RESUMEN
Isoprenoids are a class of natural products with more than 55,000 members. All isoprenoids are constructed from two precursors, isopentenyl diphosphate and its isomer dimethylallyl diphosphate. Two of the most important discoveries in isoprenoid biosynthetic studies in recent years are the elucidation of a second isoprenoid biosynthetic pathway [the methylerythritol phosphate (MEP) pathway] and a modified mevalonic acid (MVA) pathway. In this review, we summarize mechanistic insights on the MEP pathway enzymes. Because many isoprenoids have important biological activities, the need to produce them in sufficient quantities for downstream research efforts or commercial application is apparent. Recent advances in both MVA and MEP pathway-based synthetic biology are also illustrated by reviewing the landmark work of artemisinic acid and taxadien-5α-ol production through microbial fermentations.
Asunto(s)
Vías Biosintéticas/fisiología , Eritritol/metabolismo , Hemiterpenos/biosíntesis , Terpenos/metabolismo , Catálisis , Humanos , Compuestos OrganofosforadosRESUMEN
Folates are indispensable for plant development, but their molecular mode of action remains elusive. We synthesized a probe, "5-F-THF-Dayne," comprising 5-formyl-tetrahydrofolate (THF) coupled to a photoaffinity tag. Exploiting this probe in an affinity proteomics study in Arabidopsis thaliana, we retrieved 51 hits. Thirty interactions were independently validated with in vitro expressed proteins to bind 5-F-THF with high or low affinity. Interestingly, the interactors reveal associations beyond one-carbon metabolism, covering also connections to nitrogen (N) metabolism, carbohydrate metabolism/photosynthesis, and proteostasis. Two of the interactions, one with the folate biosynthetic enzyme DIHYDROFOLATE REDUCTASE-THYMIDYLATE SYNTHASE 1 (AtDHFR-TS1) and another with N metabolism-associated glutamine synthetase 1;4 (AtGLN1;4), were further characterized. In silico and experimental analyses revealed G35/K36 and E330 as key residues for the binding of 5-F-THF in AtDHFR-TS1 and AtGLN1;4, respectively. Site-directed mutagenesis of AtGLN1;4 E330, which co-localizes with the ATP-binding pocket, abolished 5-F-THF binding as well as AtGLN1;4 activity. Furthermore, 5-F-THF was noted to competitively inhibit the activities of AtDHFR-TS1 and AtGLN1;4. In summary, we demonstrated a regulatory role for 5-F-THF in N metabolism, revealed 5-F-THF-mediated feedback regulation of folate biosynthesis, and identified a total of 14 previously unknown high-affinity binding cellular targets of 5-F-THF. Together, this sets a landmark toward understanding the role of folates in plant development.
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Arabidopsis/metabolismo , Carbono/metabolismo , Ácido Fólico/biosíntesis , Leucovorina/metabolismo , Nitrógeno/metabolismo , Proteoma/metabolismo , Proteínas de Plantas/metabolismoRESUMEN
The methylerythritol phosphate (MEP) pathway is responsible for producing isoprenoids, metabolites with essential functions in the bacterial kingdom and plastid-bearing organisms including plants and Apicomplexa. Additionally, the MEP-pathway intermediate methylerythritol cyclodiphosphate (MEcPP) serves as a plastid-to-nucleus retrograde signal. A suppressor screen of the high MEcPP accumulating mutant plant (ceh1) led to the isolation of 3 revertants (designated Rceh1-3) resulting from independent intragenic substitutions of conserved amino acids in the penultimate MEP-pathway enzyme, hydroxymethylbutenyl diphosphate synthase (HDS). The revertants accumulate varying MEcPP levels, lower than that of ceh1, and exhibit partial or full recovery of MEcPP-mediated phenotypes, including stunted growth and induced expression of stress response genes and the corresponding metabolites. Structural modeling of HDS and ligand docking spatially position the substituted residues at the MEcPP binding pocket and cofactor binding domain of the enzyme. Complementation assays confirm the role of these residues in suppressing the ceh1 mutant phenotypes, albeit to different degrees. In vitro enzyme assays of wild type and HDS variants exhibit differential activities and reveal an unanticipated mismatch between enzyme kinetics and the in vivo MEcPP levels in the corresponding Rceh lines. Additional analyses attribute the mismatch, in part, to the abundance of the first and rate-limiting MEP-pathway enzyme, DXS, and further suggest MEcPP as a rheostat for abundance of the upstream enzyme instrumental in fine-tuning of the pathway flux. Collectively, this study identifies critical residues of a key MEP-pathway enzyme, HDS, valuable for synthetic engineering of isoprenoids, and as potential targets for rational design of antiinfective drugs.
