Role of a ribosomal RNA phosphate oxygen during the EF-G-triggered hydrolysis

Elongation factor-catalyzed GTP hydrolysis is a key reaction during the ribosomal elongation cycle. Recent crystal structures of G proteins, such as elongation factor G (EF-G) bound to the ribosome, as well as many biochemical studies, provide evidence that the direct interaction of translational GTPases (trGTPases) with the sarcin-ricin loop (SRL) of ribosomal RNA (rRNA) is pivotal for hydrolysis. However, the precise mechanism remains elusive and is intensively debated. Based on the close proximity of the phosphate oxygen of A2662 of the SRL to the supposedly catalytic histidine of EF-G (His87), we probed this interaction by an atomic mutagenesis approach. We individually replaced either of the two nonbridging phosphate oxygens at A2662 with a methyl group by the introduction of a methylphosphonate instead of the natural phosphate in fully functional, reconstituted bacterial ribosomes. Our major finding was that only one of the two resulting diastereomers, the SP methylphosphonate, was compatible with efficient GTPase activation on EF-G. The same trend was observed for a second trGTPase, namely EF4 (LepA). In addition, we provide evidence that the negative charge of the A2662 phosphate group must be retained for uncompromised activity in GTP hydrolysis. (1) In summary, our data strongly corroborate that the nonbridging proSP phosphate oxygen at the A2662 of the SRL is critically involved in the activation of GTP hydrolysis. A mechanistic scenario is supported in which positioning of the catalytically active, protonated His87 through electrostatic interactions with the A2662 phosphate group and H-bond networks are key features of ribosome-triggered activation of trGTPases.

Reactions of CF3-enones with arenes under superelectrophilic activation: a pathway to trans-1,3-diaryl-1-CF3-indanes, new cannabinoid receptor ligands

4-Aryl-1,1,1-trifluorobut-3-en-2-ones ArCH[double bond, length as m-dash]CHCOCF3 (CF3-enones) react with arenes in excess of Brønsted superacids (TfOH, FSO3H) to give, stereoselectively, trans-1,3-diaryl-1-trifluoromethyl indanes in 35-85% yields. The reaction intermediates, the O-protonated ArCH[double bond, length as m-dash]CHC(OH(+))CF3 and the O,C-diprotonated ArHC(+)CH2C(OH(+))CF3 species, have been studied by means of (1)H, (13)C, (19)F NMR, and DFT calculations. Both types of the cations may participate in the reaction, depending on their electrophilicity and electron-donating properties of the arenes. The formation of CF3-indanes is a result of cascade reaction of protonated CF3-enones to form chemo-, regio- and stereoselectively three new C-C bonds. The obtained trans-1,3-diaryl-1-trifluoromethyl indanes were investigated as potential ligands for cannabinoid receptors CB1 and CB2 types. The most potent compound showed sub-micromolar affinity for both receptor subtypes with a 6-fold selectivity toward the CB2 receptor with no appreciable cytotoxicity toward SHSY5Y cells.

Signal relay between two integral membrane proteins: The sensory rhodopsin I/HtrI transducer molecular complex

The molecular complex of sensory rhodopsin I (SRI) and its transducer HtrI mediate color-sensitive phototaxis in the archaeon Halobacterium salinarum. Orange light causes an attractant response by a one-photon reaction and white light causes a repellent response by a two-photon reaction. Three aspects of this molecular complex were explored: (i) We determined the stoichiometry of SRI and HtrI to be 2:2 by gene fusion analysis. A SRI-HtrI fusion protein was expressed in H. salinarum and shown to mediate 1-photon and 2-photon phototaxis responses comparable to wild-type complex. Disulfide crosslinking demonstrated that the fusion protein is a homodimer in the membrane. Measurement of photochemical reaction kinetics and pH titration of absorption spectra established that both SRI domains are complexed to HtrI in the fusion protein, and therefore the stoichiometry is 2:2. (ii) Cytoplasmic channel closure of SRI by HtrI, an important aspect of their interaction, was investigated by incremental HtrI truncation. We found that binding of the membrane-embedded portion of HtrI is insufficient for channel closure, whereas cytoplasmic extension of the second HtrI transmembrane helix by 13 residues blocks proton conduction through the channel as well as full-length HtrI. The closure activity is localized to 5 specific residues, each of which incrementally contributes to reduction of proton conductivity. Moreover, these same residues in the dark incrementally and proportionally increase the pKa of the Asp76 counterion to the protonated Schiff base chromophore. We conclude that this critical region of HtrI alters the dark conformation of SRI as well as light-induced channel opening. (iii) We developed a procedure for reconstituting HtrI-free SRI and the SRI/HtrI complex into liposomes, which exhibit photocycles with opened and closed cytoplasmic channels, respectively, as in the membrane. This opens the way for study of the light-induced conformational change and the interaction in vitro by fluorescence and spin-labeling. Single-cysteine mutations were introduced into helix F of SRI, labeled with a nitroxide spin probe and a fluorescence probe, reconstituted into proteoliposomes, and light-induced conformational changes detected in the complex. The probe signals can now be used as the readout of signaling to analyze mutants and the kinetics of signal relay. ^

