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1.
J Am Chem Soc ; 146(2): 1388-1395, 2024 Jan 17.
Artículo en Inglés | MEDLINE | ID: mdl-38176024

RESUMEN

Site-specific covalent conjugation offers a powerful tool to identify and understand protein-protein interactions. In this study, we discover that sulfur fluoride exchange (SuFEx) warheads effectively crosslink the Escherichia coli acyl carrier protein (AcpP) with its partner BioF, a key pyridoxal 5'-phosphate (PLP)-dependent enzyme in the early steps of biotin biosynthesis by targeting a tyrosine residue proximal to the active site. We identify the site of crosslink by MS/MS analysis of the peptide originating from both partners. We further evaluate the BioF-AcpP interface through protein crystallography and mutational studies. Among the AcpP-interacting BioF surface residues, three critical arginine residues appear to be involved in AcpP recognition so that pimeloyl-AcpP can serve as the acyl donor for PLP-mediated catalysis. These findings validate an evolutionary gain-of-function for BioF, allowing the organism to build biotin directly from fatty acid biosynthesis through surface modifications selective for salt bridge formation with acidic AcpP residues.


Asunto(s)
Biotina , Fluoruros , Compuestos de Azufre , Espectrometría de Masas en Tándem , Biotina/metabolismo , Escherichia coli/metabolismo , Ácidos Grasos/metabolismo
2.
Nat Commun ; 15(1): 389, 2024 Jan 09.
Artículo en Inglés | MEDLINE | ID: mdl-38195598

RESUMEN

Structural biology efforts using cryogenic electron microscopy are frequently stifled by specimens adopting "preferred orientations" on grids, leading to anisotropic map resolution and impeding structure determination. Tilting the specimen stage during data collection is a generalizable solution but has historically led to substantial resolution attenuation. Here, we develop updated data collection and image processing workflows and demonstrate, using multiple specimens, that resolution attenuation is negligible or significantly reduced across tilt angles. Reconstructions with and without the stage tilted as high as 60° are virtually indistinguishable. These strategies allowed the reconstruction to 3 Å resolution of a bacterial RNA polymerase with preferred orientation, containing an unnatural nucleotide for studying novel base pair recognition. Furthermore, we present a quantitative framework that allows cryo-EM practitioners to define an optimal tilt angle during data acquisition. These results reinforce the utility of employing stage tilt for data collection and provide quantitative metrics to obtain isotropic maps.


Asunto(s)
Benchmarking , Sistemas de Computación , Microscopía por Crioelectrón , Anisotropía , Recolección de Datos
3.
Chem Sci ; 14(39): 10925-10933, 2023 Oct 11.
Artículo en Inglés | MEDLINE | ID: mdl-37829009

RESUMEN

Protein-reactive natural products such as the fungal metabolite cerulenin are recognized for their value as therapeutic candidates, due to their ability to selectively react with catalytic residues within a protein active site or a complex of protein domains. Here, we explore the development of fatty-acid and polyketide-synthase probes by synthetically modulating cerulenin's functional moieties. Using a mechanism-based approach, we reveal unique reactivity within cerulenin and adapt it for fluorescent labeling and crosslinking of fatty-acid and iterative type-I polyketide synthases. We also describe two new classes of silylcyanohydrin and silylhemiaminal masked crosslinking probes that serve as new tools for activity and structure studies of these biosynthetic pathways.

4.
Biochemistry ; 62(21): 3050-3060, 2023 11 07.
Artículo en Inglés | MEDLINE | ID: mdl-37813856

RESUMEN

Over the past decade, advances in genomics have identified thousands of additional protein-coding small open reading frames (smORFs) missed by traditional gene finding approaches. These smORFs encode peptides and small proteins, commonly termed micropeptides or microproteins. Several of these newly discovered microproteins have biological functions and operate through interactions with proteins and protein complexes within the cell. CYREN1 is a characterized microprotein that regulates double-strand break repair in mammalian cells through interaction with Ku70/80 heterodimer. Ku70/80 binds to and stabilizes double-strand breaks and recruits the machinery needed for nonhomologous end join repair. In this study, we examined the biochemical properties of CYREN1 to better understand and explain its cellular protein interactions. Our findings support that CYREN1 is an intrinsically disordered microprotein and this disordered structure allows it to enriches several proteins, including a newly discovered interaction with SF3B1 via a distinct short linear motif (SLiMs) on CYREN1. Since many microproteins are predicted to be disordered, CYREN1 is an exemplar of how microproteins interact with other proteins and reveals an unknown scaffolding function of this microprotein that may link NHEJ and splicing.