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Sustitución de Aminoácidos , Proteínas de Arabidopsis/genética , Arabidopsis/metabolismo , Enzimas/genética , Oxidorreductasas/genética , Terpenos/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Vías Biosintéticas , Núcleo Celular/metabolismo , Enzimas/metabolismo , Eritritol/análogos & derivados , Eritritol/metabolismo , Ligandos , Simulación del Acoplamiento Molecular , Oxidorreductasas/metabolismo , Plantas Modificadas Genéticamente , Plastidios/genética , Plastidios/metabolismoRESUMEN
BACKGROUND: The anticancer drug camptothecin (CPT), first isolated from Camptotheca acuminata, was subsequently discovered in unrelated plants, including Ophiorrhiza pumila. Unlike known monoterpene indole alkaloids, CPT in C. acuminata is biosynthesized via the key intermediate strictosidinic acid, but how O. pumila synthesizes CPT has not been determined. RESULTS: In this study, we used nontargeted metabolite profiling to show that 3α-(S)-strictosidine and 3-(S), 21-(S)-strictosidinic acid coexist in O. pumila. After identifying the enzymes OpLAMT, OpSLS, and OpSTR as participants in CPT biosynthesis, we compared these enzymes to their homologues from two other representative CPT-producing plants, C. acuminata and Nothapodytes nimmoniana, to elucidate their phylogenetic relationship. Finally, using labelled intermediates to resolve the CPT biosynthesis pathway in O. pumila, we showed that 3α-(S)-strictosidine, not 3-(S), 21-(S)-strictosidinic acid, is the exclusive intermediate in CPT biosynthesis. CONCLUSIONS: In our study, we found that O. pumila, another representative CPT-producing plant, exhibits metabolite diversity in its central intermediates consisting of both 3-(S), 21-(S)-strictosidinic acid and 3α-(S)-strictosidine and utilizes 3α-(S)-strictosidine as the exclusive intermediate in the CPT biosynthetic pathway, which differs from C. acuminata. Our results show that enzymes likely to be involved in CPT biosynthesis in O. pumila, C. acuminata, and N. nimmoniana have evolved divergently. Overall, our new data regarding CPT biosynthesis in O. pumila suggest evolutionary divergence in CPT-producing plants. These results shed new light on CPT biosynthesis and pave the way towards its industrial production through enzymatic or metabolic engineering approaches.
