Comparative functional genomic study of substrate specificity evolution of the SABATH family of methyltransferases in plants

Background The plant SABATH protein family is composed of a group of related small molecule methyltransferases (MTs) that catalyze the S-adenosyl-L-methionine dependent methylation of a variety of plant small molecular weight metabolites encompassing widely divergent structures. Some of these substrates are important plant hormones and signaling molecules, such as indole-3-acetic acid (IAA), jasmonic acid (JA) and salicylic acid (SA). Methylating these compounds may have important impacts on plant growth and development. In the previous paper, we presented Indole-3-acetic acid (IAA) methyltransferase (IAMT) as an evolutionarily ancient member of the SABATH family in higher plants. Whether the IAMT exists in less evolutionarily advanced plants is still unknown. Materials and methods To further understand the evolution of the SABATH family in land plants, we undertook an integrated functional genomic approach to identify and characterize SABATH genes in a lower plant moss (Physcomitrella patens). Four putative moss SABATH genes were identified using bioinformatics tools. Results and conclusion Enzymatic assay displayed none of them had IAMT activity, suggesting that IAMT might be evolved after the divergence of lower and higher plants. However, one of them, PpSABATH1, showed methyltransferase activity with a number of compounds containing sulhydryl or selenohydryl groups. Tobacco plants overexpressing the PpSABATH1 gene under the control of CaMV35S promoter exhibited an enhanced tolerance to thiobenzoic acid. Together with these results, we hypothesize that IAMT, the evolutionarily ancient member of the SABATH family in higher plants, evolved from a sulfur methyltransferase

Comparative functional genomic study of substrate specificity evolution of the SABATH family of methyltransferases in plants

Background The plant SABATH protein family is composed of a group of related small molecule methyltransferases (MTs) that catalyze the S-adenosyl-L-methionine dependent methylation of a variety of plant small molecular weight metabolites encompassing widely divergent structures. Some of these substrates are important plant hormones and signaling molecules, such as indole-3-acetic acid (IAA), jasmonic acid (JA) and salicylic acid (SA). Methylating these compounds may have important impacts on plant growth and development. In the previous paper, we presented Indole-3-acetic acid (IAA) methyltransferase (IAMT) as an evolutionarily ancient member of the SABATH family in higher plants. Whether the IAMT exists in less evolutionarily advanced plants is still unknown. Materials and methods To further understand the evolution of the SABATH family in land plants, we undertook an integrated functional genomic approach to identify and characterize SABATH genes in a lower plant moss (Physcomitrella patens). Four putative moss SABATH genes were identified using bioinformatics tools. Results and conclusion Enzymatic assay displayed none of them had IAMT activity, suggesting that IAMT might be evolved after the divergence of lower and higher plants. However, one of them, PpSABATH1, showed methyltransferase activity with a number of compounds containing sulhydryl or selenohydryl groups. Tobacco plants overexpressing the PpSABATH1 gene under the control of CaMV35S promoter exhibited an enhanced tolerance to thiobenzoic acid. Together with these results, we hypothesize that IAMT, the evolutionarily ancient member of the SABATH family in higher plants, evolved from a sulfur methyltransferase

Biochemical and structural characterization of the Arabidopsis bifunctional enzyme dethiobiotin synthetase-diaminopelargonic acid aminotransferase: evidence for substrate channeling in biotin synthesis.

International audienceDiaminopelargonic acid aminotransferase (DAPA-AT) and dethiobiotin synthetase (DTBS) catalyze the antepenultimate and the penultimate steps, respectively, of biotin synthesis. Whereas DAPA-AT and DTBS are encoded by distinct genes in bacteria, in biotin-synthesizing eukaryotes (plants and most fungi), both activities are carried out by a single enzyme encoded by a bifunctional gene originating from the fusion of prokaryotic monofunctional ancestor genes. In few angiosperms, including Arabidopsis thaliana, this chimeric gene (named BIO3-BIO1) also produces a bicistronic transcript potentially encoding separate monofunctional proteins that can be produced following an alternative splicing mechanism. The functional significance of the occurrence of a bifunctional enzyme in biotin synthesis pathway in eukaryotes and the relative implication of each of the potential enzyme forms (bifunctional versus monofunctional) in the plant biotin pathway are unknown. In this study, we demonstrate that the BIO3-BIO1 fusion protein is the sole protein form produced by the BIO3-BIO1 locus in Arabidopsis. The enzyme catalyzes both DAPA-AT and DTBS reactions in vitro and is targeted to mitochondria in vivo. Our biochemical and kinetic characterizations of the pure recombinant enzyme show that in the course of the reaction, the DAPA intermediate is directly transferred from the DAPA-AT active site to the DTBS active site. Analysis of several structures of the enzyme crystallized in complex with and without its ligands reveals key structural elements involved for acquisition of bifunctionality and brings, together with mutagenesis experiments, additional evidences for substrate channeling

