RESUMO
OBJECTIVES: Cox10 is an enzyme required for the activity of cytochrome c oxidase. Humans who lack at least one functional copy of Cox10 have a form of Leigh Syndrome, a genetic disease that is usually fatal in infancy. As more human genomes are sequenced, new alleles are being discovered; whether or not these alleles encode functional proteins remains unclear. Thus, we set out to measure the phenotypes of many human Cox10 variants by expressing them in yeast cells. RESULTS: We successfully expressed the reference sequence and 25 variants of human Cox10 in yeast. We quantitated the ability of these variants to support growth on nonfermentable media and directly measured cytochrome c oxidase activity. 11 of these Cox10 variants supported approximately half or more the cytochrome c oxidase activity compared to the reference sequence. All of the strains containing those 11 variants also grew robustly using a nonfermentable carbon source. Cells expressing the other variants showed low cytochrome c oxidase activity and failed to grow on nonfermentable media.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons , Doença de Leigh , Fenótipo , Doença de Leigh/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Humanos , Saccharomyces cerevisiae/genética , Variação Genética , Proteínas de Membrana , Alquil e Aril TransferasesRESUMO
Terpenoids play a crucial role in plant growth and development, as well as in regulating resistance mechanisms. Terpene synthase (TPS) serves as the final step in the synthesis process of terpenoids. However, a comprehensive bioinformatics analysis of the TPS gene family in Triticum plants had not previously been systematically undertaken. In this study, a total of 531 TPS members were identified in Triticum plants. The evolutionary tree divided the TPS proteins into five subfamilies: Group1, Group2, Group3, Group4, and Group5. The results of the duplication events analysis showed that TD and WGD were major driving forces during the evolution of the TPS family. The cis-element analysis showed that the TPS genes were related to plant growth and development and environmental stress. Moreover, the GO annotation displayed that the biological function of TPS was relatively conserved in wheat plants. The RNA-seq data showed that the rice and wheat TPS genes responded to low-temperature stress and exhibited significantly different expression patterns. This research shed light on the functions of TPSs in responding to abiotic stress and demonstrated their modulatory potential during root development. These findings provide a foundation for further and deeper investigation of the TPSs' functions in Triticum plants.
Assuntos
Alquil e Aril Transferases , Evolução Molecular , Regulação da Expressão Gênica de Plantas , Família Multigênica , Filogenia , Proteínas de Plantas , Triticum , Triticum/genética , Triticum/crescimento & desenvolvimento , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Estresse Fisiológico/genética , Genoma de Planta , Oryza/genética , Oryza/crescimento & desenvolvimento , Perfilação da Expressão GênicaRESUMO
Fungal secondary metabolite (SM) biosynthetic gene clusters (BGCs) containing dimethylallyltryptophan synthases (DMATSs) produce structurally diverse prenylated indole alkaloids with wide-ranging activities that have vast potential as human therapeutics. To discover new natural products produced by DMATSs, we mined the Department of Energy Joint Genome Institute's MycoCosm database for DMATS-containing BGCs. We found a DMATS BGC in Aspergillus homomorphus CBS 101889, which also contains a nonribosomal peptide synthetase (NRPS). This BGC appeared to have a previously unreported combination of genes, which suggested the cluster might make novel SMs. We refactored this BGC with highly inducible promoters into the model fungus Aspergillus nidulans. The expression of this refactored BGC in A. nidulans resulted in the production of eight tryptophan-containing diketopiperazines, six of which are new to science. We have named them homomorphins A-F (2, 4-8). Perhaps even more intriguingly, to our knowledge, this is the first discovery of C4-prenylated tryptophan-containing diketopiperazines and their derivatives. In addition, the NRPS from this BGC is the first described that has the ability to promiscuously combine tryptophan with either of two different amino acids, in this case, l-valine or l-allo-isoleucine.