Asunto(s)
Vías Biosintéticas , Evolución Biológica , Camptotecina , Humanos , Magnoliopsida , FilogeniaRESUMEN
Nitrate is one of the major inorganic nitrogen sources for microbes. Many bacterial and archaeal lineages have the capacity to express assimilatory nitrate reductase (NAS), which catalyzes the rate-limiting reduction of nitrate to nitrite. Although a nitrate assimilatory pathway in mycobacteria has been proposed and validated physiologically and genetically, the putative NAS enzyme has yet to be identified. Here, we report the characterization of a novel NAS encoded by Mycolicibacterium smegmatis Msmeg_4206, designated NasN, which differs from the canonical NASs in its structure, electron transfer mechanism, enzymatic properties, and phylogenetic distribution. Using sequence analysis and biochemical characterization, we found that NasN is an NADPH-dependent, diflavin-containing monomeric enzyme composed of a canonical molybdopterin cofactor-binding catalytic domain and an FMN-FAD/NAD-binding, electron-receiving/transferring domain, making it unique among all previously reported hetero-oligomeric NASs. Genetic studies revealed that NasN is essential for aerobic M. smegmatis growth on nitrate as the sole nitrogen source and that the global transcriptional regulator GlnR regulates nasN expression. Moreover, unlike the NADH-dependent heterodimeric NAS enzyme, NasN efficiently supports bacterial growth under nitrate-limiting conditions, likely due to its significantly greater catalytic activity and oxygen tolerance. Results from a phylogenetic analysis suggested that the nasN gene is more recently evolved than those encoding other NASs and that its distribution is limited mainly to Actinobacteria and Proteobacteria. We observed that among mycobacterial species, most fast-growing environmental mycobacteria carry nasN, but that it is largely lacking in slow-growing pathogenic mycobacteria because of multiple independent genomic deletion events along their evolution.
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Coenzimas/metabolismo , Flavina-Adenina Dinucleótido/metabolismo , Metaloproteínas/metabolismo , Mycobacterium smegmatis/enzimología , NAD/metabolismo , Nitrato-Reductasa/metabolismo , Nitratos/metabolismo , Pteridinas/metabolismo , Electrones , Regulación Bacteriana de la Expresión Génica , Cofactores de Molibdeno , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/crecimiento & desarrollo , Nitrato-Reductasa/química , Nitrato-Reductasa/genética , Nitritos/metabolismo , Filogenia , Receptores de Neurotransmisores/metabolismoRESUMEN
Cholesterol plays essential roles in animal development and disease progression. Here, we characterize the evolutionary pattern of the canonical cholesterol biosynthesis pathway (CBP) in the animal kingdom using both genome-wide analyses and functional experiments. CBP genes in the basal metazoans were inherited from their last common eukaryotic ancestor and evolutionarily conserved for cholesterol biosynthesis. The genomes of both the basal metazoans and deuterostomes retain almost the full set of CBP genes, while Cnidaria and many protostomes have independently experienced multiple massive losses of CBP genes that might be due to the geologic events during the Ediacaran period, such as the appearance of an exogenous sterol supply and the frequent perturbation of ocean oxygenation. Meanwhile, the indispensable utilization processes of cholesterol potentially strengthened the maintenance of the complete set of CBP genes in vertebrates. These results strengthen both biotic and abiotic roles in the macroevolution of a biosynthesis pathway in animals.
RESUMEN
Cancer cells rely on fatty acid synthase (FASN), a key enzyme for de novo biosynthesis of long chain fatty acids, to sustain their proliferative potential and drive invasion. Unfortunately, conventional FASN assays are technically inadequate for discerning otherwise elusive FASN activity in complex biological milieux, which has hindered progress in the functional study of FASN and development of its inhibitors. Here, we describe a chemical probe with unprecedented selectivity and sensitivity for the labeling of active FASN in living cells, thus demonstrating a new analytical modality for visualizing endogenous FASN activity and exploring opportunities for drug discovery.