Asunto(s)
Péptidos , Proteínas , Animales , Proteínas/genética , Péptidos/genética , Sistemas de Lectura Abierta , Mamíferos/genética , Micropéptidos
5.
bioRxiv ; 2023 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-37503021

RESUMEN

Structural biology efforts using cryogenic electron microscopy are frequently stifled by specimens adopting "preferred orientations" on grids, leading to anisotropic map resolution and impeding structure determination. Tilting the specimen stage during data collection is a generalizable solution but has historically led to substantial resolution attenuation. Here, we develop updated data collection and image processing workflows and demonstrate, using multiple specimens, that resolution attenuation is negligible or significantly reduced across tilt angles. Reconstructions with and without the stage tilted as high as 60° are virtually indistinguishable. These strategies allowed the reconstruction to 3 Å resolution of a bacterial RNA polymerase with preferred orientation. Furthermore, we present a quantitative framework that allows cryo-EM practitioners to define an optimal tilt angle for dataset acquisition. These data reinforce the utility of employing stage tilt for data collection and provide quantitative metrics to obtain isotropic maps.

6.
Nat Chem Biol ; 16(8): 850-856, 2020 08.
Artículo en Inglés | MEDLINE | ID: mdl-32284603

RESUMEN

In plants, phenylalanine biosynthesis occurs via two compartmentally separated pathways. Overexpression of petunia chorismate mutase 2 (PhCM2), which catalyzes the committed step of the cytosolic pathway, increased flux in cytosolic phenylalanine biosynthesis, but paradoxically decreased the overall levels of phenylalanine and phenylalanine-derived volatiles. Concomitantly, the levels of auxins, including indole-3-acetic acid and its precursor indole-3-pyruvic acid, were elevated. Biochemical and genetic analyses revealed the existence of metabolic crosstalk between the cytosolic phenylalanine biosynthesis and tryptophan-dependent auxin biosynthesis mediated by an aminotransferase that uses a cytosolic phenylalanine biosynthetic pathway intermediate, phenylpyruvate, as an amino acceptor for auxin formation.


Asunto(s)
Ácidos Indolacéticos/química , Ácidos Indolacéticos/metabolismo , Fenilalanina/biosíntesis , Vías Biosintéticas/genética , Citosol/metabolismo , Indoles , Fenilalanina/metabolismo , Ácidos Fenilpirúvicos/metabolismo , Plantas/metabolismo , Triptófano
7.
ACS Chem Biol ; 14(8): 1767-1779, 2019 08 16.
Artículo en Inglés | MEDLINE | ID: mdl-31268677

RESUMEN

A bifurcation of the mevalonate (MVA) pathway was recently discovered in bacteria of the Chloroflexi phylum. In this alternative route for the biosynthesis of isopentenylpyrophosphate (IPP), the penultimate step is the decarboxylation of (R)-mevalonate 5-phosphate ((R)-MVAP) to isopentenyl phosphate (IP), which is followed by the ATP-dependent phosphorylation of IP to IPP catalyzed by isopentenyl phosphate kinase (IPK). Notably, the decarboxylation reaction is catalyzed by mevalonate 5-phosphate decarboxylase (MPD), which shares considerable sequence similarity with mevalonate diphosphate decarboxylase (MDD) of the classical MVA pathway. We show that an enzyme originally annotated as an MDD from the Chloroflexi bacterium Anaerolinea thermophila possesses equal catalytic efficiency for (R)-MVAP and (R)-mevalonate 5-diphosphate ((R)-MVAPP). Further, the molecular basis for this dual specificity is revealed by near atomic-resolution X-ray crystal structures of A. thermophila MPD/MDD bound to (R)-MVAP or (R)-MVAPP. These findings, when combined with sequence and structural comparisons of this bacterial enzyme, functional MDDs, and several putative MPDs, delineate key active-site residues that confer substrate specificity and functionally distinguish MPD and MDD enzyme classes. Extensive sequence analyses identified functional MPDs in the halobacteria class of archaea that had been annotated as MDDs. Finally, no eukaryotic MPD candidates were identified, suggesting the absence of the alternative MVA (altMVA) pathway in all eukaryotes, including, paradoxically, plants, which universally encode a structural and functional homologue of IPK. Additionally, we have developed a viable engineered strain of Saccharomyces cerevisiae as an in vivo metabolic model and a synthetic biology platform for enzyme engineering and terpene biosynthesis in which the classical MVA pathway has been replaced with the altMVA pathway.