Arabidopsis heterotrimeric G-protein regulates cell wall defense and resistance to necrotrophic fungi

The Arabidopsis heterotrimeric G-protein controls defense responses to necrotrophic and vascular fungi. The agb1 mutant impaired in the Gβ subunit displays enhanced susceptibility to these pathogens. Gβ/AGB1 forms an obligate dimer with either one of the Arabidopsis Gγ subunits (γ1/AGG1 and γ2/AGG2). Accordingly, we now demonstrate that the agg1 agg2 double mutant is as susceptible as agb1 plants to the necrotrophic fungus Plectosphaerella cucumerina. To elucidate the molecular basis of heterotrimeric G-protein-mediated resistance, we performed a comparative transcriptomic analysis of agb1-1 mutant and wild-type plants upon inoculation with P. cucumerina. This analysis, together with metabolomic studies, demonstrated that G-protein-mediated resistance was independent of defensive pathways required for resistance to necrotrophic fungi, such as the salicylic acid, jasmonic acid, ethylene, abscisic acid, and tryptophan-derived metabolites signaling, as these pathways were not impaired in agb1 and agg1 agg2 mutants. Notably, many mis-regulated genes in agb1 plants were related with cell wall functions, which was also the case in agg1 agg2 mutant. Biochemical analyses and Fourier Transform InfraRed (FTIR) spectroscopy of cell walls from G-protein mutants revealed that the xylose content was lower in agb1 and agg1 agg2 mutants than in wild-type plants, and that mutant walls had similar FTIR spectratypes, which differed from that of wild-type plants. The data presented here suggest a canonical functionality of the Gβ and Gγ1/γ2 subunits in the control of Arabidopsis immune responses and the regulation of cell wall composition.

Caracterización funcional de los genes sgb (suppressors of agb1-2 susceptibility to pathogens): Nuevos reguladores de la respuesta de inmunidad innata de Arabidopsis thaliana