Asunto(s)
Ácido Graso Sintasas/análisis , Colorantes Fluorescentes/química , Imagen Óptica , Ácido Graso Sintasas/metabolismo , Células HeLa , Humanos , Estructura Molecular , Tamaño de la Partícula , Propiedades de SuperficieRESUMEN
The AdhR regulatory protein is an activator of σ54-dependent transcription of adhA1 and adhA2 genes, which are required for alcohol synthesis in Clostridium beijerinckii Here, we identified the signal perceived by AdhR and determined the regulatory mechanism of AdhR activity. By assaying the activity of AdhR in N-terminally truncated forms, a negative control mechanism of AdhR activity was identified in which the central AAA+ domain is subject to repression by the N-terminal GAF and PAS domains. Binding of Fe2+ to the GAF domain was found to relieve intramolecular repression and stimulate the ATPase activity of AdhR, allowing the AdhR to activate transcription. This control mechanism enables AdhR to regulate transcription of adhA1 and adhA2 in response to cellular redox status. The mutants deficient in AdhR or σ54 showed large shifts in intracellular redox state indicated by the NADH/NAD+ ratio under conditions of increased electron availability or oxidative stress. We demonstrated that the Fe2+-activated transcriptional regulator AdhR and σ54 control alcohol synthesis to maintain redox homeostasis in clostridial cells. Expression of N-terminally truncated forms of AdhR resulted in improved solvent production by C. beijerinckiiIMPORTANCE Solventogenic clostridia are anaerobic bacteria that can produce butanol, ethanol, and acetone, which can be used as biofuels or building block chemicals. Here, we show that AdhR, a σ54-dependent transcriptional activator, senses the intracellular redox status and controls alcohol synthesis in Clostridium beijerinckii AdhR provides a new example of a GAF domain coordinating a mononuclear non-heme iron to sense and transduce the redox signal. Our study reveals a previously unrecognized functional role of σ54 in control of cellular redox balance and provides new insights into redox signaling and regulation in clostridia. Our results reveal AdhR as a novel engineering target for improving solvent production by C. beijerinckii and other solventogenic clostridia.
Asunto(s)
Proteínas Bacterianas/genética , Clostridium beijerinckii/genética , Compuestos Ferrosos/metabolismo , Proteostasis , Factores de Transcripción/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Clostridium beijerinckii/metabolismo , Oxidación-Reducción , Alineación de Secuencia , Factores de Transcripción/química , Factores de Transcripción/metabolismoRESUMEN
Copper ions play an important role in ethylene receptor biogenesis and proper function. The copper transporter RESPONSIVE-TO-ANTAGONIST1 (RAN1) is essential for copper ion transport in Arabidopsis thaliana. However it is still unclear how copper ions are delivered to RAN1 and how copper ions affect ethylene receptors. There is not a specific copper chelator which could be used to explore these questions. Here, by chemical genetics, we identified a novel small molecule, triplin, which could cause a triple response phenotype on dark-grown Arabidopsis seedlings through ethylene signaling pathway. ran1-1 and ran1-2 are hypersensitive to triplin. Adding copper ions in growth medium could partially restore the phenotype on plant caused by triplin. Mass spectrometry analysis showed that triplin could bind copper ion. Compared to the known chelators, triplin acts more specifically to copper ion and it suppresses the toxic effects of excess copper ions on plant root growth. We further showed that mutants of ANTIOXIDANT PROTEIN1 (ATX1) are hypersensitive to tiplin, but with less sensitivity comparing with the ones of ran1-1 and ran1-2. Our study provided genetic evidence for the first time that, copper ions necessary for ethylene receptor biogenesis and signaling are transported from ATX1 to RAN1. Considering that triplin could chelate copper ions in Arabidopsis, and copper ions are essential for plant and animal, we believe that, triplin not only could be useful for studying copper ion transport of plants, but also could be useful for copper metabolism study in animal and human.