Asunto(s)
Proteínas Bacterianas/metabolismo , Carboxiliasas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Carboxiliasas/química , Carboxiliasas/genética , Catálisis , Dominio Catalítico , Chloroflexi/enzimología , Descarboxilación , Ácido Mevalónico/análogos & derivados , Ácido Mevalónico/metabolismo , Unión Proteica , Ingeniería de Proteínas , Saccharomyces cerevisiae/genética , Especificidad por Sustrato
8.
J Antibiot (Tokyo) ; 69(7): 524-33, 2016 07.
Artículo en Inglés | MEDLINE | ID: mdl-27328867

RESUMEN

The plant terpene synthase (TPS) family is responsible for the biosynthesis of a variety of terpenoid natural products possessing diverse biological functions. TPSs catalyze the ionization and, most commonly, rearrangement and cyclization of prenyl diphosphate substrates, forming linear and cyclic hydrocarbons. Moreover, a single TPS often produces several minor products in addition to a dominant product. We characterized the catalytic profiles of Hyoscyamus muticus premnaspirodiene synthase (HPS) and compared it with the profile of a closely related TPS, Nicotiana tabacum 5-epi-aristolochene synthase (TEAS). The profiles of two previously studied HPS and TEAS mutants, each containing nine interconverting mutations, dubbed HPS-M9 and TEAS-M9, were also characterized. All four TPSs were compared under varying temperature and pH conditions. In addition, we solved the X-ray crystal structures of TEAS and a TEAS quadruple mutant complexed with substrate and products to gain insight into the enzymatic features modulating product formation. These informative structures, along with product profiles, provide new insight into plant TPS catalytic promiscuity.


Asunto(s)
Hyoscyamus/enzimología , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Sesquiterpenos/metabolismo , Dominio Catalítico , Estabilidad de Enzimas/genética , Concentración de Iones de Hidrógeno , Hyoscyamus/genética , Mutación , Proteínas de Plantas/genética , Temperatura
9.
Proc Natl Acad Sci U S A ; 113(14): 3797-802, 2016 Apr 05.
Artículo en Inglés | MEDLINE | ID: mdl-27001835

RESUMEN

Halogenated pyrroles (halopyrroles) are common chemical moieties found in bioactive bacterial natural products. The halopyrrole moieties of mono- and dihalopyrrole-containing compounds arise from a conserved mechanism in which a proline-derived pyrrolyl group bound to a carrier protein is first halogenated and then elaborated by peptidic or polyketide extensions. This paradigm is broken during the marine pseudoalteromonad bacterial biosynthesis of the coral larval settlement cue tetrabromopyrrole (1), which arises from the substitution of the proline-derived carboxylate by a bromine atom. To understand the molecular basis for decarboxylative bromination in the biosynthesis of 1, we sequenced two Pseudoalteromonas genomes and identified a conserved four-gene locus encoding the enzymes involved in its complete biosynthesis. Through total in vitro reconstitution of the biosynthesis of 1 using purified enzymes and biochemical interrogation of individual biochemical steps, we show that all four bromine atoms in 1 are installed by the action of a single flavin-dependent halogenase: Bmp2. Tetrabromination of the pyrrole induces a thioesterase-mediated offloading reaction from the carrier protein and activates the biosynthetic intermediate for decarboxylation. Insights into the tetrabrominating activity of Bmp2 were obtained from the high-resolution crystal structure of the halogenase contrasted against structurally homologous halogenase Mpy16 that forms only a dihalogenated pyrrole in marinopyrrole biosynthesis. Structure-guided mutagenesis of the proposed substrate-binding pocket of Bmp2 led to a reduction in the degree of halogenation catalyzed. Our study provides a biogenetic basis for the biosynthesis of 1 and sets a firm foundation for querying the biosynthetic potential for the production of 1 in marine (meta)genomes.


