RESUMO
Hydrazine (N2H4) and hypochlorite (ClO-) are both reactive chemical substances extensively utilized across various industrial domains. Excessive hydrazine (N2H4) and hypochlorite (ClO-) can pose significant risks to the environment, ecosystems, and human health. In order to assess and control the environmental hazard caused by N2H4 and ClO-, there is an imperative need for efficient methods capable of rapid and precise detection of these contaminants. This paper introduces a novel dual-responsive colorimetric/fluorescent probe (MDT) for the detection of N2H4 and ClO- in environmental and biological samples. The probe exhibits turn-on fluorescent responses to N2H4 or ClO- with low detection limits (N2H4: 8 nM; ClO-: 15 nM), large Stokes shifts (N2H4: 175 nm; ClO-: 203 nm), short response time (N2H4: 4 min; ClO-: 5 s) and broad pH range (5-10). In practical applications, MDT has been successfully employed in detecting N2H4 and ClO- in water and soil samples from diverse locations. Test strips loaded with MDT offer a visual and convenient means to track N2H4 vapor and quantify N2H4 and ClO- concentrations in solutions. Finally, MDT has been utilized for sensing N2H4 and ClO- in Arabidopsis thaliana roots and living zebrafish. This study presents a promising tool for monitoring N2H4 and ClO- in the environment and living organisms.
Assuntos
Colorimetria , Corantes Fluorescentes , Humanos , Animais , Corantes Fluorescentes/química , Colorimetria/métodos , Ácido Hipocloroso , Peixe-Zebra , Ecossistema , Espectrometria de Fluorescência/métodos , HidrazinasRESUMO
Plants can contain biosynthetic gene clusters (BGCs) that nominally resemble those found in microbes. However, while horizontal gene transmission is often observed in microbes, plants are limited to vertical gene transmission, implying that their BGCs may exhibit distinct inheritance patterns. Rice (Oryza sativa) contains two unlinked BGCs involved in diterpenoid phytoalexin metabolism, with one clearly required for momilactone biosynthesis, while the other is associated with production of phytocassanes. Here, in the process of elucidating momilactone biosynthesis, genetic evidence was found demonstrating a role for a cytochrome P450 (CYP) from the other "phytocassane" BGC. This CYP76M8 acts after the CYP99A2/3 from the "momilactone" BGC, producing a hemiacetal intermediate that is oxidized to the eponymous lactone by a short-chain alcohol dehydrogenase also from this BGC. Thus, the "momilactone" BGC is not only incomplete, but also fractured by the need for CYP76M8 to act in between steps catalyzed by enzymes from this BGC. Moreover, as supported by similar activity observed with orthologs from the momilactone-producing wild-rice species Oryza punctata, the presence of CYP76M8 in the other "phytocassane" BGC indicates interdependent evolution of these two BGCs, highlighting the distinct nature of BGC assembly in plants.
Assuntos
Evolução Biológica , Vias Biossintéticas/genética , Diterpenos/metabolismo , Família Multigênica , Oryza/genética , Diterpenos/química , Regulação da Expressão Gênica de Plantas , Oxirredução , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMO
Andrographis paniculata (Burm.f.) Wall. ex Nees is widely used as medicinal herb in Southern and Southeastern Asia and andrographolide is its main medicinal constituent. Based on the structure of andrographolide, it has been proposed that cytochrome P450 enzymes play vital roles on its biosynthesis. NADPH:cytochrome P450 reductase (CPR) is the most important redox partner of multiple P450s. In this study, three CPRs were identified in the genomic data of A. paniculata (namely ApCPR1, ApCPR2, and ApCPR3), and their coding regions were cloned. They varied from 62% to 70% identities to each other at the amino acid sequence level. ApCPR1 belongs to Class I of dicotyledonous CPR while both ApCPR2 and ApCPR3 are grouped to Class II. The recombinant enzymes ApCPR1 and ApCPR2 reduced cytochrome c and ferricyanide in an NADPH-dependent manner. In yeast, they supported the activity of CYP76AH1, a ferruginol-forming enzyme. However, ApCPR3 did not show any enzymatic activities either in vitro or in vivo. Quantitative real-time PCR analysis showed that both ApCPR1 and ApCPR2 expressed in all tissues examined, but ApCPR2 showed higher expression in leaves. Expression of ApCPR2 was inducible by MeJA and its pattern matched with andrographolide accumulation. Present investigation suggested ApCPR2 involves in the biosynthesis of secondary metabolites including andrographolide.