Un porcentaje importante de las pérdidas de la producción agrícola se deben a las enfermedades que causan en los cultivos los hongos necrótrofos y vasculares. Para mejorar la productividad agrícola es necesario tener un conocimiento detallado de las bases genéticas y moleculares que regulan la resistencia de las plantas a este tipo de patógenos. En Arabidopsis thaliana la resistencia frente a patógenos necrótrofos, como el hongo Plectosphaerella cucumerina BMM (PcBMM), es genéticamente compleja y depende de la activación coordinada de distintas rutas de señalización, como las reguladas por las hormonas ácido salicílico (SA), ácido jasmónico (JA), etileno (ET) y ácido abscísico (ABA), así como de la síntesis de compuestos antimicrobianos derivados del Triptófano y de la integridad de la pared celular (Llorente et al., 2005, Hernández-Blanco et al., 2007; Delgado-Cerezo et al., 2012). Uno de los componentes claves en la regulación de la resistencia de las plantas a patógenos (incluidos hongos necrótrofos y biótrofos) es la proteína G heterotrimérica, un complejo proteico formado por tres subunidades (Gα, Gβ y Gγ), que también regula distintos procesos del desarrollo vegetal. En Arabidopsis hay un gen que codifica para la subunidad α (GPA1), otro para la β (AGB1), y tres genes para la subunidad γ (AGG1, AGG2 y AGG3). El complejo GPA1-AGB1-AGG (1-3) se activa y disocia tras la percepción de una señal específica, actuando el dímero AGB1-AGG1/2 como un monómero funcional que regula las respuestas de defensa (Delgado-Cerezo et al., 2012). Estudios transcriptómicos y análisis bioquímicos de la pared celular en los que se comparaban los mutantes agb1-2 y agg1 agg2, y plantas silvestres (Col-0) revelaron que la resistencia mediada por Gβ-Gγ1/2 no es dependiente de rutas de defensa previamente caracterizadas, y sugieren que la proteína G podría modular la composición/estructura (integridad) de la pared celular (Delgado-Cerezo et al., 2012). Recientemente, se ha demostrado que AGB1 es un componente fundamental de la respuesta inmune mediada por Pathogen- Associated Molecular Patterns (PTI), ya que los mutantes agb1-2 son incapaces de activar tras el tratamiento con PAMPs respuestas de inmunidad, como la producción de especies reactivas de oxígeno (ROS; Liu et al., 2013). Dada la importancia de la proteína G heterotrimérica en la regulación de la respuestas de defensa (incluida la PTI) realizamos un escrutinio de mutantes supresores de la susceptibilidad de agb1-2 al hongo necrótrofo, PcBMM, para identificar componentes adicionales de las rutas de señalización reguladas por AGB1. En este escrutinio se aislaron cuatro mutantes sgb (suppressors of agb1-2 susceptibility to pathogens), dos de los cuales, sgb10 y sgb11, se han caracterizado en la presente Tesis Doctoral. El mutante sgb10 es un segundo alelo nulo del gen MKP1 (At3g55270) que codifica la MAP quinasa-fosfatasa 1 (Bartels et al., 2009). Este mutante presenta lesiones espontáneas en plantas adultas y una activación constitutiva de las principales rutas de defensa (SA, JA y ET, y de metabolitos secundarios, como la camalexina), que explicaría su elevada resistencia a PcBMM y Pseudomonas syringae. Estudios epistáticos sugieren que la resistencia mediada por SGB10 no es dependiente, si no complementaria a la regulada por AGB1. El mutante sgb10 es capaz de restablecer en agb1-2 la producción de ROS y otras respuestas PTI (fosforilación de las MAPK6/3/4/11) tras el tratamiento con PAMPs tan diversos como flg22, elf18 y quitina, lo que demuestra el papel relevante de SGB10/MKP1 y de AGB1 en PTI. El mutante sgb11 se caracteriza por presentar un fenotipo similar a los mutantes irregular xylem (e.g. irx1) afectado en pared celular secundaria: irregularidades en las células xilemáticas, reducción en el tamaño de la roseta y altura de planta, y hojas con un mayor contenido de clorofila. La resistencia de sgb11 a PcBMM es independiente de agb1-2, ya que la susceptibilidad del doble mutante sgb11 agb1-2 es intermedia entre la de agb1-2 y sgb11. El mutante sgb11 no revierte la deficiente PTI de agb1-2 tras el tratamiento con flg22, lo que indica que está alterado en una ruta distinta de la regulada por SGB10. sgb11 presenta una sobreactivación de la ruta del ácido abscísico (ABA), lo que podría explicar su resistencia a PcBMM. La mutación sgb11 ha sido cartografiada en el cromosoma III de Arabidopsis entre los marcadores AthFUS6 (81,64cM) y nga6 (86,41cM) en un intervalo de aproximadamente 200 kb, que comprende genes, entre los que no se encuentra