Asunto(s)
Flavinas/metabolismo , Halogenación/fisiología , Pseudoalteromonas/enzimología , Pseudoalteromonas/metabolismo , Pirroles/química , Secuencia de Aminoácidos , Animales , Antozoos/metabolismo , Organismos Acuáticos/metabolismo , Proteínas Bacterianas/metabolismo , Sitios de Unión/genética , Bromo/química , Cristalografía por Rayos X , Pseudoalteromonas/genética
10.
Plant Cell ; 26(9): 3709-27, 2014 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-25217505

RESUMEN

The enzymes cinnamoyl-CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) catalyze the two key reduction reactions in the conversion of cinnamic acid derivatives into monolignol building blocks for lignin polymers in plant cell walls. Here, we describe detailed functional and structural analyses of CCRs from Medicago truncatula and Petunia hybrida and of an atypical CAD (CAD2) from M. truncatula. These enzymes are closely related members of the short-chain dehydrogenase/reductase (SDR) superfamily. Our structural studies support a reaction mechanism involving a canonical SDR catalytic triad in both CCR and CAD2 and an important role for an auxiliary cysteine unique to CCR. Site-directed mutants of CAD2 (Phe226Ala and Tyr136Phe) that enlarge the phenolic binding site result in a 4- to 10-fold increase in activity with sinapaldehyde, which in comparison to the smaller coumaraldehyde and coniferaldehyde substrates is disfavored by wild-type CAD2. This finding demonstrates the potential exploitation of rationally engineered forms of CCR and CAD2 for the targeted modification of monolignol composition in transgenic plants. Thermal denaturation measurements and structural comparisons of various liganded and unliganded forms of CCR and CAD2 highlight substantial conformational flexibility of these SDR enzymes, which plays an important role in the establishment of catalytically productive complexes of the enzymes with their NADPH and phenolic substrates.


Asunto(s)
Oxidorreductasas de Alcohol/química , Aldehído Oxidorreductasas/química , Lignina/biosíntesis , Medicago truncatula/enzimología , Petunia/enzimología , Propanoles/metabolismo , Oxidorreductasas de Alcohol/metabolismo , Aldehído Oxidorreductasas/metabolismo , Sitios de Unión , Biocatálisis , Clonación Molecular , Cristalografía por Rayos X , Cisteína/metabolismo , Disulfuros/metabolismo , Ésteres/metabolismo , Cinética , Ligandos , Lignina/química , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , NADP/metabolismo , Propanoles/química , Homología Estructural de Proteína , Especificidad por Sustrato , Temperatura
11.
Angew Chem Int Ed Engl ; 53(15): 3932-6, 2014 Apr 07.
Artículo en Inglés | MEDLINE | ID: mdl-24615769

RESUMEN

The ability to reversibly control protein structure and function with light would offer high spatiotemporal resolution for investigating biological processes. To confer photoresponsiveness on general proteins, we genetically incorporated a set of photoswitchable click amino acids (PSCaas), which contain both a reversible photoswitch and an additional click functional group for further modifications. Orthogonal tRNA-synthetases were evolved to genetically encode PSCaas bearing azobenzene with an alkene, keto, or benzyl chloride group in E. coli and in mammalian cells. After incorporation into calmodulin, the benzyl chloride PSCaa spontaneously generated a covalent protein bridge by reacting with a nearby cysteine residue through proximity-enabled bioreactivity. The resultant azobenzene bridge isomerized in response to light, thereby changing the conformation of calmodulin. These genetically encodable PSCaas will prove valuable for engineering photoswitchable bridges into proteins for reversible optogenetic regulation.


Asunto(s)
Aminoácidos/química , Escherichia coli/metabolismo , Química Clic , Código Genético , Conformación Molecular , Optogenética , Ingeniería de Proteínas
12.
Nature ; 503(7477): 552-556, 2013 Nov 28.
Artículo en Inglés | MEDLINE | ID: mdl-24162851

RESUMEN

Flavoproteins catalyse a diversity of fundamental redox reactions and are one of the most studied enzyme families. As monooxygenases, they are universally thought to control oxygenation by means of a peroxyflavin species that transfers a single atom of molecular oxygen to an organic substrate. Here we report that the bacterial flavoenzyme EncM catalyses the peroxyflavin-independent oxygenation-dehydrogenation dual oxidation of a highly reactive poly(ß-carbonyl). The crystal structure of EncM with bound substrate mimics and isotope labelling studies reveal previously unknown flavin redox biochemistry. We show that EncM maintains an unexpected stable flavin-oxygenating species, proposed to be a flavin-N5-oxide, to promote substrate oxidation and trigger a rare Favorskii-type rearrangement that is central to the biosynthesis of the antibiotic enterocin. This work provides new insight into the fine-tuning of the flavin cofactor in offsetting the innate reactivity of a polyketide substrate to direct its efficient electrocyclization.


