RESUMEN
The oomycete Phytophthora infestans is a damaging crop pathogen and a model organism to study plant-pathogen interactions. We report the discovery of a family of copper-dependent lytic polysaccharide monooxygenases (LPMOs) in plant pathogenic oomycetes and its role in plant infection by P. infestans We show that LPMO-encoding genes are up-regulated early during infection and that the secreted enzymes oxidatively cleave the backbone of pectin, a charged polysaccharide in the plant cell wall. The crystal structure of the most abundant of these LPMOs sheds light on its ability to recognize and degrade pectin, and silencing the encoding gene in P. infestans inhibits infection of potato, indicating a role in host penetration. The identification of LPMOs as virulence factors in pathogenic oomycetes opens up opportunities in crop protection and food security.
Asunto(s)
Oxigenasas de Función Mixta/metabolismo , Pectinas/metabolismo , Phytophthora infestans/enzimología , Enfermedades de las Plantas/parasitología , Solanum lycopersicum/parasitología , Solanum tuberosum/parasitología , Cobre , Oxigenasas de Función Mixta/química , Oxigenasas de Función Mixta/genética , Modelos Moleculares , Oxidación-Reducción , Phytophthora infestans/genética , Phytophthora infestans/patogenicidad , Hojas de la Planta/parasitología , Polisacáridos/metabolismo , Conformación Proteica , Dominios Proteicos , Factores de Virulencia/química , Factores de Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
This study engineered ß-carotene ketolase CrtW and ß-carotene hydroxylase CrtZ to improve biosynthesis of astaxanthin in Escherichia coli. Firstly, crtW was randomly mutated to increase CrtW activities on conversion from ß-carotene to astaxanthin. A crtW* mutant with A6T, T105A and L239M mutations has improved 5.35-fold astaxanthin production compared with the wild-type control. Secondly, the expression levels of crtW* and crtZ on chromosomal were balanced by simultaneous modulation RBS regions of their genes using RBS library. The strain RBS54 selected from RBS library, directed the pathway exclusively towards the desired product astaxanthin as predominant carotenoid (99%). Lastly, the number of chromosomal copies of the balanced crtW-crtZ cassette from RBS54 was increased using a Cre-loxP based technique, and a strain with 30 copies of the crtW*-crtZ cassette was selected. This final strain DL-A008 had a 9.8-fold increase of astaxanthin production compared with the wild-type control. Fed-batch fermentation showed that DL-A008 produced astaxanthin as predominant carotenoid (99%) with a specific titer of 0.88 g·L-1 without addition of inducer. In conclusion, through constructing crtW mutation, balancing the expression levels between crtW* and crtZ, and increasing the copy number of the balanced crtW*-crtZ cassette, the activities of ß-carotene ketolase and ß-carotene hydroxylase were improved for conversion of ß-carotene to astaxanthin with higher efficiency. The series of conventional and novel metabolic engineering strategies were designed and applied to construct the astaxanthin hetero-producer strain of E. coli, possibly offering a general approach for the construction of stable hetero-producer strains for other natural products.
Asunto(s)
Escherichia coli/metabolismo , Ingeniería Metabólica/métodos , Oxigenasas de Función Mixta/genética , Oxigenasas/genética , Vías Biosintéticas , Carotenoides/química , Carotenoides/metabolismo , Oxigenasas de Función Mixta/química , Oxigenasas/química , Xantófilas/química , Xantófilas/metabolismoRESUMEN
OBJECTIVES: Establish a complete and efficient method for the preparation of cis-5-hydroxy-L-pipecolic acids (cis-5HPA), including biotransformation and isomers separation and purification. RESULTS: For non-heme Fe(II)/α-KG-dependent dioxygenases, α-ketoglutarate (α-KG) has great influence on the stability of Fe(II) ions, which is also the basic of the hydroxylation reaction to the substrate. L-pipecolic acids (L-Pip) was converted to cis-5HPA by whole-cell catalysis in water, which can reduce the loss of Fe(II) ions. 120 mM L-Pip can be transformed to 93% via cell and Fe(II) ions continuous supplementation under the reaction system optimization (the molar ratio of ascorbic acid/FeSO4·7H2O and α-KG/L-Pip were 8:1 and 1:1, respectively). After the catalytic reaction, the amino protection strategy was adopted to improve the resolution of isomer products on silica gel chromatography, and the amino protected cis-5HPA was obtained with a yield of 86.7%. CONCLUSIONS: We established a method which is promising to be used for cis-5HPA largescale preparation. It also provides a suitable reference for this type of enzyme-catalyzed reaction and the hydroxy pipecolic acid isomers separation.