ninguno previamente descrito como IRX. El aislamiento y caracterización de SGB11 apoya la relevancia de la proteína G heterotrimérica en la regulación de la interconexión entre integridad de la pared celular e inmunidad. ABSTRACT A significant percentage of agricultural losses are due to diseases caused by necrotrophic and vascular fungi. To enhance crop yields is necessary to have a detailed knowledge of the genetic and molecular bases regulating plant resistance to these pathogens. Arabidopsis thaliana resistance to necrotrophic pathogens, such as Plectosphaerella cucumerina BMM (PcBMM) fungus, is genetically complex and depends on the coordinated activation of various signaling pathways. These include those regulated by salicylic acid (SA), jasmonic acid (JA), ethylene (ET) and abscisic acid (ABA) hormones and the synthesis of tryptophan-derived antimicrobial compounds and cell wall integrity (Llorente et al., 2005, Hernández-Blanco et al., 2007; Delgado-Cerezo et al., 2012). One key component in the regulation of plant resistance to pathogens (including biotrophic and necrotrophic fungi) is the heterotrimeric G-protein. This protein complex is formed by three subunits (Gα, Gβ and Gγ), which also regulates various plant developmental processes. In Arabidopsis only one gene encodes for subunits α (GPA1) and β (AGB1), and three genes for subunit γ (AGG1, AGG2 y AGG3). The complex GPA1- AGB1-AGG(1-3) is activated and dissociates after perception of an specific signal, AGB1- AGG1/2 acts as a functional monomer regulating defense responses (Delgado-Cerezo et al., 2012). Comparative transcriptomic studies and biochemical analyses of the cell wall of agb1-2 and agg1agg2 mutant and wild plants (Col-0), showed that Gβ-Gγ1/2-mediated resistance is not dependent on previously characterized defense pathways. In addition, it suggests that G protein may modulate the composition/structure (integrity) of the plant cell wall (Delgado-Cerezo et al., 2012). Recently, it has been shown that AGB1 is a critical component of the immune response mediated by Pathogen-Associated Molecular Patterns (PTI), as agb1-2 mutants are unable to activate immune responses such as oxygen reactive species (ROS) production after PAMPs treatment (Liu et al., 2013). Considering the importance of the heterotrimeric G protein in regulation of defense responses (including PTI), we performed a screening for suppressors of agb1-2 susceptibility to the necrotrophic fungus PcBMM. This would allow the identification of additional components of the signaling pathways regulated by AGB1. In this search four sgb mutants (suppressors of agb1-2 susceptibility to pathogens) were isolated, two of which, sgb10 and sgb11, have been characterized in this PhD thesis. sgb10 mutant is a second null allele of MKP1 gene (At3g55270), which encodes the MAP kinase-phosphatase 1 (Bartels et al., 2009). This mutant exhibits spontaneous lesions in adult plants and a constitutive activation of the main defense pathways (SA, JA and ET, and secondary metabolites, such as camalexin), which explains its high resistance to Pseudomonas syringae and PcBMM. Epistatic studies suggest that SGB10- mediated resistance is not dependent, but complementary to the regulated by AGB1. The sgb10 mutant is able to restore agb1-2 ROS production and other PTI responses (MAPK6/3/4/11 phosphorylation) upon treatment with PAMPs as diverse as, flg22, elf18 and chitin, demonstrating the relevant role of SGB10/MKP1 and AGB1 in PTI. sgb11 mutant is characterized by showing a similar phenotype to irregular xylem mutants (e.g. irx1), affected in secondary cell wall: irregular xylems cells, rosette size reduction and plant height, and higher chlorophyll content on leaves. The resistance of sgb11 to PcBMM is independent of agb1-2, as susceptibility of the double mutant agb1-2sgb11 is intermediate between agb1-2 and sgb11. The sgb11 mutant does not revert the deficient PTI response in agb1-2 after flg22 treatment, indicating that is altered in a pathway different to the one regulated by SGB10. sgb11 presents an over-activation of the abscisic acid pathway (ABA), which could explain its resistance to PcBMM. The sgb11 mutation has been mapped on chromosome III of Arabidopsis, between AthFUS6 (81.64 cM) and nga6 (86.41 cM) markers, in 200 kb interval, which does not include previously known IRX genes. The isolation and characterization of SGB11 supports the importance of heterotrimeric G protein in the regulation of the interconnection between the cell wall integrity and immunity.

