leaves, linked to a non-synonymous polymorphism (G1199A) in the magnesium chelatase I subunit (CsCHLI). Homozygous CsCHLIA plants exhibited an albino phenotype due to defects in magnesium protoporphyrin IX and chlorophylls in the leaves. Biochemical assays revealed that CsCHLI mutations did not affect subcellular localization or interactions with CsCHLIG and CsCHLD. However, combining CsCHLIA with CsCHLIG significantly reduced ATPase activity. RNA-seq analysis tentatively indicated that CsCHLI inhibited photosynthesis and enhanced photoinhibition, which in turn promoted protein degradation and increased the amino acid levels in chlorotic leaves. RT-qPCR and enzyme activity assays confirmed the crucial role of asparagine synthetase and arginase in asparagine and arginine accumulation, with levels increasing over 90-fold in chlorotic leaves. Therefore, this study provides insights into the genetic mechanism underlying tea chlorosis and the relationship between chlorophyll biosynthesis and amino acid metabolism.
Asunto(s)
Anemia Hipocrómica , Camellia sinensis , Liasas , Camellia sinensis/genética , Camellia sinensis/metabolismo , Clorofila/metabolismo , Té/metabolismo , Aminoácidos/metabolismo , Mutación , Anemia Hipocrómica/metabolismo , Hojas de la Planta/genética , Hojas de la Planta/metabolismoRESUMEN
L-ergothioneine is widely distributed among various microbes to regulate their physiology and pathogenicity within complex environments. One of the key steps in the ergothioneine-biosynthesis pathway, the C-S bond cleavage reaction, uses the pyridoxal 5'-phosphate dependent C-S lyase to produce the final product L-ergothioneine. Here, we present the crystallographic structure of the ergothioneine-biosynthesis C-S lyase EgtE from Mycobacterium smegmatis (MsEgtE) represents the first published structure of ergothioneine-biosynthesis C-S lyases in bacteria and shows the effects of active site residues on the enzymatic reaction. The MsEgtE and the previously reported ergothioneine-biosynthesis C-S lyase Egt2 from Neurospora crassa (NcEgt2) fold similarly. However, discrepancies arise in terms of substrate recognition, as observed through sequence and structure comparison of MsEgtE and NcEgt2. The structural-based sequence alignment of the ergothioneine-biosynthesis C-S lyase from fungi and bacteria shows clear distinctions among the recognized substrate residues, but Arg348 is critical and an extremely conserved residue for substrate recognition. The α14 helix is exclusively found in the bacteria EgtE, which represent the most significant difference between bacteria EgtE and fungi Egt2, possibly resulting from the convergent evolution of bacteria and fungi.