Amélioration et automatisation des étapes de préparation des cristaux de protéines à la diffraction aux rayons X

Crystallography is from far the most contributing technique for the structure analysis of macromolecules at atomic resolution. In this thesis, instrumentation development issues to improve and accelerate experimental procedures for X-ray diffraction experiments are tackled. Indeed the preparation steps of protein crystals for X-ray diffraction data collection are the main causes of forming a bottleneck towards automated pipelines from protein crystallization to structure resolution. Firstly, an emerging method in today macromolecular crystallography is the room temperature in situ X-ray diffraction of protein crystal samples in their crystallization drops, with proven benefits in crystal screening and also structure resolution. However, it requires a great number of crystals to be centered and diffracted in a row. Thus a fully automated system providing a solution to this requirement is presented and assessed in this manuscript as one of the results of this PhD studies. Secondly, in this manuscript, studies and developments on automating harvesting, cryo-protecting and flash-cooling steps of protein crystals preparation for X-ray diffraction are reported, as well as assessment experiments and results. With a new robotic approach, crystals are manipulated with a micro-gripper on a 6-axis robotic arm to prepare and to analyze crystals with 360 rotation possibility for cryo-temperature single wavelength X-ray diffraction. Lysozyme and NikA Fe-EDTA protein crystals has been prepared and diffracted with this new method. Structural comparisons show no differences between the new methodology and the manual one, while robustness, repeatability and experimental time are significantly improved. At last, different integration scenarios of the presented methodologies, highlights their interest in fully automated macromolecular crystallography pipelines.La cristallographie est la technique qui contribue le plus à l'analyse des structures des macromolécules biologiques à la résolution atomique. Dans ce manuscrit de thèse nous abordons des développements instrumentaux pour l'amélioration et l'accélération des étapes expérimentales dans la procédure de mesure de la diffraction aux rayons X. En effet, les étapes de préparation des cristaux de protéine à la diffraction aux rayons X constituent la cause principale du goulot d'étranglement dans les plateformes à haut débit de la cristallisation des protéines jusqu'à la résolution des structures. Premièrement, la diffraction in situ aux rayons X des cristaux à la température ambiante, dans les plaques de cristallisation, est une méthodologie émergeante dans la cristallographie des protéines avec des capacités bénéfiques dans le criblage des cristaux mais aussi dans la résolution de structures. Cependant, un grand nombre de cristaux devront être centrés puis analysés par la diffraction aux rayons X automatiquement l'un à la suite de l'autre. Ainsi, un système automatisé répondant à cette exigence est présenté et évalué dans ce manuscrit comme étant l'un des résultats des études menées au cours de cette thèse. Deuxièmement, des études et des développements d'automatisation des étapes d'extraction et de micromanipulation, de cryo-protection et de congélation rapide pour la préparation des cristaux à la diffraction aux rayons X sont décrits dans ce manuscrit, ainsi que les résultats des expériences et des évaluations. Avec une approche nouvelle, les cristaux sont manipulés grâce à une micro-pince montée sur un bras robotique 6-axes pour les préparer et les analyser avec la possibilité de rotation de 360 pour la diffraction aux rayons X à longueur d'onde constate et à température cryogénique. Des cristaux des protéines lysozyme et NikA Fe-EDTA ont été préparés et diffractés avec cette nouvelle méthode. La comparaison structurale ne montre pas de différence entre la nouvelle méthode et celle manuelle, cependant la robustesse, la répétabilité et le gain de temps d'expériences sont significativement améliorés. Finalement, différents scénarios d'intégration des méthodologies présentées, met en évidence leurs intérêts dans les plateformes tout automatisés de cristallographie des macromolécules biologiques.SAVOIE-SCD - Bib.électronique (730659901) / SudocGRENOBLE1/INP-Bib.électronique (384210012) / SudocGRENOBLE2/3-Bib.électronique (384219901) / SudocSudocFranceF