YUCCA8 and YUCCA9 overexpression reveals a link between auxin signaling and lignification through the induction of ethylene biosynthesis.

Auxin is associated with the regulation of virtually every aspect of plant growth and development. Many previous genetic and biochemical studies revealed that, among the proposed routes for the production of auxin, the so-called indole-3-pyruvic acid (IPA) pathway is the main source for indole-3-acetic acid (IAA) in plants. The IPA pathway involves the action of 2 classes of enzymes, tryptophan-pyruvate aminotransferases (TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS 1(TAA1)/TRYPTOPHAN AMINOTRANSFERASE RELATED (TAR)) and flavin monooxygenases (YUCCA). Both enzyme classes appear to be encoded by small gene families in Arabidopsis consisting of 5 and 11 members, respectively. We recently showed that it is possible to induce transcript accumulation of 2 YUCCA genes, YUC8 and YUC9, by methyl jasmonate treatment. Both gene products were demonstrated to contribute to auxin biosynthesis in planta.1 Here we report that the overexpression of YUC8 as well as YUC9 led to strong lignification of plant aerial tissues. Furthermore, new evidence indicates that this abnormally strong secondary growth is linked to increased levels of ethylene production.

Amino acid availability in ruminants of cereals and cereal co-products

Microbial corrected in situ estimatesof the ruminal undegraded fraction (RU) and intestinal effectivedigestibility (IED) of amino acids (AA), except tryptophan, of rye, wheat and corn grains, wheat bran, wheat and barley distilled dried grains and corn gluten feed were measured on three rumen- and duodenum-cannulated wethers using 15N-labelling techniques and considering ruminal rates of particle comminution and outflow.

Functional characterization of YODA, a mitogen-activated protein kinase kinase kinase (MAP3K) that regulates a novel innate immunity pathway in Arabidopsis thaliana