Assuntos
Aspergillus nidulans , Aspergillus , Dicetopiperazinas , Peptídeo Sintases , Triptofano , Triptofano/metabolismo , Triptofano/química , Dicetopiperazinas/química , Aspergillus nidulans/genética , Aspergillus nidulans/metabolismo , Aspergillus/química , Peptídeo Sintases/metabolismo , Peptídeo Sintases/genética , Estrutura Molecular , Família Multigênica , Alcaloides Indólicos/química , Alcaloides Indólicos/metabolismo , Alquil e Aril Transferases/metabolismo , Alquil e Aril Transferases/genéticaRESUMO
It is unknown why roses are terpene-rich, what the terpene biosynthetic pathways in roses are, and why only a few rose species produce the major components of rose essential oil. Here, we assembled two high-quality chromosome-level genomes for Rosa rugosa and Rosa multiflora. We also re-sequenced 132 individuals from the F1 progeny of Rosa chinensis and Rosa wichuraiana and 36 of their related species. Comparative genomics revealed that expansions of the 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) and terpene synthases (TPSs) gene families led to the enrichment of terpenes in rose scent components. We constructed a terpene biosynthesis network and discovered a TPS-independent citronellol biosynthetic pathway in roses through gene functional identification, genome-wide association studies (GWASs), and multi-omic analysis. Heterologous co-expression of rose citronellol biosynthetic genes in Nicotiana benthamiana led to citronellol production. Our genomic and metabolomic analyses suggested that the copy number of NUDX1-1a determines the citronellol content in different rose species. Our findings not only provide additional genome and gene resources and reveal the evolution of the terpene biosynthetic pathways but also present a nearly complete scenario for terpenoid metabolism that will facilitate the breeding of fragrant roses and the production of rose oil.
Assuntos
Vias Biossintéticas , Rosa , Terpenos , Rosa/genética , Rosa/metabolismo , Terpenos/metabolismo , Vias Biossintéticas/genética , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Estudo de Associação Genômica Ampla , Odorantes , Evolução Molecular , Genoma de Planta , Monoterpenos Acíclicos/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMO
Dendrobium loddigesii, a member of the Orchidaceae family, is a valuable horticultural crop known for its aromatic qualities. However, the mechanisms responsible for the development of its aromatic characteristics remain poorly understood. To elucidate these underlying mechanisms, we assembled the first chromosome-level reference genome of D. loddigesii using PacBio HiFi-reads, Illumina short-reads, and Hi-C data. The assembly comprises 19 pseudochromosomes with N50 contig and N50 scaffold sizes of 55.15 and 89.94 Mb, respectively, estimating the genome size to be 1.68 Gb, larger than that of other sequenced Dendrobium species. During the flowering stages, we conducted a comprehensive analysis combining volatilomics and transcriptomics to understand the characteristics and biosynthetic mechanisms pathways of the floral scent. Our findings emphasize the significant contribution of aromatic terpenoids, especially monoterpenoids, in defining the floral aroma. Furthermore, we identified two crucial terpene synthase (TPS) genes that play a key role in maintaining the aroma during flowering. Through the integration volatilomics data with catalytic assays of DlTPSbs proteins, we identified specific compounds responsible for the aromatic characteristics of D. loddigesii. This integrated analysis of the genome, transcriptome, and volatilome, offers valuable insights into the development and preservation of D. loddigesii's aromatic characteristics, setting the stage for further exploration of the botanical perfumer hypothesis.