Asunto(s)
Proteínas Bacterianas/metabolismo , Flavinas/metabolismo , Flavoproteínas/metabolismo , Oxigenasas de Función Mixta/metabolismo , Streptomyces/enzimología , Antibacterianos/biosíntesis , Proteínas Bacterianas/química , Biocatálisis , Hidrocarburos Aromáticos con Puentes/metabolismo , Cristalografía por Rayos X , Ciclización , Flavoproteínas/química , Marcaje Isotópico , Oxigenasas de Función Mixta/química , Modelos Químicos , Modelos Moleculares , Oxidación-Reducción , Policétidos/metabolismo , Conformación Proteica , Streptomyces/metabolismo , Especificidad por Sustrato
13.
Chembiochem ; 14(16): 2100-5, 2013 Nov 04.
Artículo en Inglés | MEDLINE | ID: mdl-24019075

RESUMEN

Unnatural amino acids (UAAs) containing conjugated ring systems are of interest for their optical properties. Until now, such bulky and planar UAAs could not be incorporated into proteins using the pyrrolysyl tRNA/synthetase shuttling system. Using the "small-intelligent" approach to construct a highly diverse library, we evolved novel synthetases specific for two such UAAs and incorporated them into proteins in E. coli and mammalian cells.


Asunto(s)
Aminoácidos/metabolismo , Aminoacil-ARNt Sintetasas/metabolismo , Biblioteca de Genes , Aminoácidos/química , Animales , Evolución Molecular Dirigida , Escherichia coli/genética , Código Genético , Lisina/análogos & derivados , Lisina/química , Lisina/metabolismo , Mioglobina/genética , Mioglobina/metabolismo , Especificidad por Sustrato , Ballenas/genética
14.
Nat Prod Rep ; 29(10): 1238-50, 2012 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-22850796

RESUMEN

The addition of a methyl moiety to a small chemical is a common transformation in the biosynthesis of natural products across all three domains of life. These methylation reactions are most often catalysed by S-adenosyl-L-methionine (SAM)-dependent methyltransferases (MTs). MTs are categorized based on the electron-rich, methyl accepting atom, usually O, N, C, or S. SAM-dependent natural product MTs (NPMTs) are responsible for the modification of a wide array of structurally distinct substrates, including signalling and host defense compounds, pigments, prosthetic groups, cofactors, cell membrane and cell wall components, and xenobiotics. Most notably, methylation modulates the bioavailability, bioactivity, and reactivity of acceptor molecules, and thus exerts a central role on the functional output of many metabolic pathways. Our current understanding of the structural enzymology of NPMTs groups these phylogenetically diverse enzymes into two MT-superfamily fold classes (class I and class III). Structural biology has also shed light on the catalytic mechanisms and molecular bases for substrate specificity for over fifty NPMTs. These biophysical-based approaches have contributed to our understanding of NPMT evolution, demonstrating how a widespread protein fold evolved to accommodate chemically diverse methyl acceptors and to catalyse disparate mechanisms suited to the physiochemical properties of the target substrates. This evolutionary diversity suggests that NPMTs may serve as starting points for generating new biocatalysts.