Asunto(s)
Ácidos Cetoglutáricos/química , Oxigenasas de Función Mixta/química , Ácidos Pipecólicos/química , Prolina/química , Hidroxilación , Isomerismo , Oxidación-ReducciónRESUMEN
Naphthalene, as a component of crude oil, is a common environmental pollutant. Biochemical and genetic aspects of naphthalene catabolism have been examined in most detail in the bacteria of Pseudomonas genus. In pseudomonads, the key intermediate in naphthalene degradation is salicylate. In this study, we investigated the ability of Rhodococcus opacus strain 3D to utilize naphthalene as a sole carbon and energy source. The characteristic feature of this strain is the inability to grow in the mineral medium supplemented with salicylate (typical intermediate of naphthalene degradation in Gram-negative bacteria). The absence of salicylate hydroxylase activity and salicylate accumulation in the course of R. opacus 3D cultivation in the mineral medium supplemented with naphthalene indicated existence of an alternative pathway of naphthalene oxidation. At the same time, R. opacus 3D was able to use monoaromatic compounds (salts of gentisic, ortho-phthalic, and 2-hydroxycinnamic acids and coumarin) as growth substrates. Based on the analysis of enzymatic activities, identification of the reaction intermediates, genetic determinants, and growth substrates, we concluded that R. opacus 3D carries out naphthalene degradation through an alternative pathway via formation of ortho-phthalic acid, which is untypical for pseudomonads. Using mass spectrometry, we showed for the first time that salicylic acid associate formed in trace amounts in the process of naphthalene degradation is not further metabolized and accumulated in the growth medium in a form of a dimer.
Asunto(s)
Cinamatos/química , Naftalenos/química , Ácidos Ftálicos/química , Rhodococcus/metabolismo , Aguas del Alcantarillado , Carbono/química , Dimerización , Espectrometría de Masas , Redes y Vías Metabólicas , Oxigenasas de Función Mixta/química , Pseudomonas/metabolismo , Salicilatos/química , Aguas Residuales , Contaminantes Químicos del Agua/análisis , Contaminación del Agua , Purificación del Agua/métodosRESUMEN
The selective hydroxylation of C-H bonds is of great interest to the synthetic community. Both homogeneous catalysts and enzymes offer complementary means to tackle this challenge. Herein, we show that biotinylated Fe(TAML)-complexes (TAML = Tetra Amido Macrocyclic Ligand) can be used as cofactors for incorporation into streptavidin to assemble artificial hydroxylases. Chemo-genetic optimization of both cofactor and streptavidin allowed optimizing the performance of the hydroxylase. Using H2O2 as oxidant, up to â¼300 turnovers for the oxidation of benzylic C-H bonds were obtained. Upgrading the ee was achieved by kinetic resolution of the resulting benzylic alcohol to afford up to >98% ee for (R)-tetralol. X-ray analysis of artificial hydroxylases highlights critical details of the second coordination sphere around the Fe(TAML) cofactor.