Assuntos
Dendrobium , Flores , Dendrobium/genética , Dendrobium/metabolismo , Flores/genética , Flores/metabolismo , Odorantes , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Transcriptoma/genética , Genoma de Planta , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Regulação da Expressão Gênica de Plantas , MultiômicaRESUMO
In mammals, cobalamin is an essential cofactor that is delivered by a multitude of chaperones in an elaborate trafficking pathway to two client enzymes, methionine synthase and methylmalonyl-CoA mutase (MMUT). Rhodibalamins, the rhodium analogs of cobalamins, have been described as antimetabolites due to their ability to inhibit bacterial growth. In this study, we have examined the reactivity of adenosylrhodibalamin (AdoRhbl) with two key human chaperones, MMACHC (also known as CblC) and adenosyltransferase (MMAB, also known as ATR), and with the human and Mycobacterium tuberculosis MMUT. We demonstrate that while AdoRhbl binds tightly to all four proteins, the Rh-carbon bond is resistant to homolytic (on MMAB and MMUT) as well as heterolytic (on MMACHC) rupture. On the other hand, MMAB catalyzes Rh-carbon bond formation, converting rhodi(I)balamin in the presence of ATP to AdoRhbl. We report the first crystal structure of a rhodibalamin (AdoRhbl) bound to a B12 protein, i.e., MMAB, in the presence of triphosphate, which shows a weakened but intact Rh-carbon bond. The structure provides insights into how MMAB cleaves the corresponding Co-carbon bond in a sacrificial homolytic reaction that purportedly functions as a cofactor sequestration strategy. Collectively, the study demonstrates that while the noble metal substitution of cobalt by rhodium sets up structural mimicry, it compromises chemistry, which could be exploited for targeting human and bacterial B12 chaperones and enzymes.
Assuntos
Vitamina B 12 , Vitamina B 12/metabolismo , Vitamina B 12/química , Vitamina B 12/análogos & derivados , Humanos , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/metabolismo , Metilmalonil-CoA Mutase/metabolismo , Metilmalonil-CoA Mutase/química , Ródio/química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/química , Mimetismo Molecular , Modelos Moleculares , Alquil e Aril TransferasesRESUMO
Lilac (Syringa oblata) is a well-known horticultural plant, and its aromatic heartwood is widely utilized in Traditional Mongolian Medicine for treating angina. However, limited research on the dynamic changes and mechanisms of aromatic substance formation during heartwood development hinders the analysis and utilization of its medicinal components. In this study, volatile metabolome analysis revealed that sesquiterpenes are the primary metabolites responsible for the aroma in heartwood, with cadinane and eremophilane types being the most prevalent. Among the identified sesquiterpene synthases, SoSTPS1-5 exhibited significantly increased expression in heartwood formation and was selected for further investigation. Molecular docking simulations predicted multiple amino acid binding sites and confirmed its ability to catalyze the formation of eremophilane, copaene, cadinane, germacrane, and elemane-type sesquiterpenes from FPP (farnesyl pyrophosphate). Co-expression and promoter analysis suggested a transcriptional regulatory network primarily involving WRKY transcription factors. Additionally, aiotic and biotic stress inducers, such as Ag+, Fusarium oxysporum, and especially MeJA, were found to activate the expression of SoSTPS1-5 and promote sesquiterpene accumulation. This study provides insights into the basis of medicinal substance formation and the potential mechanisms of sesquiterpene accumulation in lilac heartwood, laying a foundation for future research on the biosynthesis and utilization of its medicinal components.
Assuntos
Proteínas de Plantas , Sesquiterpenos , Sesquiterpenos/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Simulação de Acoplamento Molecular , Regulação da Expressão Gênica de Plantas , Alquil e Aril Transferases/metabolismo , Alquil e Aril Transferases/genética , Madeira/metabolismoRESUMO
New diterpenoids are accessible from non-natural FPP derivatives as substrates for an enzymatic elongation cyclization cascade using the geranylgeranyl pyrophosphate synthase (GGPPS) from Streptomyces cyaneofuscatus and the spata-13,17-diene synthase (SpS) from Streptomyces xinghaiensis. This approach led to four new biotransformation products including three new cyclododecane cores and a macrocyclic ether. For the first time, a 1,12-terpene cyclization was observed when shifting the central olefinic double bond toward the geminial methyl groups creating a nonconjugated 1,4-diene.