Assuntos
Andrographis/enzimologia , NADPH-Ferri-Hemoproteína Redutase/genética , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Sequência de Aminoácidos , Andrographis/genética , Andrographis/metabolismo , Biocatálise , Clonagem Molecular , Diterpenos/metabolismo , Genômica , NADPH-Ferri-Hemoproteína Redutase/químicaRESUMO
Electrochemical H2 generation from H2O has been focused on the exploration of non-noble metals as well as earth-rich catalysts. In our practical work, we provide a simple cost-efficient fabrication process to prepare large Mo sheets via the controlled equilibrium between sublimation of MoO3 and reduction of H2. Porous MoP sheets were synthesized from the obtained Mo sheets as the Mo source and template which exhibit notable activity in the hydrogen evolution reaction with a low onset potential of -88 mV vs RHE, small Tafel value of 54.5 mV/dec, and strong catalytic stability. With Mo sheets as the universal Mo source and template, MoS2 and Mo2C sheets were synthesized by a similar process, and the corresponding catalytic activities were calculated by density functional theory.
RESUMO
Rice (Oryza sativa) produces a variety of labdane-related diterpenoids as phytoalexins and allelochemicals. The production of these important natural products has been partially elucidated. However, the oxidases responsible for production of the keto groups found in many of these diterpenoids have largely remained unknown. Only one short-chain alcohol dehydrogenase/reductases (SDRs), which has been proposed to catalyze the last step in such a pathway, has been characterized to date. While rice contains >220 SDRs, only the transcription of five has been shown to be induced by the fungal cell wall elicitor chitin. This includes the momilactone A synthase (OsMAS/SDR110C-MS1), with the other four all falling in the same SDR110C family, further suggesting roles in diterpenoid biosynthesis. Here, biochemical characterization with simplified substrate analogs was first used to indicate potential functions, which were then supported by further analyses with key biosynthetic intermediates. Kinetic studies were then employed to further clarify these roles. Surprisingly, OsSDR110C-MS2 more efficiently catalyzes the final oxidation to produce momilactone A that was previously assigned to OsMAS/SDR110C-MS1, and we speculate that this latter SDR may have an alternative function instead. Conversely, two of these SDRs clearly appear to act in oryzalexin biosynthesis, with OsSDR110C-MI3 readily oxidizing the 3α-hydroxyl of oryzalexin D, while OsSDR110C-MS3 can also oxidize the accompanying 7ß-hydroxyl. Together, these SDRs then serve to produce oryzalexins A-C from oryzalexin D, essentially completing elucidation of the biosynthesis of this family of rice phytoalexins.
Assuntos
Álcool Desidrogenase/metabolismo , Oryza/enzimologia , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Álcool Desidrogenase/genética , Diterpenos/metabolismo , Oryza/genética , Feromônios/metabolismo , Proteínas de Plantas/genética , Sesquiterpenos/metabolismo , FitoalexinasRESUMO
The substrate specificity of enzymes from natural products' metabolism is a topic of considerable interest, with potential biotechnological use implicit in the discovery of promiscuous enzymes. However, such studies are often limited by the availability of substrates and authentic standards for identification of the resulting products. Here, a modular metabolic engineering system is used in a combinatorial biosynthetic approach toward alleviating this restriction. In particular, for studies of the multiply reactive cytochrome P450, ent-kaurene oxidase (KO), which is involved in production of the diterpenoid plant hormone gibberellin. Many, but not all, plants make a variety of related diterpenes, whose structural similarity to ent-kaurene makes them potential substrates for KO. Use of combinatorial biosynthesis enabled analysis of more than 20 such potential substrates, as well as structural characterization of 12 resulting unknown products, providing some insight into the underlying structure-function relationships. These results highlight the utility of this approach for investigating the substrate specificity of enzymes from complex natural products' biosynthesis.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Giberelinas/biossíntese , Sondas Moleculares , Filogenia , Plantas/enzimologia , Plantas/metabolismo , Especificidade por SubstratoRESUMO
The oxygenation reactions catalyzed by cytochromes P450 (CYPs) play critical roles in plant natural products biosynthesis. At the same time, CYPs are one of most challenging enzymes to functionally characterize due to the difficulty of recombinantly expressing these membrane-associated monooxygenases. In the course of investigating rice diterpenoid biosynthesis, we have developed a synthetic biology approach for functional expression of relevant CYPs in Escherichia coli. In certain cases, activity was observed for only one of two closely related paralogs although it seems clear that related reactions are required for production of the known diterpenoids. Here, we report that optimization of the recombinant expression system enabled characterization of not only these previously recalcitrant CYPs, but also discovery of additional activity relevant to rice diterpenoid biosynthesis. Of particular interest, CYP701A8 was found to catalyze 3ß-hydroxylation of syn-pimaradiene, which is presumably relevant to momilactone biosynthesis, while CYP71Z6 & 7 were found to catalyze multiple reactions, with CYP71Z6 catalyzing the production of 2α,3α-dihydroxy-ent-isokaurene via 2α-hydroxy-ent-isokaurene, and CYP71Z7 catalyzing the production of 3α-hydroxy-ent-cassadien-2-one via 2α-hydroxy-ent-cassadiene and ent-cassadien-2-one, which may be relevant to oryzadione and phytocassane biosynthesis, respectively.