Las cascadas de señalización mediadas por proteína quinasas activadas por mitógeno (MAP quinasas) son capaces de integrar y transducir señales ambientales en respuestas celulares. Entre estas señales se encuentran los PAMPs/MAMPs (Pathogen/Microbe-Associated Molecular Patterns), que son moléculas de patógenos o microorganismos, o los DAMPs (Damaged-Associated Molecular Patterns), que son moléculas derivadas de las plantas producidas en respuesta a daño celular. Tras el reconocimiento de los PAMPs/DAMPs por receptores de membrana denominados PRRs (Pattern Recognition Receptors), como los receptores con dominio quinasa (RLKs) o los receptores sin dominio quinasa (RLPs), se activan respuestas moleculares, incluidas cascadas de MAP quinasas, que regulan la puesta en marcha de la inmunidad activada por PAMPs (PTI). Esta Tesis describe la caracterización funcional de la MAP quinasa quinasa quinasa (MAP3K) YODA (YDA), que actúa como un regulador clave de la PTI en Arabidopsis. Se ha descrito previamente que YDA controla varios procesos de desarrollo, como la regulación del patrón estomático, la elongación del zigoto y la arquitectura floral. Hemos caracterizado un alelo mutante hipomórfico de YDA (elk2 o yda11) que presenta una elevada susceptibilidad a patógenos biótrofos y necrótrofos. Notablemente, plantas que expresan una forma constitutivamente activa de YDA (CA-YDA), con una deleción en el dominio N-terminal, presentan una resistencia de amplio espectro frente a diferentes tipos de patógenos, incluyendo hongos, oomicetos y bacterias, lo que indica que YDA juega un papel importante en la regulación de la resistencia de las plantas a patógenos. Nuestros datos indican que esta función es independiente de las respuestas inmunes mediadas por los receptores previamente caracterizados FLS2 y CERK1, que reconocen los PAMPs flg22 y quitina, respectivamente, y que están implicados en la resistencia de Arabidopsis frente a bacterias y hongos. Hemos demostrado que YDA controla la resistencia frente al hongo necrótrofo Plectosphaerella cucumerina y el patrón estomático mediante su interacción genética con la RLK ERECTA (ER), un PRR implicado en la regulación de estos procesos. Por el contrario, la interacción genética entre ER y YDA en la regulación de otros procesos de desarrollo es aditiva en lugar de epistática. Análisis genéticos indicaron que MPK3, una MAP quinasa que funciona aguas abajo de YDA en el desarrollo estomático, es un componente de la ruta de señalización mediada por YDA para la resistencia frente a P. cucumerina, lo que sugiere que el desarrollo de las plantas y la PTI comparten el módulo de transducción de MAP quinasas asociado a YDA. Nuestros experimentos han revelado que la resistencia mediada por YDA es independiente de las rutas de señalización reguladas por las hormonas de defensa ácido salicílico, ácido jasmónico, ácido abscísico o etileno, y también es independiente de la ruta de metabolitos secundarios derivados del triptófano, que están implicados en inmunidad vegetal. Además, hemos demostrado que respuestas asociadas a PTI, como el aumento en la concentración de calcio citoplásmico, la producción de especies reactivas de oxígeno, la fosforilación de MAP quinasas y la expresión de genes de defensa, no están afectadas en el mutante yda11. La expresión constitutiva de la proteína CA-YDA en plantas de Arabidopsis no provoca un aumento de las respuestas PTI, lo que sugiere la existencia de mecanismos de resistencia adicionales regulados por YDA que son diferentes de los regulados por FLS2 y CERK1. En línea con estos resultados, nuestros datos transcriptómicos revelan una sobre-representación en plantas CA-YDA de genes de defensa que codifican, por ejemplo, péptidos antimicrobianos o reguladores de muerte celular, o proteínas implicadas en la biogénesis de la pared celular, lo que sugiere una conexión potencial entre la composición e integridad de la pared celular y la resistencia de amplio espectro mediada por YDA. Además, análisis de fosfoproteómica indican la fosforilación diferencial de proteínas relacionadas con la pared celular en plantas CA-YDA en comparación con plantas silvestres. El posible papel de la ruta ER-YDA en la regulación de la integridad de la pared celular está apoyado por análisis bioquímicos y glicómicos de las paredes celulares de plantas er, yda11 y CA-YDA, que revelaron cambios significativos en la composición de la pared celular de estos genotipos en comparación con la de plantas silvestres. En resumen, nuestros datos indican que ER y YDA forman parte de una nueva ruta de inmunidad que regula la integridad de la pared celular y respuestas defensivas, confiriendo una resistencia de amplio espectro frente a patógenos. ABSTRACT Plant mitogen-activated protein kinase (MAPK) cascades transduce environmental signals and developmental cues into cellular responses. Among these signals are the pathogen- or microbe-associated molecular patterns (PAMPs or MAMPs) and the damage-associated molecular patterns (DAMPs). These PAMPs/DAMPs, upon recognition by plant pattern recognition receptors (PRRs), such as Receptor-Like Kinases (RLKs) and Receptor-Like Proteins (RLPs), activate molecular responses, including MAPK cascades, which regulate the onset of PAMP-triggered immunity (PTI). This Thesis describes the functional characterization of the MAPK kinase kinase (MAP3K) YODA (YDA) as a key regulator of Arabidopsis PTI. YDA has been previously described to control several developmental processes, such as stomatal patterning, zygote elongation and inflorescence architecture. We characterized a hypomorphic, non-embryo lethal mutant allele of YDA (elk2 or yda11) that was found to be highly susceptible to biotrophic and necrotrophic pathogens. Remarkably, plants expressing a constitutive active form of YDA (CA-YDA), with a deletion in the N-terminal domain, showed broad-spectrum resistance to different types of pathogens, including fungi, oomycetes and bacteria, indicating that YDA plays a relevant function in plant resistance to pathogens. Our data indicated that this function is independent of the immune responses regulated by the well characterized FLS2 and CERK1 RLKs, which are the PRRs recognizing flg22 and chitin PAMPs, respectively, and are required for Arabidopsis resistance to bacteria and fungi. We demonstrate that YDA controls resistance to the necrotrophic fungus Plectosphaerella cucumerina and stomatal patterning by genetically interacting with ERECTA (ER) RLK, a PRR involved in regulating these processes. In contrast, the genetic interaction between ER and YDA in the regulation of other ER-associated developmental processes was additive, rather than epistatic. Genetic analyses indicated that MPK3, a MAP kinase that functions downstream of YDA in stomatal development, also regulates plant resistance to P. cucumerina in a YDA-dependent manner, suggesting that the YDA-associated MAPK transduction module is shared in plant development and PTI. Our experiments revealed that YDA-mediated resistance was independent of signalling pathways regulated by defensive hormones like salicylic acid, jasmonic acid, abscisic acid or ethylene, and of the tryptophan-derived metabolites pathway, which are involved in plant immunity. In addition, we showed that PAMP-mediated PTI responses, such as the increase of cytoplasmic Ca2+ concentration, reactive oxygen species (ROS) burst, MAPK phosphorylation, and expression of defense-related genes are not impaired in the yda11 mutant. Furthermore, the expression of CA-YDA protein does not result in enhanced PTI responses, further suggesting the existence of additional mechanisms of resistance regulated by YDA that differ from those regulated by the PTI receptors FLS2 and CERK1. In line with these observations, our transcriptomic data revealed the over-representation in CA-YDA plants of defensive genes, such as those encoding antimicrobial peptides and cell death regulators, and genes encoding cell wall-related proteins, suggesting a potential link between plant cell wall composition and integrity and broad spectrum resistance mediated by YDA. In addition, phosphoproteomic data revealed an over-representation of genes encoding wall-related proteins in CA-YDA plants in comparison with wild-type plants. The putative role of the ER-YDA pathway in regulating cell wall integrity was further supported by biochemical and glycomics analyses of er, yda11 and CA-YDA cell walls, which revealed significant changes in the cell wall composition of these genotypes compared with that of wild-type plants. In summary, our data indicate that ER and YDA are components of a novel immune pathway that regulates cell wall integrity and defensive responses, which confer broad-spectrum resistance to pathogens.