Assuntos
Alquil e Aril Transferases , Dimetilaliltranstransferase , Diterpenos , Streptomyces , Diterpenos/química , Diterpenos/metabolismo , Dimetilaliltranstransferase/metabolismo , Dimetilaliltranstransferase/química , Streptomyces/enzimologia , Streptomyces/química , Alquil e Aril Transferases/metabolismo , Alquil e Aril Transferases/química , Estrutura Molecular , Ciclização , Fosfatos de Poli-Isoprenil/química , Fosfatos de Poli-Isoprenil/metabolismo , BiotransformaçãoRESUMO
BACKGROUND: Isopentenyltransferases (IPT) serve as crucial rate-limiting enzyme in cytokinin synthesis, playing a vital role in plant growth, development, and resistance to abiotic stress. RESULTS: Compared to the wild type, transgenic creeping bentgrass exhibited a slower growth rate, heightened drought tolerance, and improved shade tolerance attributed to delayed leaf senescence. Additionally, transgenic plants showed significant increases in antioxidant enzyme levels, chlorophyll content, and soluble sugars. Importantly, this study uncovered that overexpression of the MtIPT gene not only significantly enhanced cytokinin and auxin content but also influenced brassinosteroid level. RNA-seq analysis revealed that differentially expressed genes (DEGs) between transgenic and wild type plants were closely associated with plant hormone signal transduction, steroid biosynthesis, photosynthesis, flavonoid biosynthesis, carotenoid biosynthesis, anthocyanin biosynthesis, oxidation-reduction process, cytokinin metabolism, and wax biosynthesis. And numerous DEGs related to growth, development, and stress tolerance were identified, including cytokinin signal transduction genes (CRE1, B-ARR), antioxidase-related genes (APX2, PEX11, PER1), Photosynthesis-related genes (ATPF1A, PSBQ, PETF), flavonoid synthesis genes (F3H, C12RT1, DFR), wax synthesis gene (MAH1), senescence-associated gene (SAG20), among others. CONCLUSION: These findings suggest that the MtIPT gene acts as a negative regulator of plant growth and development, while also playing a crucial role in the plant's response to abiotic stress.
Assuntos
Agrostis , Alquil e Aril Transferases , Citocininas , Secas , Folhas de Planta , Senescência Vegetal , Plantas Geneticamente Modificadas , Agrostis/genética , Agrostis/fisiologia , Agrostis/metabolismo , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Plantas Geneticamente Modificadas/genética , Senescência Vegetal/genética , Folhas de Planta/genética , Folhas de Planta/fisiologia , Citocininas/metabolismo , Regulação da Expressão Gênica de Plantas , Reguladores de Crescimento de Plantas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Estresse Fisiológico/genética , Fotossíntese/genética , Genes de Plantas , Resistência à SecaRESUMO
Eunicellane diterpenoids, containing a typical 6,10-bicycle, are bioactive compounds widely present in marine corals, but rarely found in bacteria and plants. The intrinsic macrocycle exhibits innate structural flexibility resulting in dynamic conformational changes. However, the mechanisms controlling flexibility remain unknown. The discovery of a terpene synthase, MicA, that is responsible for the biosynthesis of a nearly non-flexible eunicellane skeleton, enable us to propose a feasible theory about the flexibility in eunicellane structures. Parallel studies of all eunicellane synthases in nature discovered to date, including 2Z-geranylgeranyl diphosphate incubations and density functional theory-based Boltzmann population computations, reveale that a trans-fused bicycle with a 2Z-configuration alkene restricts conformational flexibility resulting in a nearly non-flexible eunicellane skeleton. The catalytic route and the enzymatic mechanism of MicA are also elucidated by labeling experiments, density functional theory calculations, structural analysis of the artificial intelligence-based MicA model, and mutational studies.