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Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas de Transporte de Catión/genética , Cobre/metabolismo , Factores de Transcripción/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/metabolismo , Proteínas de Transporte de Catión/metabolismo , Proteínas Transportadoras de Cobre , Etilenos/metabolismo , Regulación de la Expresión Génica de las Plantas , N-Metiltransferasa de Histona-Lisina , Humanos , Transporte Iónico/genética , Desarrollo de la Planta , Plantas Modificadas Genéticamente , Proteínas de Unión al ARN , Plantones/genética , Transducción de Señal , Tiourea/análogos & derivados , Factores de Transcripción/metabolismo , Proteína de Unión al GTP ranRESUMEN
Monoterpenoid indole alkaloids are a large (â¼3000 members) and structurally diverse class of metabolites restricted to a limited number of plant families in the order Gentianales. Tabernanthe iboga or iboga (Apocynaceae) is native to western equatorial Africa and has been used in traditional medicine for centuries. Howard Lotsof is credited with bringing iboga to the attention of Western medicine through his accidental discovery that iboga can alleviate opioid withdrawal symptoms. Since this observation, iboga has been investigated for its use in the general management of addiction. We were interested in elucidating ibogaine biosynthesis to understand the unique reaction steps en route to ibogaine. Furthermore, because ibogaine is currently sourced from plant material, these studies may help improve the ibogaine supply chain through synthetic biology approaches. Here, we used next-generation sequencing to generate the first iboga transcriptome and leveraged homology-guided gene discovery to identify the penultimate hydroxylase and final O-methyltransferase steps in ibogaine biosynthesis, herein named ibogamine 10-hydroxylase (I10H) and noribogaine-10-O-methyltransferase (N10OMT). Heterologous expression in Saccharomyces cerevisiae (I10H) or Escherichia coli (N10OMT) and incubation with putative precursors, along with HPLC-MS analysis, confirmed the predicted activities of both enzymes. Moreover, high expression levels of their transcripts were detected in ibogaine-accumulating plant tissues. These discoveries coupled with our publicly available iboga transcriptome will contribute to additional gene discovery efforts and could lead to the stabilization of the global ibogaine supply chain and to the development of ibogaine as a treatment for addiction.
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Sistema Enzimático del Citocromo P-450/metabolismo , Ibogaína/biosíntesis , Proteína O-Metiltransferasa/metabolismo , Tabernaemontana/química , Alcaloides , Catálisis , Secuenciación de Nucleótidos de Alto Rendimiento , Trastornos Relacionados con Opioides/tratamiento farmacológico , Tabernaemontana/enzimología , Tabernaemontana/metabolismo , Transcriptoma/genéticaRESUMEN
Rifampin (RIF) is a first-line drug used for the treatment of tuberculosis and other bacterial infections. Various RIF resistance mechanisms have been reported, and recently an RIF-inactivation enzyme, RIF phosphotransferase (RPH), was reported to phosphorylate RIF at its C21 hydroxyl at the cost of ATP. However, the underlying molecular mechanism remained unknown. Here, we solve the structures of RPH from Listeria monocytogenes (LmRPH) in different conformations. LmRPH comprises three domains: an ATP-binding domain (AD), an RIF-binding domain (RD), and a catalytic His-containing domain (HD). Structural analyses reveal that the C-terminal HD can swing between the AD and RD, like a toggle switch, to transfer phosphate. In addition to its catalytic role, the HD can bind to the AD and induce conformational changes that stabilize ATP binding, and the binding of the HD to the RD is required for the formation of the RIF-binding pocket. A line of hydrophobic residues forms the RIF-binding pocket and interacts with the 1-amino, 2-naphthol, 4-sulfonic acid and naphthol moieties of RIF. The R group of RIF points toward the outside of the pocket, explaining the low substrate selectivity of RPH. Four residues near the C21 hydroxyl of RIF, His825, Arg666, Lys670, and Gln337, were found to play essential roles in the phosphorylation of RIF; among these the His825 residue may function as the phosphate acceptor and donor. Our study reveals the molecular mechanism of RIF phosphorylation catalyzed by RPH and will guide the development of a new generation of rifamycins.
Asunto(s)
Listeria monocytogenes/metabolismo , Fosfotransferasas/química , Rifampin/química , Adenosina Trifosfato/metabolismo , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X , Farmacorresistencia Bacteriana , Listeria monocytogenes/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Naftoles/química , Fosfotransferasas/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Rifampin/metabolismo , Ácidos Sulfónicos/químicaRESUMEN
LRRK2-IN-1, one of the first selective inhibitors of leucine-rich repeat kinase 2 (LRRK2), was serendipitously found to exhibit potent antiproliferative activity in several types of human cancer cells. In this study, we employed a chemoproteomic strategy utilizing a photoaffinity probe to identify the cellular target(s) of LRRK2-IN-1 underlying its anticancer activity. LRRK2-IN-1 was found to induce cell cycle arrest as well as cancer cell death by specifically binding to human proliferating cell nuclear antigen (PCNA) in cancer cells. Our current findings suggest the potential of LRRK2-IN-1 as a novel pharmacological molecule for scrutinizing cell physiology and furnish a logical foundation for the future development of therapeutic reagents for cancer.