Calculated 11B–13C NMR chemical shift relationship in hypercoordinate methonium and boronium ions

The boronium-carbonium continuum was extended to include hypercoordinated protonated methanes and their boron analogs. The 11B NMR chemical shifts of the hypercoordinated hydriodo boron compounds and the 13C NMR chemical shifts of the corresponding isoelectronic and isostructural carbocations were calculated by using the GIAO-MP2 method. The data show good linear correlation between 11B and 13C NMR chemical shifts, which indicates that the same factors that determine the chemical shifts of the boron nuclei also govern the chemical shifts of carbon nuclei of these hypercoordinated hydriodo compounds.

From ab initio quantum mechanics to molecular neurobiology: A cation–π binding site in the nicotinic receptor

The nicotinic acetylcholine receptor is the prototype ligand-gated ion channel. A number of aromatic amino acids have been identified as contributing to the agonist binding site, suggesting that cation–π interactions may be involved in binding the quaternary ammonium group of the agonist, acetylcholine. Here we show a compelling correlation between: (i) ab initio quantum mechanical predictions of cation–π binding abilities and (ii) EC50 values for acetylcholine at the receptor for a series of tryptophan derivatives that were incorporated into the receptor by using the in vivo nonsense-suppression method for unnatural amino acid incorporation. Such a correlation is seen at one, and only one, of the aromatic residues—tryptophan-149 of the α subunit. This finding indicates that, on binding, the cationic, quaternary ammonium group of acetylcholine makes van der Waals contact with the indole side chain of α tryptophan-149, providing the most precise structural information to date on this receptor. Consistent with this model, a tethered quaternary ammonium group emanating from position α149 produces a constitutively active receptor.

A glutamate residue in the catalytic center of the yeast chorismate mutase restricts enzyme activity to acidic conditions

Chorismate mutase acts at the first branchpoint of aromatic amino acid biosynthesis and catalyzes the conversion of chorismate to prephenate. Comparison of the x-ray structures of allosteric chorismate mutase from the yeast Saccharomyces cerevisiae with Escherichia coli chorismate mutase/prephenate dehydratase suggested conserved active sites between both enzymes. We have replaced all critical amino acid residues, Arg-16, Arg-157, Lys-168, Glu-198, Thr-242, and Glu-246, of yeast chorismate mutase by aliphatic amino acid residues. The resulting enzymes exhibit the necessity of these residues for catalytic function and provide evidence of their localization at the active site. Unlike some bacterial enzymes, yeast chorismate mutase has highest activity at acidic pH values. Replacement of Glu-246 in the yeast chorismate mutase by glutamine changes the pH optimum for activity of the enzyme from a narrow to a broad pH range. These data suggest that Glu-246 in the catalytic center must be protonated for maximum catalysis and restricts optimal activity of the enzyme to low pH.