Assuntos
Alquil e Aril Transferases , Diterpenos , Alquil e Aril Transferases/metabolismo , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/química , Diterpenos/metabolismo , Diterpenos/química , Fosfatos de Poli-Isoprenil/metabolismo , Fosfatos de Poli-Isoprenil/química , Modelos MolecularesRESUMO
Many proteins undergo a post-translational lipid attachment, which increases their hydrophobicity, thus strengthening their membrane association properties or aiding in protein interactions. Geranylgeranyltransferase-I (GGTase-I) is an enzyme involved in a 3-step post-translational modification (PTM) pathway that attaches a 20-carbon lipid group called geranylgeranyl at the carboxy-terminal cysteine of proteins ending in a canonical CaaL motif (C-cysteine, a-aliphatic, L-often leucine, but can be phenylalanine, isoleucine, methionine, or valine). Genetic approaches involving 2 distinct reporters were employed in this study to assess Saccharomyces cerevisiae GGTase-I specificity, for which limited data exist, toward all 8,000 CXXX combinations. Orthogonal biochemical analyses and structure-based alignments were also performed to better understand the features required for optimal target interaction. These approaches indicate that yeast GGTase-I best modifies the Cxa[L/F/I/M/V] sequence that resembles but is not an exact match for the canonical CaaL motif. We also observed that minor modification of noncanonical sequences is possible. A consistent feature associated with well-modified sequences was the presence of a nonpolar a2 residue and a hydrophobic terminal residue, which are features recognized by mammalian GGTase-I. These results thus support that mammalian and yeast GGTase-I exhibit considerable shared specificity.
Assuntos
Alquil e Aril Transferases , Motivos de Aminoácidos , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato , Alquil e Aril Transferases/metabolismo , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Sequência de Aminoácidos , Modelos MolecularesRESUMO
Terpene synthases (TPSs) are enzymes responsible for catalyzing the production of diverse terpenes, the largest class of secondary metabolites in plants. Here, we identified 107 TPS gene loci encompassing 92 full-length TPS genes in upland cotton (Gossypium hirsutum L.). Phylogenetic analysis showed they were divided into six subfamilies. Segmental duplication and tandem duplication events contributed greatly to the expansion of TPS gene family, particularly the TPS-a and TPS-b subfamilies. Expression profile analysis screened out that GhTPSs may mediate the interaction between cotton and Verticillium dahliae. Three-dimensional structures and subcellular localizations of the two selected GhTPSs, GhTPS6 and GhTPS47, which belong to the TPS-a subfamily, demonstrated similarity in protein structures and nucleus and cytoplasm localization. Virus-induced gene silencing (VIGS) of the two GhTPSs yielded plants characterized by increased wilting and chlorosis, more severe vascular browning, and higher disease index than control plants. Additionally, knockdown of GhTPS6 and GhTPS47 led to the down-regulation of cotton terpene synthesis following V. dahliae infection, indicating that these two genes may positively regulate resistance to V. dahliae through the modulation of disease-resistant terpene biosynthesis. Overall, our study represents a comprehensive analysis of the G. hirsutum TPS gene family, revealing their potential roles in defense responses against Verticillium wilt.
Assuntos
Alquil e Aril Transferases , Resistência à Doença , Gossypium , Filogenia , Doenças das Plantas , Proteínas de Plantas , Gossypium/genética , Gossypium/microbiologia , Gossypium/enzimologia , Gossypium/metabolismo , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Doenças das Plantas/microbiologia , Doenças das Plantas/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Resistência à Doença/genética , Regulação da Expressão Gênica de Plantas , Ascomicetos , VerticilliumRESUMO
Terpenes comprise the largest class of natural products and are used in applications spanning the areas of medicine, cosmetics, fuels, flavorings, and more. Copalyl diphosphate synthase from the Penicillium genus is the first bifunctional terpene synthase identified to have both prenyltransferase and class II cyclase activities within the same polypeptide chain. Prior studies of bifunctional terpene synthases reveal that these systems achieve greater catalytic efficiency by channeling geranylgeranyl diphosphate between the prenyltransferase and cyclase domains. A molecular-level understanding of substrate transit phenomena in these systems is highly desirable, but a long disordered polypeptide segment connecting the prenyltranferase and cyclase domains thwarts the crystallization of full-length enzymes. Accordingly, these systems are excellent candidates for structural analysis using cryo-electron microscopy (cryo-EM). Notably, these systems form hexameric or octameric oligomers, so the quaternary structure of the full-length enzyme may influence substrate transit between catalytic domains. Here, we describe methods for the preparation of bifunctional hexameric copalyl diphosphate synthase from Penicillium fellutanum (PfCPS). We also outline approaches for the preparation of cryo-EM grids, data collection, and data processing to yield two-dimensional and three-dimensional reconstructions.
