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

A β Strand Lock Exchange for Signal Transduction in TonB-Dependent Transducers on the Basis of a Common Structural Motif

SummaryTransport of molecules larger than 600 Da across the outer membrane involves TonB-dependent receptors and TonB-ExbB-ExbD of the inner membrane. The transport is energy consuming, and involves direct interactions between a short N-terminal sequence of receptor, called the TonB box, and TonB. We solved the structure of the ferric pyoverdine (Pvd-Fe) outer membrane receptor FpvA from Pseudomonas aeruginosa in its apo form. Structure analyses show that residues of the TonB box are in a β strand which interacts through a mixed four-stranded β sheet with the periplasmic signaling domain involved in interactions with an inner membrane sigma regulator. In this conformation, the TonB box cannot form a four-stranded β sheet with TonB. The FhuA-TonB or BtuB-TonB structures show that the TonB-FpvA interactions require a conformational change which involves a β strand lock-exchange mechanism. This mechanism is compatible with movements of the periplasmic domain deduced from crystallographic analyses of FpvA, FpvA-Pvd, and FpvA-Pvd-Fe

Tyrosine metabolism: identification of a key residue in the acquisition of prephenate aminotransferase activity by 1β aspartate aminotransferase

International audienceAlternative routes for the post-chorismate branch of the biosynthetic pathway leading to tyrosine exist, the 4-hydroxyphenylpyruvate or the arogenate route. The arogenate route involves the transamination of prephenate into arogenate. In a previous study, we found that, depending on the microorganisms possessing the arogenate route, three different aminotransferases evolved to perform prephenate transamination, that is, 1β aspartate aminotransferase (1β AAT), N-succinyl-l,l-diaminopimelate aminotransferase, and branched-chain aminotransferase. The present work aimed at identifying molecular determinant(s) of 1β AAT prephenate aminotransferase (PAT) activity. To that purpose, we conducted X-ray crystal structure analysis of two PAT competent 1β AAT from Arabidopsis thaliana and Rhizobium meliloti and one PAT incompetent 1β AAT from R. meliloti. This structural analysis supported by site-directed mutagenesis, modeling, and molecular dynamics simulations allowed us to identify a molecular determinant of PAT activity in the flexible N-terminal loop of 1β AAT. Our data reveal that a Lys/Arg/Gln residue in position 12 in the sequence (numbering according to Thermus thermophilus 1β AAT), present only in PAT competent enzymes, could interact with the 4-hydroxyl group of the prephenate substrate, and thus may have a central role in the acquisition of PAT activity by 1β AAT

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

Host-parasite co-metabolic activation of antitrypanosomal aminomethyl-benzoxaboroles

<div><p>Recent development of benzoxaborole-based chemistry gave rise to a collection of compounds with great potential in targeting diverse infectious diseases, including human African Trypanosomiasis (HAT), a devastating neglected tropical disease. However, further medicinal development is largely restricted by a lack of insight into mechanism of action (MoA) in pathogenic kinetoplastids. We adopted a multidisciplinary approach, combining a high-throughput forward genetic screen with functional group focused chemical biological, structural biology and biochemical analyses, to tackle the complex MoAs of benzoxaboroles in <i>Trypanosoma brucei</i>. We describe an oxidative enzymatic pathway composed of host semicarbazide-sensitive amine oxidase and a trypanosomal aldehyde dehydrogenase TbALDH3. Two sequential reactions through this pathway serve as the key underlying mechanism for activating a series of 4-aminomethylphenoxy-benzoxaboroles as potent trypanocides; the methylamine parental compounds as pro-drugs are transformed first into intermediate aldehyde metabolites, and further into the carboxylate metabolites as effective forms. Moreover, comparative biochemical and crystallographic analyses elucidated the catalytic specificity of TbALDH3 towards the benzaldehyde benzoxaborole metabolites as xenogeneic substrates. Overall, this work proposes a novel drug activation mechanism dependent on both host and parasite metabolism of primary amine containing molecules, which contributes a new perspective to our understanding of the benzoxaborole MoA, and could be further exploited to improve the therapeutic index of antimicrobial compounds.</p></div