Asunto(s)
Alcoholes Bencílicos/metabolismo , Biotina/metabolismo , Hierro/metabolismo , Oxigenasas de Función Mixta/metabolismo , Estreptavidina/metabolismo , Alcoholes Bencílicos/química , Biotina/química , Hidroxilación , Hierro/química , Oxigenasas de Función Mixta/química , Modelos Moleculares , Estructura Molecular , Estereoisomerismo , Estreptavidina/químicaRESUMEN
In previous work, we identified a triple mutant of the castor (Ricinus communis) stearoyl-Acyl Carrier Protein desaturase (T117R/G188L/D280K) that, in addition to introducing a double bond into stearate to produce oleate, performed an additional round of oxidation to convert oleate to a trans allylic alcohol acid. To determine the contributions of each mutation, in this work we generated individual castor desaturase mutants carrying residue changes corresponding to those in the triple mutant and investigated their catalytic activities. We observed that T117R, and to a lesser extent D280K, accumulated a novel product, namely erythro-9,10-dihydroxystearate, that we identified via its methyl ester through gas chromatography-mass spectrometry and comparison with authentic standards. The use of 18O2 labeling showed that the oxygens of both hydroxyl moieties originate from molecular oxygen rather than water. Incubation with an equimolar mixture of 18O2 and 16O2 demonstrated that both hydroxyl oxygens originate from a single molecule of O2, proving the product is the result of dioxygenase catalysis. Using prolonged incubation, we discovered that wild-type castor desaturase is also capable of forming erythro-9,10-dihydroxystearate, which presents a likely explanation for its accumulation to â¼0.7% in castor oil, the biosynthetic origin of which had remained enigmatic for decades. In summary, the findings presented here expand the documented constellation of di-iron enzyme catalysis to include a dioxygenase reactivity in which an unactivated alkene is converted to a vicinal diol.
Asunto(s)
Dioxigenasas/metabolismo , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Ricinus/enzimología , Ácidos Esteáricos/metabolismo , Aceite de Ricino/química , Catálisis , Dioxigenasas/química , Cromatografía de Gases y Espectrometría de Masas , Oxigenasas de Función Mixta/química , Mutación , Ácido Oléico/química , Ácido Oléico/metabolismo , Oxidación-Reducción , Oxígeno/metabolismo , Propanoles/metabolismo , Ricinus/genética , Ricinus/metabolismo , Ácidos Esteáricos/químicaRESUMEN
A 61.3 kDa Phenol hydroxylase (PheA) was purified and characterized from Pseudomonas sp. KZNSA (PKZNSA). Cell free extract of the isolate grown in mineral salt medium supplemented with 600 ppm phenol showed 21.58 U/mL of PheA activity with a specific activity of 7.67 U/mg of protein. The enzyme was purified to 1.6-fold with a total yield of 33.6%. The purified PheA was optimally active at pH 8 and temperature 30 °C, with ≈95% stability at pH 7.5 and temperature 30 °C after 2 h. The Lineweaver-Burk plot showed the vmax and Km values of 4.04 µM/min and 4.03 µM, respectively, for the substrate phenol. The ES-MS data generated from the tryptic digested fragments of pure protein and PCR amplification of a ≈600 bp gene from genomic DNA of PKZNSA lead to the determination of complete amino acid and nucleotide sequence of PheA. Bioinformatics tools and homology modelling studies indicated that PheA from PKZNSA is likely a probable protein kinase UbiB (2-octaprenylphenol hydroxylase) involving Lys and Asp at positions 153 and 288 for binding and active site, respectively. Characterization and optimization of PheA activity may be useful for a better understanding of 2,4-dichlorophenol degradation by this organism and for potential industrial application of the enzyme.
Asunto(s)
Oxigenasas de Función Mixta/química , Oxigenasas de Función Mixta/aislamiento & purificación , Modelos Moleculares , Pseudomonas/enzimología , Secuencia de Aminoácidos , Secuencia de Bases , Fenómenos Biofísicos , Inhibidores Enzimáticos/farmacología , Estabilidad de Enzimas/efectos de los fármacos , Concentración de Iones de Hidrógeno , Iones , Metales/farmacología , Filogenia , Pseudomonas/genética , ARN Ribosómico 16S/genética , Especificidad por Sustrato/efectos de los fármacos , TemperaturaRESUMEN
Neutron crystallography is a powerful method to determine the positions of H atoms in macromolecular structures. However, it is sometimes hard to judge what would constitute a chemically reasonable model, and the geometry of H atoms depends more on the surroundings (for example the formation of hydrogen bonds) than heavy atoms, so that the empirical geometry information for the H atoms used to supplement the experimental data is often less accurate. These problems may be reduced by using quantum-mechanical calculations. A method has therefore been developed to combine quantum-mechanical calculations with joint crystallographic refinement against X-ray and neutron data. A first validation of this method is provided by re-refining the structure of the galectin-3 carbohydrate-recognition domain in complex with lactose. The geometry is improved, in particular for water molecules, for which the method leads to better-resolved hydrogen-bonding interactions. The method has also been applied to the active copper site of lytic polysaccharide monooxygenase and shows that the protonation state of the amino-terminal histidine residue can be determined.