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Antiparkinsonianos/farmacología , Benzodiazepinonas/farmacología , Antígeno Nuclear de Célula en Proliferación/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , Proliferación Celular/efectos de los fármacos , Reposicionamiento de Medicamentos , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Concentración 50 Inhibidora , Células Jurkat , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/antagonistas & inhibidores , Sondas Moleculares/química , Enfermedad de Parkinson/tratamiento farmacológico , Etiquetas de Fotoafinidad/química , Proteómica/métodosRESUMEN
Antisense transcription emerges as a key regulator of important biological processes in the human malaria parasite Plasmodium falciparum. RNA-processing factors, however, remain poorly characterized in this pathogen. Here, we purified the multiprotein RNA exosome complex of malaria parasites by affinity chromatography, using HA-tagged PfRrp4 and PfDis3 as the ligands. Seven distinct core exosome subunits (PfRrp41, PfMtr3, PfRrp42, PfRrp45, PfRrp4, PfRrp40, PfCsl4) and two exoribonuclease proteins PfRrp6 and PfDis3 are identified by mass spectrometry. Western blot analysis detects Dis3 and Rrp4 predominantly in the cytoplasmic fraction during asexual blood stage development. An inducible gene knock out of the PfDis3 subunit reveals the upregulation of structural and coding RNA, but the vast majority belongs to antisense RNA. Furthermore, we detect numerous types of cryptic unstable transcripts (CUTs) linked to virulence gene families including antisense RNA in the rif gene family. Our work highlights the limitations of steady-state RNA analysis to predict transcriptional activity and link the RNA surveillance machinery directly with post-transcriptional control and gene expression in malaria parasites.
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Complejo Multienzimático de Ribonucleasas del Exosoma/genética , Plasmodium falciparum/genética , Proteínas Protozoarias/metabolismo , Citoplasma/genética , Citoplasma/metabolismo , Complejo Multienzimático de Ribonucleasas del Exosoma/metabolismo , Regulación de la Expresión Génica , Técnicas de Inactivación de Genes , Plasmodium falciparum/patogenicidad , Proteínas Protozoarias/genética , ARN sin Sentido/metabolismo , Proteínas de Unión al ARN/genéticaRESUMEN
Terpene synthases (TPSs) are responsible for the extremely diversified and complex structure of terpenoids. Amorpha-4,11-diene synthase (ADS) has a high (90%) fidelity in generating the sesquiterpene precursor for the biosynthesis of artemisinin, an antimalarial drug, however, little is known about how active site residues of ADS are involved in carbocation rearrangement and cyclization reactions. Here, we identify seven residues that are key to most of the catalytic steps in ADS. By structural modeling and amino acid sequence alignments of ADS with two functionally relevant sesquiterpene synthases from Artemisia annua, we performed site-directed mutagenesis and found that a single substitution, T296V, impaired the ring closure activity almost completely, and tetra-substitutions (L374Y/L404V/L405I/G439S) led to an enzyme generating 80% monocyclic bisabolyl-type sesquiterpenes, whereas a double mutant (T399L/T447G) showed compromised activity in regioselective deprotonation to yield 34.7 and 37.7% normal and aberrant deprotonation products, respectively. Notably, Thr296, Leu374, Gly439, Thr399, and Thr447, which play a major role in directing catalytic cascades, are located around conserved metal-binding motifs and function through impacting the folding of the substrate/intermediate, implying that residues surrounding the two motifs could be valuable targets for engineering TPS activity. Using this knowledge, we substantially increased amorpha-4,11-diene production in a near-additive manner by engineering Thr399 and Thr447 for product release. Our results provide new insight for the rational design of enzyme activity using synthetic biology.