Assuntos
Alquil e Aril Transferases , Microscopia Crioeletrônica , Penicillium , Penicillium/enzimologia , Alquil e Aril Transferases/química , Alquil e Aril Transferases/metabolismo , Alquil e Aril Transferases/isolamento & purificação , Microscopia Crioeletrônica/métodos , Diterpenos/metabolismo , Diterpenos/química , Proteínas Fúngicas/química , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/metabolismo , Dimetilaliltranstransferase/metabolismo , Dimetilaliltranstransferase/química , Dimetilaliltranstransferase/genética , Dimetilaliltranstransferase/isolamento & purificaçãoRESUMO
Prenyltransferases are terpene synthases that combine 5-carbon precursor molecules into linear isoprenoids of varying length that serve as substrates for terpene cyclases, enzymes that catalyze fascinating cyclization reactions to form diverse terpene natural products. Terpenes and their derivatives comprise the largest class of natural products and have myriad functions in nature and diverse commercial uses. An emerging class of bifunctional terpene synthases contains both prenyltransferase and cyclase domains connected by a disordered linker in a single polypeptide chain. Fusicoccadiene synthase from Phomopsis amygdali (PaFS) is one of the most well-characterized members of this subclass and serves as a model system for the exploration of structure-function relationships. PaFS has been structurally characterized using a variety of biophysical techniques. The enzyme oligomerizes to form a stable core of six or eight prenyltransferase domains that produce a 20-carbon linear isoprenoid, geranylgeranyl diphosphate (GGPP), which then transits to the cyclase domains for the generation of fusicoccadiene. Cyclase domains are in dynamic equilibrium between randomly splayed-out and prenyltransferase-associated positions; cluster channeling is implicated for GGPP transit from the prenyltransferase core to the cyclase domains. In this chapter, we outline the methods we are developing to interrogate the nature of cluster channeling in PaFS, including enzyme activity and product analysis assays, approaches for engineering the linker segment connecting the prenyltransferase and cyclase domains, and structural analysis by cryo-EM.
Assuntos
Alquil e Aril Transferases , Alquil e Aril Transferases/metabolismo , Alquil e Aril Transferases/química , Alquil e Aril Transferases/genética , Dimetilaliltranstransferase/metabolismo , Dimetilaliltranstransferase/química , Dimetilaliltranstransferase/genética , Diterpenos/metabolismo , Diterpenos/química , Ensaios Enzimáticos/métodos , Fosfatos de Poli-Isoprenil/metabolismo , Fosfatos de Poli-Isoprenil/química , CiclizaçãoRESUMO
Terpene Synthases (TPS) catalyze the formation of multicyclic, complex terpenes and terpenoids from linear substrates. Molecular docking is an important research tool that can further our understanding of TPS multistep mechanisms and guide enzyme design. Standard docking programs are not well suited to tackle the unique challenges of TPS, like the many chemical steps which form multiple stereo-centers, the weak dispersion interactions between the isoprenoid chain and the hydrophobic region of the active site, description of carbocation intermediates, and finding mechanistically meaningful sets of docked poses. To address these and other unique challenges, we developed the multistate, multiscale docking program EnzyDock and used it to study many TPS and other enzymes. In this review we discuss the unique challenges of TPS, the special features of EnzyDock developed to address these challenges and demonstrate its successful use in ongoing research on the bacterial TPS CotB2.