Asunto(s)
Cristalografía por Rayos X/métodos , Galectina 3/química , Oxigenasas de Función Mixta/química , Neutrones , Polisacáridos/química , Conformación Proteica , Proteínas Sanguíneas , Dominio Catalítico , Galectinas , Humanos , Modelos Moleculares , Estructura Molecular , Teoría CuánticaRESUMEN
KEY MESSAGE: A p-coumaroyl CoA 2'-hydroxylase responsible for the formation of coumarin lactone ring was identified from Peucedanum praeruptorum Dunn and functionally characterized in vitro. Coumarins are important plant secondary metabolites with a variety of biological activities. Ortho-hydroxylation of cinnamates leads to the formation of coumarin lactone ring and is generally thought to be a key step in coumarin biosynthesis. However, ortho-hydroxylases, especially p-coumaroyl CoA 2'-hydroxylase (C2'H) responsible for the biosynthesis of the most common coumarin skeleton, have received insufficient attention. Here, a putative ortho-hydroxylase PpC2'H was isolated from P. praeruptorum Dunn, a traditional Chinese medicinal herb rich in coumarins. Expression profile indicated that PpC2'H exhibited the highest transcript level in roots and could be up-regulated by MeJA elicitation. Subcellular localization of PpC2'H was demonstrated to be cytosol in planta. In order to functionally characterize PpC2'H, the purified recombinant protein was incubated with various potential substrates. HPLC-ESI-MS analysis indicated that PpC2'H catalyzed the conversion of p-coumaroyl CoA into hydroxylated intermediate, which then underwent spontaneous lactonization to generate umbelliferone. Our data also showed that light would promote the spontaneous process. In addition, based on homology modeling and site-directed mutagenesis, amino acid residues Phe-130, Lys-141, Asn-207, His-224, Asp-226, His-282 and Phe-298 were verified essential for enzymatic activity. These findings provide insight into structure-function relationship of this pivotal ortho-hydroxylase and also contribute to elucidating the biosynthetic mechanism of coumarin skeleton.
Asunto(s)
Apiaceae/enzimología , Vías Biosintéticas , Cumarinas/metabolismo , Oxigenasas de Función Mixta/metabolismo , Secuencia de Aminoácidos , Vías Biosintéticas/genética , Cromatografía Líquida de Alta Presión , Cumarinas/química , ADN Complementario/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Cinética , Luz , Oxigenasas de Función Mixta/química , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Especificidad de Órganos/genética , Especificidad de Órganos/efectos de la radiación , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Protoplastos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Alineación de Secuencia , Espectrometría de Masa por Ionización de Electrospray , Homología Estructural de Proteína , Fracciones Subcelulares/enzimología , Transcriptoma/genética , Transcriptoma/efectos de la radiaciónRESUMEN
The availability of catalytically active peptidylglycine α-amidating monooxygenase (PAM) should provide the means to examine its potential use for the chemienzymatic synthesis of bioactive peptides for the purpose of pharmacological studies. Hypoglycemic activity is one of the most important features of insulin derivatives. Insulin glargine amide was found to show a time/effect profile which is distinctly more flat and thus more advantageous than insulin glargine itself. The aim of the study was to obtain recombinant PAM and use it for insulin analogue amidation. We stably expressed a recombinant PAM in CHO dhfr-cells in culture. Recombinant PAM was partially purified by fractional ammonium sulphate precipitation and ion-exchange chromatography. The enzyme was used to modify glycine-extended A22(G)-B31(K)-B32(R) human insulin analogue (GKR). Alpha-amidated insulin was analyzed by HPLC and mass spectrometry. Hypoglycemic activity of amidated and non-amidated insulin was compared. The pharmacodynamic effect was based on glucose concentration measurement in Wistar rats with hyperglycemia induced by streptozotocin. The overall glycemic profile up to 36 h was evaluated after subcutaneous single dosing at a range of 2.5-7.5 U/kg b.w. The experiment on rats confirmed with a statistical significance (P < 0.05) hypoglycemic activity of GKR-NH2 in comparison to a control group receiving 0.9% NaCl. Characteristics for GKR-NH2 profile was a rather fast beginning of action (0.5-2.0 h) and quite prolonged return to initial values. GKR-NH2 is a candidate for a hypoglycemic drug product in diabetes care. In addition, this work also provides a valuable alternative method for preparing any other recombinant bioactive peptides with C-terminal amidation.