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Transferasas Alquil y Aril/metabolismo , Artemisia annua/enzimología , Sesquiterpenos/metabolismo , Transferasas Alquil y Aril/química , Transferasas Alquil y Aril/genética , Dominio Catalítico , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación/genética , Sesquiterpenos Policíclicos , Conformación Proteica , Sesquiterpenos/químicaRESUMEN
BACKGROUND: Huperzia serrata (H. serrata) is an economically important traditional Chinese herb with the notably medicinal value. As a representative member of the Lycopodiaceae family, the H. serrata produces various types of effectively bioactive lycopodium alkaloids, especially the huperzine A (HupA) which is a promising drug for Alzheimer's disease. Despite their medicinal importance, the public genomic and transcriptomic resources are very limited and the biosynthesis of HupA is largely unknown. Previous studies on comparison of 454-ESTs from H. serrata and Phlegmariurus carinatus predicted putative genes involved in lycopodium alkaloid biosynthesis, such as lysine decarboxylase like (LDC-like) protein and some CYP450s. However, these gene annotations were not carried out with further biochemical characterizations. To understand the biosynthesis of HupA and its regulation in H. serrata, a global transcriptome analysis on H. Serrata tissues was performed. RESULTS: In this study, we used the Illumina Highseq4000 platform to generate a substantial RNA sequencing dataset of H. serrata. A total of 40.1 Gb clean data was generated from four different tissues: root, stem, leaf, and sporangia and assembled into 181,141 unigenes. The total length, average length, N50 and GC content of unigenes were 219,520,611 bp, 1,211 bp, 2,488 bp and 42.51%, respectively. Among them, 105,516 unigenes (58.25%) were annotated by seven public databases (NR, NT, Swiss-Prot, KEGG, COG, Interpro, GO), and 54 GO terms and 3,391 transcription factors (TFs) were functionally classified, respectively. KEGG pathway analysis revealed that 72,230 unigenes were classified into 21 functional pathways. Three types of candidate enzymes, LDC, CAO and PKS, responsible for the biosynthesis of precursors of HupA were all identified in the transcripts. Four hundred and fifty-seven CYP450 genes in H. serrata were also analyzed and compared with tissue-specific gene expression. Moreover, two key classes of CYP450 genes BBE and SLS, with 23 members in total, for modification of the lycopodium alkaloid scaffold in the late two stages of biosynthesis of HupA were further evaluated. CONCLUSION: This study is the first report of global transcriptome analysis on all tissues of H. serrata, and critical genes involved in the biosynthesis of precursors and scaffold modifications of HupA were discovered and predicted. The transcriptome data from this work not only could provide an important resource for further investigating on metabolic pathways in H. serrata, but also shed light on synthetic biology study of HupA.
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Alcaloides/biosíntesis , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Huperzia/genética , Huperzia/metabolismo , Transcriptoma , Alcaloides/metabolismo , Biología Computacional/métodos , Bases de Datos Genéticas , Ontología de Genes , Secuenciación de Nucleótidos de Alto Rendimiento , Huperzia/clasificación , Redes y Vías Metabólicas , Anotación de Secuencia Molecular , Filogenia , Reproducibilidad de los Resultados , SesquiterpenosRESUMEN
Despite the fact that multiple artemisinin-alkylated proteins in Plasmodium falciparum have been identified in recent studies, the alkylation mechanism and accurate binding site of artemisinin-protein interaction have remained elusive. Here, we report the chemical-probe-based enrichment of the artemisinin-binding peptide and characterization of the artemisinin-binding site of P. falciparum translationally controlled tumor protein (TCTP). A peptide fragment within the N-terminal region of TCTP was enriched and found to be alkylated by an artemisinin-derived probe. MS2 fragments showed that artemisinin could alkylate multiple amino acids from Phe12 to Tyr22 of TCTP, which was supported by labeling experiments upon site-directed mutagenesis and computational modeling studies. Taken together, the "capture-and-release" strategy affords consolidated advantages previously unavailable in artemisinin-protein binding site studies, and our results deepened the understanding of the mechanism of protein alkylation via heme-activated artemisinin.