Assuntos
Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Diterpenos/metabolismo , Oryza/enzimologia , Escherichia coli/genética , Escherichia coli/metabolismo , Oryza/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Natural products biosynthesis often requires the action of multiple CYPs (cytochromes P450), whose ability to introduce oxygen, increasing solubility, is critical for imparting biological activity. In previous investigations of rice diterpenoid biosynthesis, we characterized CYPs that catalyse alternative hydroxylation of ent-sandaracopimaradiene, the precursor to the rice oryzalexin antibiotic phytoalexins. In particular, CYP76M5, CYP76M6 and CYP76M8 were all shown to carry out C-7ß hydroxylation, whereas CYP701A8 catalyses C-3α hydroxylation, with oxy groups found at both positions in oryzalexins A-D, suggesting that these may act consecutively in oryzalexin biosynthesis. In the present paper, we report that, although CYP701A8 only poorly reacts with 7ß-hydroxy-ent-sandaracopimaradiene, CYP76M6 and CYP76M8 readily react with 3α-hydroxy-ent-sandaracopimaradiene. Notably, their activity yields distinct products, resulting from hydroxylation at C-9ß by CYP76M6 or C-7ß by CYP76M8, on different sides of the core tricyclic ring structure. Thus CYP76M6 and CYP76M8 have distinct non-redundant roles in orzyalexin biosynthesis. Moreover, the resulting 3α,7ß- and 3α,9ß-diols correspond to oryzalexins D and E respectively. Accordingly, the results of the present study complete the functional identification of the biosynthetic pathway underlying the production of these bioactive phytoalexins. In addition, the altered regiochemistry catalysed by CYP76M6 following C-3α hydroxylation has some implications for its active-site configuration, offering further molecular insight.
Assuntos
Antibacterianos/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Diterpenos/metabolismo , Oryza/enzimologia , Proteínas de Plantas/metabolismo , Antibacterianos/química , Ligação Competitiva , Domínio Catalítico , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/genética , Diterpenos/química , Retículo Endoplasmático/enzimologia , Retículo Endoplasmático/metabolismo , Ligação de Hidrogênio , Hidroxilação , Isoenzimas/metabolismo , Cinética , Oryza/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Plastídeos/enzimologia , Plastídeos/metabolismo , Conformação Proteica , Transporte Proteico , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Sesquiterpenos/química , Sesquiterpenos/metabolismo , Estereoisomerismo , FitoalexinasRESUMO
Two of the most agriculturally important cereal crop plants are wheat (Triticum aestivum) and rice (Oryza sativa). Rice has been shown to produce a number of diterpenoid natural products as phytoalexins and/or allelochemicals--specifically, labdane-related diterpenoids, whose biosynthesis proceeds via formation of an eponymous labdadienyl/copalyl diphosphate (CPP) intermediate (e.g., the ent-CPP of gibberellin phytohormone biosynthesis). Similar to rice, wheat encodes a number of CPP synthases (CPS), and the three CPS characterized to date (TaCPS1-3) all have been suggested to produce ent-CPP. However, several of the downstream diterpene synthases will only react with CPP intermediate of normal or syn, but not ent, stereochemistry, as described in the accompanying report. Investigation of additional CPS did not resolve this issue, as the only other functional synthase (TaCPS4) also produced ent-CPP. Chiral product characterization of all the TaCPS then established that TaCPS2 uniquely produces normal, rather than ent-, CPP, thus, providing a suitable substrate source for the downstream diterpene synthases. Notably, TaCPS2 is most homologous to the similarly stereochemically differentiated syn-CPP synthase from rice (OsCPS4), while the non-inducible TaCPS3 and TaCPS4 cluster with the rice OsCPS1 required for gibberellin phytohormone biosynthesis, as well as with a barley (Hordeum vulgare) CPS (HvCPS1) that also is characterized here as similarly producing ent-CPP. These results suggest that diversification of labdane-related diterpenoid metabolism beyond the ancestral gibberellins occurred early in cereal evolution, and included the type of stereochemical variation demonstrated here.