Assuntos
Alquil e Aril Transferases , Domínio Catalítico , Simulação de Acoplamento Molecular , Alquil e Aril Transferases/metabolismo , Alquil e Aril Transferases/química , Alquil e Aril Transferases/genética , Terpenos/metabolismo , Terpenos/química , Software , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genéticaRESUMO
Expression and purification of membrane-bound proteins remains a challenge and limits enzymology efforts, contributing to a substantial knowledge gap in the biochemical functions of many proteins found in nature. Accordingly, the study of bacterial UbiA terpene synthases (TSs) has been limited due to the experimental hurdles required to purify active enzymes for characterization in vitro. Previous work employed the use of microsomes or crude membrane fractions to test enzyme activity; however, these methods can be labor intensive, require access to an ultracentrifuge, or may not be suitable for all membrane-bound TSs. We detail here an alternative strategy for the in vivo expression and biochemical characterization of the membrane associated UbiA TSs by employing a precursor overproduction system in Escherichia coli.
Assuntos
Alquil e Aril Transferases , Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , Alquil e Aril Transferases/metabolismo , Alquil e Aril Transferases/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
Two terpene synthases from the pleuromutilin producing fungus Clitopilus passeckerianus were functionally characterised. The first enzyme CpTS1 produces the new diterpene clitopilene with a novel 6-6-5-5 tetracyclic skeleton, while the second enzyme CpTS2 makes the new sesquiterpene isopentalenene. The CpTS1 reaction mechanism was studied in depth using experimental and theoretical approaches.
Assuntos
Alquil e Aril Transferases , Diterpenos , Alquil e Aril Transferases/metabolismo , Diterpenos/química , Diterpenos/metabolismo , Sesquiterpenos/química , Sesquiterpenos/metabolismo , PleuromutilinasRESUMO
MAIN CONCLUSION: CaTPS2 and CaTPS3 were significantly expressed in flowers of Curcuma alismatifolia 'Shadow' and demonstrated bifunctional enzyme activity, CaTPS2 generated linalool and nerolidol as products, and CaTPS3 catalyzed ß-myrcene and ß-farnesene formation. This study presents the discovery and functional characterization of floral terpene synthase (TPS) genes in Curcuma alismatifolia 'Shadow', a cultivar renowned for its unique fragrance. Addressing the gap in understanding the genetic basis of floral scent in this species, we identified eight TPS genes through comprehensive transcriptome sequencing. Among these, CaTPS2 and CaTPS3 were significantly expressed in floral tissues and demonstrated bifunctional enzyme activity corresponding to the major volatile compounds detected in 'Shadow'. Functional analyses, including in vitro assays complemented with rigorous controls and alternative identification methods, elucidated the roles of these TPS genes in terpenoid biosynthesis. In vitro studies were conducted via heterologous expression in E. coli, followed by purification of the recombinant protein using affinity chromatography, enzyme assays were performed with GPP/FPP as the substrate, and volatile products were inserted into the GC-MS for analysis. Partially purified recombinant protein of CaTPS2 catalyzed GPP and FPP to produce linalool and nerolidol, respectively, while partially purified recombinant protein of CaTPS3 generated ß-myrcene and ß-farnesene with GPP and FPP as substrates, respectively. Real-time quantitative PCR further validated the expression patterns of these genes, correlating with terpenoid accumulation in different plant tissues. Our findings illuminate the molecular mechanisms underpinning floral fragrance in C. alismatifolia and provide a foundation for future genetic enhancements of floral scent in ornamental plants. This study, therefore, contributes to the broader understanding of terpenoid biosynthesis in plant fragrances, paving the way for biotechnological applications in horticulture plant breeding.