Asunto(s)
Amidina-Liasas/biosíntesis , Hipoglucemiantes/química , Insulina/análogos & derivados , Insulina/química , Oxigenasas de Función Mixta/biosíntesis , Proteínas Recombinantes/biosíntesis , Amidina-Liasas/química , Amidina-Liasas/aislamiento & purificación , Animales , Glucemia , Células CHO , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Cricetinae , Cricetulus , Diabetes Mellitus Experimental/tratamiento farmacológico , Evaluación Preclínica de Medicamentos , Femenino , Hipoglucemiantes/farmacología , Insulina/farmacología , Masculino , Oxigenasas de Función Mixta/química , Oxigenasas de Función Mixta/aislamiento & purificación , Ratas Wistar , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
Stearoyl-acyl carrier protein desaturase (SAD), locating in the plastid stroma, is an important fatty acid biosynthetic enzyme in higher plants. SAD catalyzes desaturation of stearoyl-ACP to oleyl-ACP and plays a key role in determining the homeostasis between saturated fatty acids and unsaturated fatty acids, which is an important player in cold acclimation in plants. Here, four new full-length cDNA of SADs (ScoSAD, SaSAD, ScaSAD and StSAD) were cloned from four Solanum species, Solanum commersonii, S. acaule, S. cardiophyllum and S. tuberosum, respectively. The ORF of the four SADs were 1182 bp in length, encoding 393 amino acids. A sequence alignment indicated 13 amino acids varied among the SADs of three wild species. Further analysis showed that the freezing tolerance and cold acclimation capacity of S. commersonii are similar to S. acaule and their SAD amino acid sequences were identical but differed from that of S. cardiophyllum, which is sensitive to freezing. Furthermore, the sequence alignments between StSAD and ScoSAD indicated that only 7 different amino acids at residues were found in SAD of S. tuberosum (Zhongshu8) against the protein sequence of ScoSAD. A phylogenetic analysis showed the three wild potato species had the closest genetic relationship with the SAD of S. lycopersicum and Nicotiana tomentosiformis but not S. tuberosum. The SAD gene from S. commersonii (ScoSAD) was cloned into multiple sites of the pBI121 plant binary vector and transformed into the cultivated potato variety Zhongshu 8. A freeze tolerance analysis showed overexpression of the ScoSAD gene in transgenic plants significantly enhanced freeze tolerance in cv. Zhongshu 8 and increased their linoleic acid content, suggesting that linoleic acid likely plays a key role in improving freeze tolerance in potato plants. This study provided some new insights into how SAD regulates in the freezing tolerance and cold acclimation in potato.
Asunto(s)
Genes de Plantas , Oxigenasas de Función Mixta/genética , Solanum tuberosum/genética , Adaptación Fisiológica/genética , Secuencia de Aminoácidos , Clonación Molecular , ADN Complementario/genética , Evolución Molecular , Congelación , Oxigenasas de Función Mixta/química , Datos de Secuencia Molecular , Plantas Modificadas Genéticamente , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Solanum tuberosum/enzimología , Solanum tuberosum/fisiologíaRESUMEN
We determined the molecular weight and some properties of multiple forms of phenol oxidase from tea leaves and four other perennial plants. It was shown that multiple high- and low-molecular forms of phenol oxidase differed in substrate specificity. Low-molecular forms of the enzyme mostly demonstrated hydroxylase activity, while high-molecular forms showed catechol oxidase activity. It was revealed that the withering stage of black tea production is accompanied by the formation of only high-molecular forms of phenol oxidase, which possess catechol oxidase activity crucial for the procurement of oxidative reactions and the quality of the product.