Crystal structure of the cold-active aminopeptidase from Colwellia psychrerythraea, a close structural homologue of the human bifunctional leukotriene A4 hydrolase

peer reviewedThe crystal structure of a cold-active aminopeptidase (ColAP) from Colwellia psychrerythraea strain 34H has been determined, extending the number of crystal structures of the M1 metallopeptidase family to four among the 436 members currently identified. In agreement with their sequence similarity, the overall structure of ColAP displayed a high correspondence with leukotriene A4 hydrolase (LTA4H), a human bifunctional enzyme that converts leukotriene A4 (LTA4) in the potent chemoattractant leukotriene B4. Indeed, both enzymes are composed of three domains, an N-terminal saddle-like domain, a catalytic thermolysin-like domain, and a less conserved C-terminal alpha-helical flat spiral domain. Together, these domains form a deep cavity harboring the zinc binding site formed by residues included in the conserved HEXXHX(18)H motif. A detailed structural comparison of these enzymes revealed several plausible determinants of ColAP cold adaptation. The main differences involve specific amino acid substitutions, loop content and solvent exposure, complexity and distribution of ion pairs, and differential domain flexibilities. Such elements may act synergistically to allow conformational flexibility needed for an efficient catalysis in cold environments. Furthermore, the region of ColAP corresponding to the aminopeptidase active site of LTA4H is much more conserved than the suggested LTA4 substrate binding region. This observation supports the hypothesis that this region of the LTA4H active site has evolved in order to fit the lipidic substrate

Analytical ultracentrifugation and preliminary X-ray studies of the chloroplast envelope quinone oxidoreductase homologue from Arabidopsis thaliana.

International audienceQuinone oxidoreductases reduce a broad range of quinones and are widely distributed among living organisms. The chloroplast envelope quinone oxidoreductase homologue (ceQORH) from Arabidopsis thaliana binds NADPH, lacks a classical N-terminal and cleavable chloroplast transit peptide, and is transported through the chloroplast envelope membrane by an unknown alternative pathway without cleavage of its internal chloroplast targeting sequence. To unravel the fold of this targeting sequence and its substrate specificity, ceQORH from A. thaliana was overexpressed in Escherichia coli, purified and crystallized. Crystals of apo ceQORH were obtained and a complete data set was collected at 2.34 Å resolution. The crystals belonged to space group C2221, with two molecules in the asymmetric unit

Cyclic olefin homopolymer-based microfluidics for protein crystallization and in situ X-ray diffraction

A cyclic olefin homopolymer-based microfluidics system has been established for protein crystallization and in situ X-ray diffraction

Structure of the Mg-Chelatase Cofactor GUN4 Reveals a Novel Hand-Shaped Fold for Porphyrin Binding

In plants, the accumulation of the chlorophyll precursor Mg-protoporphyrin IX (Mg-Proto) in the plastid regulates the expression of a number of nuclear genes with functions related to photosynthesis. Analysis of the plastid-to-nucleus signaling activity of Mg-Proto in Arabidopsis thaliana led to the discovery of GUN4, a novel porphyrin-binding protein that also dramatically enhances the activity of Mg-chelatase, the enzyme that synthesizes Mg-Proto. GUN4 may also play a role in both photoprotection and the cellular shuttling of tetrapyrroles. Here we report a 1.78-Å resolution crystal structure of Synechocystis GUN4, in which the porphyrin-binding domain adopts a unique three dimensional fold with a “cupped hand” shape. Biophysical and biochemical analyses revealed the specific site of interaction between GUN4 and Mg-Proto and the energetic determinants for the GUN4 • Mg-Proto interaction. Our data support a novel protective function for GUN4 in tetrapyrrole trafficking. The combined structural and energetic analyses presented herein form the physical-chemical basis for understanding GUN4 biological activity, including its role in the stimulation of Mg-chelatase activity, as well as in Mg-Proto retrograde signaling