Assuntos
Monoterpenos Acíclicos , Alquil e Aril Transferases , Curcuma , Flores , Sesquiterpenos , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Flores/genética , Flores/enzimologia , Flores/metabolismo , Sesquiterpenos/metabolismo , Monoterpenos Acíclicos/metabolismo , Curcuma/genética , Curcuma/enzimologia , Curcuma/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Regulação da Expressão Gênica de Plantas , Terpenos/metabolismo , Compostos Orgânicos Voláteis/metabolismo , Filogenia , OdorantesRESUMO
Isoprenoid metabolism and its derivatives took part in photosynthesis, growth regulation, signal transduction, and plant defense to biotic and abiotic stresses. However, how aluminum (Al) stress affects the isoprenoid metabolism and whether isoprenoid metabolism plays a vital role in the Citrus plants in coping with Al stress remain unclear. In this study, we reported that Al-treatment-induced alternation in the volatilization rate of monoterpenes (α-pinene, ß-pinene, limonene, α-terpinene, γ-terpinene and 3-carene) and isoprene were different between Citrus sinensis (Al-tolerant) and C. grandis (Al-sensitive) leaves. The Al-induced decrease of CO2 assimilation, maximum quantum yield of primary PSII photochemistry (Fv/Fm), the lower contents of glucose and starch, and the lowered activities of enzymes involved in the mevalonic acid (MVA) pathway and 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway might account for the different volatilization rate of isoprenoids. Furthermore, the altered transcript levels of genes related to isoprenoid precursors and/or derivatives metabolism, such as geranyl diphosphate (GPP) synthase (GPPS) in GPP biosynthesis, geranylgeranyl diphosphate synthase (GGPPS), chlorophyll synthase (CHS) and GGPP reductase (GGPPR) in chlorophyll biosynthesis, limonene synthase (LS) and α-pinene synthase (APS) in limonene and α-pinene synthesis, respectively, might be responsible for the different contents of corresponding products in C. grandis and C. sinensis. Our data suggested that isoprenoid metabolism was involved in Al tolerance response in Citrus, and the alternation of some branches of isoprenoid metabolism could confer different Al-tolerance to Citrus species.
Assuntos
Alumínio , Monoterpenos Bicíclicos , Citrus , Limoneno , Fotossíntese , Folhas de Planta , Terpenos , Alumínio/toxicidade , Terpenos/metabolismo , Citrus/metabolismo , Citrus/efeitos dos fármacos , Limoneno/metabolismo , Fotossíntese/efeitos dos fármacos , Monoterpenos Bicíclicos/metabolismo , Folhas de Planta/metabolismo , Folhas de Planta/efeitos dos fármacos , Estresse Fisiológico/efeitos dos fármacos , Monoterpenos/metabolismo , Hemiterpenos/metabolismo , Cicloexenos/metabolismo , Fosfatos Açúcares/metabolismo , Butadienos/metabolismo , Eritritol/análogos & derivados , Eritritol/metabolismo , Ácido Mevalônico/metabolismo , Monoterpenos Cicloexânicos , Citrus sinensis/metabolismo , Citrus sinensis/efeitos dos fármacos , Citrus sinensis/genética , Clorofila/metabolismo , Alquil e Aril Transferases/metabolismo , Alquil e Aril Transferases/genética , VolatilizaçãoRESUMO
Primary coenzyme Q10 deficiency-1, caused by COQ2 disease-causing variants, is an autosomal recessive disorder, and genetic testing is the gold standard for diagnosing this condition. A Chinese boy with steroid-resistant nephrotic syndrome, focal segmental glomerulosclerosis, and progressive kidney insufficiency was included in the study. Electron microscopy revealed the glomerular basement membrane with irregular thickness and lamellation with diffuse effacement of foot processes in the podocytes, and swollen mitochondria with abnormal cristae in the podocytes. Coenzyme Q10 supplementation started about 3 weeks after the onset of mild kidney dysfunction did not improve the proband's kidney outcome. Proband-only whole-exome sequencing and Sanger sequencing revealed two heteroallelic COQ2 variants: a maternally inherited novel variant c.1013G > A[p.(Gly338Glu)] in exon 6 and a variant of unknown origin c.1159C > T[p.(Arg387*)] in exon 7. Subsequent long-read sequencing demonstrated these two variants were located on different alleles. Our report extends the phenotypic and genotypic spectrum of COQ2 glomerulopathy.