Asunto(s)
Productos Biológicos/metabolismo , Metiltransferasas/metabolismo , Animales , Bacterias/enzimología , Humanos , Metiltransferasas/genética , Ratones , Estructura Molecular , Plantas/enzimología , Conformación Proteica , Ratas
15.
Nature ; 485(7399): 530-3, 2012 May 13.
Artículo en Inglés | MEDLINE | ID: mdl-22622584

RESUMEN

Specialized metabolic enzymes biosynthesize chemicals of ecological importance, often sharing a pedigree with primary metabolic enzymes. However, the lineage of the enzyme chalcone isomerase (CHI) remained unknown. In vascular plants, CHI-catalysed conversion of chalcones to chiral (S)-flavanones is a committed step in the production of plant flavonoids, compounds that contribute to attraction, defence and development. CHI operates near the diffusion limit with stereospecific control. Although associated primarily with plants, the CHI fold occurs in several other eukaryotic lineages and in some bacteria. Here we report crystal structures, ligand-binding properties and in vivo functional characterization of a non-catalytic CHI-fold family from plants. Arabidopsis thaliana contains five actively transcribed genes encoding CHI-fold proteins, three of which additionally encode amino-terminal chloroplast-transit sequences. These three CHI-fold proteins localize to plastids, the site of de novo fatty-acid biosynthesis in plant cells. Furthermore, their expression profiles correlate with those of core fatty-acid biosynthetic enzymes, with maximal expression occurring in seeds and coinciding with increased fatty-acid storage in the developing embryo. In vitro, these proteins are fatty-acid-binding proteins (FAPs). FAP knockout A. thaliana plants show elevated α-linolenic acid levels and marked reproductive defects, including aberrant seed formation. Notably, the FAP discovery defines the adaptive evolution of a stereospecific and catalytically 'perfected' enzyme from a non-enzymatic ancestor over a defined period of plant evolution.


Asunto(s)
Arabidopsis/química , Biocatálisis , Evolución Molecular , Ácidos Grasos/metabolismo , Liasas Intramoleculares/química , Liasas Intramoleculares/metabolismo , Pliegue de Proteína , Arabidopsis/enzimología , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cristalografía por Rayos X , Proteínas de Unión a Ácidos Grasos/química , Proteínas de Unión a Ácidos Grasos/deficiencia , Proteínas de Unión a Ácidos Grasos/genética , Proteínas de Unión a Ácidos Grasos/metabolismo , Liasas Intramoleculares/deficiencia , Liasas Intramoleculares/genética , Ligandos , Modelos Moleculares , Fenotipo , Unión Proteica , Estereoisomerismo , Ácido alfa-Linolénico/metabolismo
16.
Stem Cells ; 29(8): 1231-40, 2011 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-21681861

RESUMEN

Although unnatural amino acids (Uaas) have been genetically encoded in bacterial, fungal, and mammalian cells using orthogonal transfer RNA (tRNA)/aminoacyl-tRNA synthetase pairs, applications of this method to a wider range of specialized cell types, such as stem cells, still face challenges. While relatively straightforward in stem cells, transient expression lacks sufficient temporal resolution to afford reasonable levels of Uaa incorporation and to allow for the study of the longer term differentiation process of stem cells. Moreover, Uaa incorporation may perturb differentiation. Here, we describe a lentiviral-based gene delivery method to stably incorporate Uaas into proteins expressed in neural stem cells, specifically HCN-A94 cells. The transduced cells differentiated into neural progenies in the same manner as the wild-type cells. By genetically incorporating a fluorescent Uaa into a voltage-dependent membrane lipid phosphatase, we show that this Uaa optically reports the conformational change of the voltage-sensitive domain in response to membrane depolarization. The method described here should be generally applicable to other stem cells and membrane proteins.


Asunto(s)
Benzofenonas/metabolismo , Diferenciación Celular , Células-Madre Neurales/metabolismo , Neuronas/metabolismo , Fenilalanina/análogos & derivados , Tirosina-ARNt Ligasa/genética , Alanina/análogos & derivados , Alanina/química , Alanina/metabolismo , Aminoacil-ARNt Sintetasas/genética , Aminoacil-ARNt Sintetasas/metabolismo , Animales , Benzofenonas/química , Línea Celular , Compuestos de Dansilo/química , Compuestos de Dansilo/metabolismo , Fluorescencia , Ingeniería Genética , Vectores Genéticos , Humanos , Lentivirus , Potenciales de la Membrana , Neuronas/citología , Técnicas de Placa-Clamp , Fenilalanina/química , Fenilalanina/genética , Fenilalanina/metabolismo , Monoéster Fosfórico Hidrolasas/química , Monoéster Fosfórico Hidrolasas/genética , Monoéster Fosfórico Hidrolasas/metabolismo , Conformación Proteica , Estructura Terciaria de Proteína , Ratas , Imagen de Lapso de Tiempo
17.
Plant Cell ; 22(12): 4114-27, 2010 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-21177481