Asunto(s)
Camellia sinensis/química , Monofenol Monooxigenasa/química , Hojas de la Planta/química , Proteínas de Plantas/química , Té/química , Catecol Oxidasa/química , Desecación , Tecnología de Alimentos , Isoenzimas/química , Oxigenasas de Función Mixta/química , Peso Molecular , Oxidación-Reducción , Especificidad por SustratoRESUMEN
Cytochrome P450 monooxygenases (P450s) are known to oxidize hydrocarbons, albeit with limited substrate specificity across classes of these compounds. Here we report a P450 monooxygenase (CYP63A2) from the model ligninolytic white rot fungus Phanerochaete chrysosporium that was found to possess a broad oxidizing capability toward structurally diverse hydrocarbons belonging to mutagenic/carcinogenic fused-ring higher-molecular-weight polycyclic aromatic hydrocarbons (HMW-PAHs), endocrine-disrupting long-chain alkylphenols (APs), and crude oil aliphatic hydrocarbon n-alkanes. A homology-based three-dimensional (3D) model revealed the presence of an extraordinarily large active-site cavity in CYP63A2 compared to the mammalian PAH-oxidizing (CYP3A4, CYP1A2, and CYP1B1) and bacterial aliphatic-hydrocarbon-oxidizing (CYP101D and CYP102A1) P450s. This structural feature in conjunction with ligand docking simulations suggested potential versatility of the enzyme. Experimental characterization using recombinantly expressed CYP63A2 revealed its ability to oxidize HMW-PAHs of various ring sizes, including 4 rings (pyrene and fluoranthene), 5 rings [benzo(a)pyrene], and 6 rings [benzo(ghi)perylene], with the highest enzymatic activity being toward the 5-ring PAH followed by the 4-ring and 6-ring PAHs, in that order. Recombinant CYP63A2 activity yielded monohydroxylated PAH metabolites. The enzyme was found to also act as an alkane ω-hydroxylase that oxidized n-alkanes with various chain lengths (C9 to C12 and C15 to C19), as well as alkyl side chains (C3 to C9) in alkylphenols (APs). CYP63A2 showed preferential oxidation of long-chain APs and alkanes. To our knowledge, this is the first P450 identified from any of the biological kingdoms that possesses such broad substrate specificity toward structurally diverse xenobiotics (PAHs, APs, and alkanes), making it a potent enzyme biocatalyst candidate to handle mixed pollution (e.g., crude oil spills).
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Oxigenasas de Función Mixta/metabolismo , Phanerochaete/metabolismo , Hidrocarburos Policíclicos Aromáticos/metabolismo , Alcanos/metabolismo , Secuencia de Aminoácidos , Dominio Catalítico , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/genética , Hidrocarburos/metabolismo , Oxigenasas de Función Mixta/química , Oxigenasas de Función Mixta/genética , Datos de Secuencia Molecular , Oxidación-Reducción , Petróleo/metabolismo , Contaminación por Petróleo , Phanerochaete/enzimología , Hidrocarburos Policíclicos Aromáticos/química , Alineación de Secuencia , Especificidad por SustratoRESUMEN
A multistep enzyme catalysis was successfully implemented to produce long-chain α,ω-dicarboxylic and ω-hydroxycarboxylic acids from renewable fatty acids and plant oils. Sebacic acid as well as ω-hydroxynonanoic acid and ω-hydroxytridec-11-enoic acid were produced from oleic and ricinoleic acid.