Etude structurale d'une Aldéhyde Déshydrogénase NADP dépendante de Streptococcus mutans

Non disponible / Not availableL'aldh de streptococcus mutans, enzyme tetramerique de 200 kda, peut être décrite comme un dimère de dimère. Le monomère se divise en trois domaines : le domaine de fixation du cofacteur, le domaine catalytique et un domaine d'oligomerisation. L'apoenzyme est en interaction avec plusieurs ions sulfate mimant les groupements phosphate du nadp et du d-g3p. L'étude structurale de l'holoenzyme a permis de mettre en évidence le nouveau mode de fixation du nadp au domaine de rossmann et les facteurs moléculaires majeurs responsables de la spécificité de cofacteur. La comparaison structurale de l'Apo et de l'holoenzyme montre deux changements locaux de conformation dus à la fixation du nadp. L'un de ceux-ci est la rotation de la chaine latérale de c284. Cette rotation positionne son groupement sulphydrile à proximité des dipôles de la chaine principale et l'oriente vers le site catalytique. Ce changement serait à l'origine de la baisse de son pka et de son changement d'accessibilité nécessaires à l'acylation. L'étude d'une seconde structure d'apoenzyme a révélé la présence d'un second ion sulfate dans le site catalytique en interaction avec l'asparagine conservée asn154 et avec le groupement nH de cys284. Cette interaction est également observée avec la fonction aldéhydique du substrat dans la structure du complexe c284s-nadp-g3p suggérant la présence d'un site oxyanion stabilisant différents intermédiaires réactionnels. La comparaison des différentes structures d'aldh a permis de mettre en évidence la flexibilité du nad(p) en plus de celle de glu250. Les différentes positions et conformations observées pour le cofacteur chez les aldh résultent probablement de son mode particulier de fixation au domaine de rossmann qui serait du à la longueur de la première boucle du domaine de rossmann des aldh, dont l'orientation serait contrainte par la présence d'une proline conservée. La flexibilité du cofacteur et du glutamate suggèrent leurs mouvements au cours de l'acte catalytique

Etude structurale d'une Aldéhyde Déshydrogénase NADP dépendante de Streptococcus mutans

NANCY1-SCD Sciences & Techniques (545782101) / SudocSudocFranceF

Crystallization of cytochrome bc1 complex

A significant fraction of the proteins in any genome are membrane proteins, and membrane proteins are vital to many fundamental processes of life. While many membrane proteins have now been crystallized in three dimensions, membrane proteins have proven considerably more difficult to crystallize than soluble proteins, and some have not yielded after extensive efforts at crystallization, e.g., the mitochondrial adenine nucleotide transporter, the bacterial chemotaxis receptor, and lactose permease.One membrane protein that crystallizes fairly readily is the mitochondrial cytochrome bc1 complex. This 11-subunit respiratory enzyme has been crystallized in at least eight different crystal forms. We here review conditions that lead to crystallization in various forms, and provide some of the history of our crystallization attempts

Crystallization of cytochrome bc1 complex

A significant fraction of the proteins in any genome are membrane proteins, and membrane proteins are vital to many fundamental processes of life. While many membrane proteins have now been crystallized in three dimensions, membrane proteins have proven considerably more difficult to crystallize than soluble proteins, and some have not yielded after extensive efforts at crystallization, e.g., the mitochondrial adenine nucleotide transporter, the bacterial chemotaxis receptor, and lactose permease.One membrane protein that crystallizes fairly readily is the mitochondrial cytochrome bc1 complex. This 11-subunit respiratory enzyme has been crystallized in at least eight different crystal forms. We here review conditions that lead to crystallization in various forms, and provide some of the history of our crystallization attempts