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Artemisininas/metabolismo , Química Clic/métodos , Plasmodium falciparum/química , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Alquilación , Sitios de Unión , Hemo/química , Espectrometría de Masas , Simulación del Acoplamiento Molecular , Mutagénesis Sitio-Dirigida , Proteínas Protozoarias/genética , Reproducibilidad de los ResultadosRESUMEN
Herein, the structure resulting from in situ turnover in a chemically challenging quaternary ammonium oxidative demethylation reaction was captured via crystallographic analysis and analyzed via single-crystal spectroscopy. Crystal structures were determined for the Rieske-type monooxygenase, stachydrine demethylase, in the unliganded state (at 1.6 Å resolution) and in the product complex (at 2.2 Å resolution). The ligand complex was obtained from enzyme aerobically cocrystallized with the substrate stachydrine (N,N-dimethylproline). The ligand electron density in the complex was interpreted as proline, generated within the active site at 100 K by the absorption of X-ray photon energy and two consecutive demethylation cycles. The oxidation state of the Rieske iron-sulfur cluster was characterized by UV-visible spectroscopy throughout X-ray data collection in conjunction with resonance Raman spectra collected before and after diffraction data. Shifts in the absorption band wavelength and intensity as a function of absorbed X-ray dose demonstrated that the Rieske center was reduced by solvated electrons generated by X-ray photons; the kinetics of the reduction process differed dramatically for the liganded complex compared to unliganded demethylase, which may correspond to the observed turnover in the crystal.
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Oxigenasas de Función Mixta/análisis , Compuestos de Amonio Cuaternario/química , Cristalografía por Rayos X , Oxigenasas de Función Mixta/metabolismo , Modelos Moleculares , Oxidación-Reducción , Prolina/análogos & derivados , Prolina/química , Prolina/metabolismo , Compuestos de Amonio Cuaternario/metabolismo , Espectrofotometría Ultravioleta , Espectrometría RamanRESUMEN
H(2)(18)O under the bridge: Recently, the deoxyxylulose phosphate (DXP) pathway was discovered to be a second pathway supplying isoprenoid biosynthetic precursors. One of steps is an IspG-catalyzed reductive deoxygenation of methylerythritol cyclodiphosphate (MEcPP) to 4-hydroxyl-3-methyl-2-(E)-1-diphosphate (HMBPP). Using [2-(13) C,(18) O]-MEcPP, we detected the positional isotopic exchange for the bridging oxygen in MEcPP.
Asunto(s)
Isomerasas Aldosa-Cetosa/química , Transferasas Alquil y Aril/química , Eritritol/análogos & derivados , Modelos Moleculares , Complejos Multienzimáticos/química , Oxidorreductasas/química , Xilosa/análogos & derivados , Isomerasas Aldosa-Cetosa/metabolismo , Transferasas Alquil y Aril/metabolismo , Catálisis , Deuterio , Eritritol/química , Eritritol/metabolismo , Estructura Molecular , Complejos Multienzimáticos/metabolismo , Oxidorreductasas/metabolismo , Isótopos de Oxígeno , Xilosa/química , Xilosa/metabolismoRESUMEN
IspG is a [4Fe-4S] cluster-containing protein, and the [4Fe-4S](+) species is proposed to be the catalytically relevant species. However, attempts reported in the literature failed to detect the [4Fe-4S](+) species. In this study, using a potent reduction system, we have successfully detected the [4Fe-4S](+) species with X-band EPR spectroscopy. In addition, we have improved the Escherichia coli IspG activity to 550 nmol min(-1) mg(-1), which is approximately 20-fold greater than that of the NADPH-Fpr-FldA system in the literature.