Asunto(s)
Ácidos Dicarboxílicos/síntesis química , Ácidos Grasos/química , Aceites de Plantas/química , Ácidos Dicarboxílicos/análisis , Ácidos Dicarboxílicos/química , Oxigenasas de Función Mixta/química , Oxigenasas de Función Mixta/metabolismo , Pseudomonas fluorescens/enzimologíaRESUMEN
Indole-3-acetic acid (IAA), a major plant auxin, is produced in both tryptophan-dependent and tryptophan-independent pathways. A major pathway in Arabidopsis thaliana generates IAA in two reactions from tryptophan. Step one converts tryptophan to indole-3-pyruvic acid (IPA) by tryptophan aminotransferases followed by a rate-limiting step converting IPA to IAA catalyzed by YUCCA proteins. We identified eight putative StYUC (Solanum tuberosum YUCCA) genes whose deduced amino acid sequences share 50%-70% identity with those of Arabidopsis YUCCA proteins. All include canonical, conserved YUCCA sequences: FATGY motif, FMO signature sequence, and FAD-binding and NADP-binding sequences. In addition, five genes were found with ~50% amino acid sequence identity to Arabidopsis tryptophan aminotransferases. Transgenic potato (Solanum tuberosum cv. Jowon) constitutively overexpressing Arabidopsis AtYUC6 displayed high-auxin phenotypes such as narrow downward-curled leaves, increased height, erect stature, and longevity. Transgenic potato plants overexpressing AtYUC6 showed enhanced drought tolerance based on reduced water loss. The phenotype was correlated with reduced levels of reactive oxygen species in leaves. The results suggest a functional YUCCA pathway of auxin biosynthesis in potato that may be exploited to alter plant responses to the environment.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Ácidos Indolacéticos/metabolismo , Oxigenasas de Función Mixta/genética , Fenotipo , Solanum tuberosum/genética , Solanum tuberosum/metabolismo , Agua/metabolismo , Secuencia de Aminoácidos , Proteínas de Arabidopsis/química , Bases de Datos Genéticas , Expresión Génica , Oxigenasas de Función Mixta/química , Datos de Secuencia Molecular , Solanum tuberosum/fisiología , Estrés Fisiológico , Triptófano-Transaminasa/genéticaRESUMEN
Oxo-sulfido- and oxo-selenido-molybdenum(VI) complexes with an ene-1,2-dithiolate ligand are generated as models of the active sites of molybdenum hydroxylases. The sulfide and selenide groups are highly reactive toward triphenylphosphine in the order of Se > S.
Asunto(s)
Complejos de Coordinación/química , Oxigenasas de Función Mixta/química , Molibdeno/química , Selenio/química , Compuestos de Sulfhidrilo/química , Dominio Catalítico , Ligandos , Oxigenasas de Función Mixta/metabolismo , Conformación Molecular , TemperaturaRESUMEN
Peptidylglycine α-hydroxylating monooxygenase (PHM) catalyzes the stereospecific hydroxylation of the Cα of C-terminal glycine-extended peptides and proteins, the first step in the activation of many peptide hormones, growth factors, and neurotransmitters. The crystal structure of the enzyme revealed two nonequivalent Cu sites (Cu(M) and Cu(H)) separated by â¼11 Å. In the resting state of the enzyme, Cu(M) is coordinated in a distorted tetrahedral geometry by one methionine, two histidines, and a water molecule. The coordination site of the water molecule is the position where external ligands bind. The Cu(H) has a planar T-shaped geometry with three histidines residues and a vacant position that could potentially be occupied by a fourth ligand. Although the catalytic mechanism of PHM and the role of the metals are still being debated, Cu(M) is identified as the metal involved in catalysis, while Cu(H) is associated with electron transfer. To further probe the role of the metals, we studied how small molecules such as nitrite (NO(2)(-)), azide (N(3)(-)), and carbon monoxide (CO) interact with the PHM copper ions. The crystal structure of an oxidized nitrite-soaked PHMcc, obtained by soaking for 20 h in mother liquor supplemented with 300 mM NaNO(2), shows that nitrite anion coordinates Cu(M) in an asymmetric bidentate fashion. Surprisingly, nitrite does not bind Cu(H), despite the high concentration used in the experiments (nitrite/protein > 1000). Similarly, azide and carbon monoxide coordinate Cu(M) but not Cu(H) in the PHMcc crystal structures obtained by cocrystallization with 40 mM NaN(3) and by soaking CO under 3 atm of pressure for 30 min. This lack of reactivity at the Cu(H) is also observed in the reduced form of the enzyme: CO binds Cu(M) but not Cu(H) in the structure of PHMcc obtained by exposure of a crystal to 3 atm CO for 15 min in the presence of 5 mM ascorbic acid (reductant). The necessity of Cu(H) to maintain its redox potential in a narrow range compatible with its role as an electron-transfer site seems to explain the lack of coordination of small molecules to Cu(H); coordination of any external ligand will certainly modify its redox potential.