Assuntos
Alquil e Aril Transferases/metabolismo , Diterpenos/metabolismo , Grão Comestível/metabolismo , Proteínas de Plantas/metabolismo , Triticum/enzimologia , Algoritmos , Alquil e Aril Transferases/genética , Sequência de Aminoácidos , Biologia Computacional , Diterpenos/química , Filogenia , Proteínas de Plantas/genética , Proteínas Recombinantes/metabolismo , Alinhamento de SequênciaRESUMO
Recent reports have revealed genomic clustering of enzymatic genes for particular biosynthetic pathways in plant specialized/secondary metabolism. Rice (Oryza sativa) carries two such clusters for production of antimicrobial diterpenoid phytoalexins, with the cluster on chromosome 2 containing four closely related/homologous members of the cytochrome P450 CYP76M subfamily (CYP76M5-8). Notably, the underlying evolutionary expansion of these CYP appears to have occurred after assembly of the ancestral biosynthetic gene cluster, suggesting separate roles. It has been demonstrated that CYP76M7 catalyzes C11α-hydroxylation of ent-cassadiene, and presumably mediates an early step in biosynthesis of the derived phytocassane class of phytoalexins. Here we report biochemical characterization of CYP76M5, -6, and -8. Our results indicate that CYP76M8 is a multifunctional/promiscuous hydroxylase, with CYP76M5 and -7 seeming to provide only redundant activity, while CYP76M6 seems to provide both redundant and novel activity, relative to CYP76M8. RNAi-mediated double knockdown of CYP76M7 and -8 suppresses elicitor inducible phytocassane production, indicating a role for these monooxygenases in phytocassane biosynthesis. In addition, our data suggests that CYP76M5, -6, and -8 may play redundant roles in production of the oryzalexin class of phytoalexins as well. Intriguingly, the preceding diterpene synthase for oryzalexin biosynthesis, unlike that for the phytocassanes, is not found in the chromosome 2 diterpenoid biosynthetic gene cluster. Accordingly, our results not only uncover a complex evolutionary history, but also further suggest some intriguing differences between plant biosynthetic gene clusters and the seemingly similar microbial operons. The implications for the underlying metabolic evolution of plants are then discussed.
Assuntos
Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Família Multigênica/genética , Oryza/enzimologia , Oryza/genética , Vias Biossintéticas/genética , Cromossomos de Plantas/genética , Diterpenos/metabolismo , Metabolismo Energético/genética , Evolução Molecular , Regulação da Expressão Gênica de Plantas/fisiologia , Técnicas de Silenciamento de Genes , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sesquiterpenos/metabolismo , FitoalexinasRESUMO
All higher plants contain an ent-kaurene oxidase (KO), as such a cytochrome P450 (CYP) 701 family member is required for gibberellin (GA) phytohormone biosynthesis. While gene expansion and functional diversification of GA-biosynthesis-derived diterpene synthases into more specialized metabolism has been demonstrated, no functionally divergent KO/CYP701 homologs have been previously identified. Rice (Oryza sativa) contains five CYP701A subfamily members in its genome, despite the fact that only one (OsKO2/CYP701A6) is required for GA biosynthesis. Here we demonstrate that one of the other rice CYP701A subfamily members, OsKOL4/CYP701A8, does not catalyze the prototypical conversion of the ent-kaurene C4α-methyl to a carboxylic acid, but instead carries out hydroxylation at the nearby C3α position in a number of related diterpenes. In particular, under conditions where OsKO2 catalyzes the expected conversion of ent-kaurene to ent-kaurenoic acid required for GA biosynthesis, OsKOL4 instead efficiently reacts with ent-sandaracopimaradiene and ent-cassadiene to produce the corresponding C3α-hydroxylated diterpenoids. These compounds are expected intermediates in biosynthesis of the oryzalexin and phytocassane families of rice antifungal phytoalexins, respectively, and can be detected in rice plants under the appropriate conditions. Thus, it appears that OsKOL4 plays a role in the more specialized diterpenoid metabolism of rice, and our results provide evidence for divergence of a KO/CYP701 family member from GA biosynthesis. This further expands the range of enzymes recruited from the ancestral GA primary pathway to the more complex and specialized labdane-related diterpenoid metabolic network found in rice.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Diterpenos/metabolismo , Genoma de Planta , Oryza/enzimologia , Proteínas de Plantas/metabolismo , Sequência de Aminoácidos , Sistema Enzimático do Citocromo P-450/genética , DNA Complementar/genética , DNA Complementar/metabolismo , Ativação Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Giberelinas/metabolismo , Hidroxilação , Oryza/genética , Oryza/metabolismo , Filogenia , Proteínas de Plantas/genética , Recombinação Genética , Sesquiterpenos/metabolismo , FitoalexinasRESUMO
Rice (Oryza sativa) contains a biosynthetic gene cluster associated with production of at least two groups of diterpenoid phytoalexins, the antifungal phytocassanes and antibacterial oryzalides. While cytochromes P450 (CYP) from this cluster are known to be involved in phytocassane production, such mono-oxygenase activity relevant to oryzalide biosynthesis was unknown. Here we report biochemical characterization demonstrating that CYP71Z6 from this cluster acts as an ent-isokaurene C2-hydroxylase that is presumably involved in the biosynthesis of oryzalides. Our results further suggest that the closely related and co-clustered CYP71Z7 likely acts as a C2-hydroxylase involved in a latter step of phytocassane biosynthesis. Thus, CYP71Z6 & 7 appear to have evolved distinct roles in rice diterpenoid metabolism, offering insight into plant biosynthetic gene cluster evolution.