RESUMEN

Lignin forms from the polymerization of phenylpropanoid-derived building blocks (the monolignols), whose modification through hydroxylation and O-methylation modulates the chemical and physical properties of the lignin polymer. The enzyme caffeic acid O-methyltransferase (COMT) is central to lignin biosynthesis. It is often targeted in attempts to engineer the lignin composition of transgenic plants for improved forage digestibility, pulping efficiency, or utility in biofuel production. Despite intensive investigation, the structural determinants of the regiospecificity and substrate selectivity of COMT remain poorly defined. Reported here are x-ray crystallographic structures of perennial ryegrass (Lolium perenne) COMT (Lp OMT1) in open conformational state, apo- and holoenzyme forms and, most significantly, in a closed conformational state complexed with the products S-adenosyl-L-homocysteine and sinapaldehyde. The product-bound complex reveals the post-methyl-transfer organization of COMT's catalytic groups with reactant molecules and the fully formed phenolic-ligand binding site. The core scaffold of the phenolic ligand forges a hydrogen-bonding network involving the 4-hydroxy group that anchors the aromatic ring and thereby permits only metahydroxyl groups to be positioned for transmethylation. While distal from the site of transmethylation, the propanoid tail substituent governs the kinetic preference of ryegrass COMT for aldehydes over alcohols and acids due to a single hydrogen bond donor for the C9 oxygenated moiety dictating the preference for an aldehyde.


Asunto(s)
Lolium/enzimología , Metiltransferasas/metabolismo , Dominio Catalítico , Cristalografía por Rayos X , Enlace de Hidrógeno , Cinética , Metiltransferasas/química , Modelos Moleculares , Relación Estructura-Actividad , Especificidad por Sustrato
18.
Plant Cell ; 22(10): 3357-73, 2010 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-20952635

RESUMEN

Cinnamoyl CoA-reductase (CCR) and caffeic acid O-methyltransferase (COMT) catalyze key steps in the biosynthesis of monolignols, which serve as building blocks in the formation of plant lignin. We identified candidate genes encoding these two enzymes in perennial ryegrass (Lolium perenne) and show that the spatio-temporal expression patterns of these genes in planta correlate well with the developmental profile of lignin deposition. Downregulation of CCR1 and caffeic acid O-methyltransferase 1 (OMT1) using an RNA interference-mediated silencing strategy caused dramatic changes in lignin level and composition in transgenic perennial ryegrass plants grown under both glasshouse and field conditions. In CCR1-deficient perennial ryegrass plants, metabolic profiling indicates the redirection of intermediates both within and beyond the core phenylpropanoid pathway. The combined results strongly support a key role for the OMT1 gene product in the biosynthesis of both syringyl- and guaiacyl-lignin subunits in perennial ryegrass. Both field-grown OMT1-deficient and CCR1-deficient perennial ryegrass plants showed enhanced digestibility without obvious detrimental effects on either plant fitness or biomass production. This highlights the potential of metabolic engineering not only to enhance the forage quality of grasses but also to produce optimal feedstock plants for biofuel production.


Asunto(s)
Aldehído Oxidorreductasas/metabolismo , Lignina/biosíntesis , Lolium/enzimología , Metiltransferasas/metabolismo , Proteínas de Plantas/metabolismo , Aldehído Oxidorreductasas/genética , Regulación de la Expresión Génica de las Plantas , Lolium/genética , Metiltransferasas/genética , Datos de Secuencia Molecular , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente/enzimología , Plantas Modificadas Genéticamente/genética , Interferencia de ARN , ARN de Planta/genética
19.
Plant J ; 54(3): 362-74, 2008 May.
Artículo en Inglés | MEDLINE | ID: mdl-18208524