Asunto(s)
Cobre/química , Oxigenasas de Función Mixta/química , Complejos Multienzimáticos/química , Azidas/química , Sitios de Unión , Catálisis , Cristalografía por Rayos X , Modelos Moleculares , Estructura Molecular , EstereoisomerismoRESUMEN
Hyoscyamine 6ß-hydroxylase (H6H; EC 1.14.11.11), an important enzyme in the biosynthesis of tropane alkaloids, catalyzes the hydroxylation of hyoscyamine to give 6ß-hydroxyhyoscyamine and its epoxidation in the biosynthetic pathway leading to scopolamine. Datura metel produces scopolamine as the predominant tropane alkaloid. The cDNA encoding H6H from D. metel (DmH6H) was cloned, heterologously expressed and biochemically characterized. The purified recombinant His-tagged H6H from D. metel (DmrH6H) was capable of converting hyoscyamine to scopolamine. The functionally expressed DmrH6H was confirmed by HPLC and ESI-MS verification of the products, 6ß-hydroxyhyoscyamine and its derivative, scopolamine; the DmrH6H epoxidase activity was low compared to the hydroxylase activity. The K(m) values for both the substrates, hyoscyamine and 2-oxoglutarate, were 50µM each. The CD (circular dichroism) spectrum of the DmrH6H indicated a preponderance of α-helicity in the secondary structure. From the fluorescence studies, Stern-Volmer constants for hyoscyamine and 2-oxoglutarate were found to be 0.14M(-1) and 0.56M(-1), respectively. These data suggested that the binding of the substrates, hyoscyamine and 2-oxoglutarate, to the enzyme induced significant conformational changes.
Asunto(s)
Atropina/metabolismo , Datura metel/enzimología , Expresión Génica , Genes de Plantas , Oxigenasas de Función Mixta/química , Proteínas de Plantas/química , Escopolamina/biosíntesis , Dicroismo Circular , ADN Complementario , Datura metel/química , Datura metel/genética , Ácidos Cetoglutáricos/metabolismo , Cinética , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/aislamiento & purificación , Oxigenasas de Función Mixta/metabolismo , Oxidorreductasas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/aislamiento & purificación , Raíces de Plantas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
The unique amino acid hypusine is formed exclusively in eIF5A by the successive action of deoxyhypusine synthase and deoxyhypusine hydroxylase (yeast Lia1, human DOHH). Although the first enzyme has been extensively studied, both Lia1 structure and the mechanism of action remain unclear. Hence, a multi-approach was used to evaluate Lia1 catalysis, metal/substrate binding, structural conformation and stability. Mutational analyses of Lia1 revealed fine differences in the mode of substrate binding between the human and yeast counterparts. Like human DOHH, recombinant Lia1 is an iron metalloenzyme. Iron is essential for enzyme activity since its loss renders the enzyme totally inactive. The separation of iron-free and iron-bound forms by gel filtration and native electrophoresis suggests differences in Lia1 tertiary structure related to the iron binding. The ability of Lia1 to undergo conformational changes prompted us to use a set of complementary spectroscopic approaches and SAXS to obtain detailed information on the processes underlying dissociation of iron from Lia1 at different levels of the protein organization. The additive effect of weak interactions, especially within the metal center, resulted in an active enzyme in a stabilized and compact three-dimensional fold. Loss of tertiary contacts upon iron displacement led to an elongated conformation of Lia1, in which the N- and C-terminal domains are no longer in close proximity to guarantee the proper orientation of the active groups within the active site pocket. Our results demonstrate an essential structural role for iron binding in addition to its contribution to the catalysis of hypusine formation in the eIF-5A precursor.
Asunto(s)
Hierro/metabolismo , Oxigenasas de Función Mixta/química , Oxigenasas de Función Mixta/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Dominio Catalítico , Estabilidad de Enzimas , Cinética , Oxigenasas de Función Mixta/genética , Datos de Secuencia Molecular , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Bacterial and Archaeal cells use selenium structurally in selenouridine-modified tRNAs, in proteins translated with selenocysteine, and in the selenium-dependent molybdenum hydroxylases (SDMH). The first two uses both require the selenophosphate synthetase gene, selD. Examining over 500 complete prokaryotic genomes finds selD in exactly two species lacking both the selenocysteine and selenouridine systems, Enterococcus faecalis and Haloarcula marismortui. Surrounding these orphan selD genes, forming bidirectional best hits between species, and detectable by Partial Phylogenetic Profiling vs. selD, are several candidate molybdenum hydroxylase subunits and accessory proteins. We propose that certain accessory proteins, and orphan selD itself, are markers through which new selenium-dependent molybdenum hydroxylases can be found.