A deletion in an indole synthase gene is responsible for the DIMBOA-deficient phenotype of bxbx maize

The biosynthesis of DIMBOA, a pesticidal secondary metabolite of maize, branches off the tryptophan pathway. We have previously demonstrated that indole is the last intermediate common to both the tryptophan and hydroxamic acid pathways. The earliest discovered mutant in the DIMBOA pathway, bxbx (benzoxazineless), is deficient in the production of DIMBOA and related compounds. This paper presents evidence that a gene identified by Kramer and Koziel [Kramer, V. C. & Koziel, M. G. (1995) Plant Mol. Biol. 27, 1183–1188] as maize tryptophan synthase α (TSA) is the site of the genetic lesion in the DIMBOA-deficient mutant maize line bxbx. We demonstrate that the TSA gene has sustained a 924-bp deletion in bxbx compared with its counterpart in wild-type maize. We report that the TSA gene maps to the same location as the bxbx mutation, on the short arm of chromosome 4. We present evidence that the very early and very high level of expression of TSA corresponds to the timing and level of DIMBOA biosynthesis but is strikingly different from the expression of the maize tryptophan synthase β (TSB) genes. We show that feeding indole to bxbx seedlings restores their ability to synthesize DIMBOA. We conclude that the maize enzyme initially named tryptophan synthase α in fact is a DIMBOA biosynthetic enzyme, and we propose that it be renamed indole synthase. This work confirms and enlarges upon the findings of Frey et al. [Frey, M. Chomet, P., Glawischniq, E., Stettner, C., Grün, S., Winklmair, A., Eisenreich, W., Bacher, A., Meeley, R. B., Briggs, S. P., Simcox, K. & Gierl, A. (1997) Science 277, 696–699], which appeared while the present paper was in review.

Yeast chorismate mutase in the R state: Simulations of the active site

The isomerization of chorismate to prephenate by chorismate mutase in the biosynthetic pathway that forms Tyr and Phe involves C5—O (ether) bond cleavage and C1—C9 bond formation in a Claisen rearrangement. Development of negative charge on the ether oxygen, stabilized by Lys-168 and Glu-246, is inferred from the structure of a complex with a transition state analogue (TSA) and from the pH-rate profile of the enzyme and the E246Q mutant. These studies imply a protonated Glu-246 well above pH 7. Here, several 500-ps molecular dynamics simulations test the stability of enzyme–TSA complexes by using a solvated system with stochastic boundary conditions. The simulated systems are (i) protonated Glu-246 (stable), (ii) deprotonated Glu-246 (unstable), (iii) deprotonated Glu-246 plus one H2O between Glu-246 and the ether oxygen (unstable), (iv) the E246Q mutant (stable), and (v) addition of OH− between protonated Glu-246 and the ether oxygen. In (v), a local conformational change of Lys-168 displaced the OH− into the solvent region, suggesting a possible rate-determining step that precedes the catalytic step. In a 500-ps simulation of the enzyme complexed with the reactant chorismate or the product prephenate, no water molecule remained near the oxygen of the ligand. Calculations using the linearized Poisson–Boltzmann equation show that the effective pKa of Glu-246 is shifted from 5.8 to 8.1 as the negative charge on the ether oxygen of the TSA is changed from −0.56 electron to −0.9 electron. Altogether, these results support retention of a proton on Glu-246 to high pH and the absence of a water molecule in the catalytic steps.

A mammalian mitochondrial drug receptor functions as a bacterial “oxygen” sensor

The rat mitochondrial outer membrane-localized benzodiazepine receptor (MBR) was expressed in wild-type and TspO− (tryptophan-rich sensory protein) strains of the facultative photoheterotroph, Rhodobacter sphaeroides 2.4.1, and was shown to retain its structure within the bacterial outer membrane as assayed by its binding properties with a variety of MBR ligands. Functionally, it was able to substitute for TspO by negatively regulating the expression of photosynthesis genes in response to oxygen. This effect was reversed pharmacologically with the MBR ligand PK11195. These results suggest a close evolutionary and functional relationship between the bacterial TspO and the MBR. This relationship provides further support for the origin of the mammalian mitochondrion from a “photosynthetic” precursor. Finally, these findings provide novel insights into the physiological role that has been obscure for the MBR in situ.

Differences between males and females in rates of serotonin synthesis in human brain

Rates of serotonin synthesis were measured in the human brain using positron emission tomography. The sensitivity of the method is indicated by the fact that measurements are possible even after a substantial lowering of synthesis induced by acute tryptophan depletion. Unlike serotonin levels in human brain, which vary greatly in different brain areas, rates of synthesis of the indolamine are rather uniform throughout the brain. The mean rate of synthesis in normal males was found to be 52% higher than in normal females; this marked difference may be a factor relevant to the lower incidence of major unipolar depression in males.