Assuntos
Vias Biossintéticas/genética , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Família Multigênica , Oryza/genética , Oryza/metabolismo , Antibacterianos/biossíntese , Biocatálise , Diterpenos/metabolismo , Duplicação Gênica , Hidroxilação , Oryza/enzimologiaRESUMO
The Arabidopsis (Arabidopsis thaliana) genome encodes nine Salt Overly Sensitive3 (SOS3)-like calcium-binding proteins (SCaBPs; also named calcineurin B-like protein [CBL]) and 24 SOS2-like protein kinases (PKSs; also named as CBL-interacting protein kinases [CIPKs]). A general regulatory mechanism between these two families is that SCaBP calcium sensors activate PKS kinases by interacting with their FISL motif. In this study, we demonstrated that phosphorylation of SCaBPs by their functional interacting PKSs is another common regulatory mechanism. The phosphorylation site serine-216 at the C terminus of SCaBP1 by PKS24 was identified by liquid chromatography-quadrupole mass spectrometry analysis. This serine residue is conserved within the PFPF motif at the C terminus of SCaBP proteins. Phosphorylation of this site of SCaBP8 by SOS2 has been determined previously. We further showed that CIPK23/PKS17 phosphorylated CBL1/SCaBP5 and CBL9/SCaBP7 and PKS5 phosphorylated SCaBP1 at the same site in vitro and in vivo. Furthermore, the phosphorylation stabilized the interaction between SCaBP and PKS proteins. This tight interaction neutralized the inhibitory effect of PKS5 on plasma membrane H(+)-ATPase activity. These data indicate that SCaBP phosphorylation by their interacting PKS kinases is a critical component of the SCaBP-PKS regulatory pathway in Arabidopsis.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/química , Sequência Conservada/genética , Dados de Sequência Molecular , Fosforilação , Ligação Proteica , ATPases Translocadoras de Prótons/metabolismo , Saccharomyces cerevisiae/metabolismo , Serina/metabolismoRESUMO
The Salt Overly Sensitive (SOS) pathway plays an important role in the regulation of Na+/K+ ion homeostasis and salt tolerance in Arabidopsis thaliana. Previously, we reported that the calcium binding proteins SOS3 and SOS3-LIKE CALCIUM BINDING PROTEIN8 (SCaBP8) nonredundantly activate the protein kinase SOS2. Here, we show that SOS2 phosphorylates SCaBP8 at its C terminus but does not phosphorylate SOS3. In vitro, SOS2 phosphorylation of SCaBP8 was enhanced by the bimolecular interaction of SOS2 and SCaBP8 and did not require calcium ions. In vivo, this phosphorylation was induced by salt stress, occurred at the membrane, stabilized the SCaBP8-SOS2 interaction, and enhanced plasma membrane Na+/H+ exchange activity. When a Ser at position 237 in the SCaBP8 protein (the SOS2 phosphorylation target) was mutated to Ala, SCaBP8 was no longer phosphorylated by SOS2 and the mutant protein could not fully rescue the salt-sensitive phenotype of the scabp8 mutant. By contrast, when Ser-237 was mutated to Asp to mimic the charge of a phosphorylated Ser residue, the mutant protein rescued the scabp8 salt sensitivity. These data demonstrate that calcium sensor phosphorylation is a critical component of SOS pathway regulation of salt tolerance in Arabidopsis.