RESUMEN

Many plants synthesize the volatile phenylpropene compounds eugenol and isoeugenol to serve in defense against herbivores and pathogens and to attract pollinators. Clarkia breweri flowers emit a mixture of eugenol and isoeugenol, while Petunia hybrida flowers emit mostly isoeugenol with small amounts of eugenol. We recently reported the identification of a petunia enzyme, isoeugenol synthase 1 (PhIGS1) that catalyzes the formation of isoeugenol, and an Ocimum basilicum (basil) enzyme, eugenol synthase 1 (ObEGS1), that produces eugenol. ObEGS1 and PhIGS1 both utilize coniferyl acetate, are 52% sequence identical, and belong to a family of NADPH-dependent reductases involved in secondary metabolism. Here we show that C. breweri flowers have two closely related proteins (96% identity), CbIGS1 and CbEGS1, that are similar to ObEGS1 (58% and 59% identity, respectively) and catalyze the formation of isoeugenol and eugenol, respectively. In vitro mutagenesis experiments demonstrate that substitution of only a single residue can substantially affect the product specificity of these enzymes. A third C. breweri enzyme identified, CbEGS2, also catalyzes the formation of eugenol from coniferyl acetate and is only 46% identical to CbIGS1 and CbEGS1 but more similar (>70%) to other types of reductases. We also found that petunia flowers contain an enzyme, PhEGS1, that is highly similar to CbEGS2 (82% identity) and that converts coniferyl acetate to eugenol. Our results indicate that plant enzymes with EGS and IGS activities have arisen multiple times and in different protein lineages.


Asunto(s)
Clarkia/enzimología , Enzimas/metabolismo , Petunia/enzimología , Proteínas de Plantas/metabolismo , Secuencia de Aminoácidos , Clarkia/genética , Clarkia/metabolismo , Electroforesis en Gel de Poliacrilamida , Enzimas/genética , Eugenol/análogos & derivados , Eugenol/química , Eugenol/metabolismo , Flores/enzimología , Flores/metabolismo , Datos de Secuencia Molecular , Estructura Molecular , Petunia/genética , Petunia/metabolismo , Filogenia , Proteínas de Plantas/clasificación , Proteínas de Plantas/genética , Homología de Secuencia de Aminoácido
20.
PLoS One ; 2(10): e993, 2007 Oct 03.
Artículo en Inglés | MEDLINE | ID: mdl-17912370

RESUMEN

Phenylpropenes, a large group of plant volatile compounds that serve in multiple roles in defense and pollinator attraction, contain a propenyl side chain. Eugenol synthase (EGS) catalyzes the reductive displacement of acetate from the propenyl side chain of the substrate coniferyl acetate to produce the allyl-phenylpropene eugenol. We report here the structure determination of EGS from basil (Ocimum basilicum) by protein x-ray crystallography. EGS is structurally related to the short-chain dehydrogenase/reductases (SDRs), and in particular, enzymes in the isoflavone-reductase-like subfamily. The structure of a ternary complex of EGS bound to the cofactor NADP(H) and a mixed competitive inhibitor EMDF ((7S,8S)-ethyl (7,8-methylene)-dihydroferulate) provides a detailed view of the binding interactions within the EGS active site and a starting point for mutagenic examination of the unusual reductive mechanism of EGS. The key interactions between EMDF and the EGS-holoenzyme include stacking of the phenyl ring of EMDF against the cofactor's nicotinamide ring and a water-mediated hydrogen-bonding interaction between the EMDF 4-hydroxy group and the side-chain amino moiety of a conserved lysine residue, Lys132. The C4 carbon of nicotinamide resides immediately adjacent to the site of hydride addition, the C7 carbon of cinnamyl acetate substrates. The inhibitor-bound EGS structure suggests a two-step reaction mechanism involving the formation of a quinone-methide prior to reduction. The formation of this intermediate is promoted by a hydrogen-bonding network that favors deprotonation of the substrate's 4-hydroxyl group and disfavors binding of the acetate moiety, akin to a push-pull catalytic mechanism. Notably, the catalytic involvement in EGS of the conserved Lys132 in preparing the phenolic substrate for quinone methide formation through the proton-relay network appears to be an adaptation of the analogous role in hydrogen bonding played by the equivalent lysine residue in other enzymes of the SDR family.


Asunto(s)
Eugenol/metabolismo , Ocimum basilicum/enzimología , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/química , Benzoquinonas/química , Sitios de Unión , Unión Competitiva , Catálisis , Cristalografía por Rayos X/métodos , Enlace de Hidrógeno , Isoflavonas/química , Lisina/química , Modelos Químicos , Conformación Molecular , NADP/química , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , UDPglucosa 4-Epimerasa/química
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