The crystal structure of the ring-hydroxylating dioxygenase from Sphingomonas CHY-1

International audienceThe ring-hydroxylating dioxygenase (RHD) from Sphingomonas CHY-1 is remarkable due to its ability to initiate the oxidation of a wide range of polycyclic aromatic hydrocarbons (PAHs), including PAHs containing four- and five-fused rings, known pollutants for their toxic nature. Although the terminal oxygenase from CHY-1 exhibits limited sequence similarity with well characterized RHDs from the naphthalene dioxygenase family, the crystal structure determined to 1.85 Å by molecular replacement revealed the enzyme to share the same global α3β3 structural pattern. The catalytic domain distinguishes itself from other bacterial non-heme Rieske iron oxygenases by a substantially larger hydrophobic substrate binding pocket, the largest ever reported for this type of enzyme. While residues in the proximal region close to the mononuclear iron atom are conserved, the central region of the catalytic pocket is shaped mainly by the side chains of three amino acids, Phe350, Phe404 ad Leu356, which contribute to the uniform trapezoidal form of the pocket. Two flexible loops, LI and LII, exposed to the solvent seem to control the substrate access to the catalytic pocket and control the pocket length. Compared with other naphthalene dioxygenases residues Leu223 and Leu226, on loop LI, are moved toward the solvent, thus elongating the catalytic pocket by at least 2 Å. An 11 Å long water channel extends from the interface between the α and β subunits to the catalytic site. The comparison of these structures with other known oxygenases suggests that the broad substrate specificity presented by the CHY-1 oxygenase is primarily due to the large size and particular topology of its catalytic pocket and provided the basis for the study of its reaction mechanism

Characterization of a naphthalene dioxygenase endowed with an exceptionally broad substrate specificity toward polycyclic aromatic hydrocarbons

International audienceIn Sphingomonas CHY-1, a single ring-hydroxylating dioxygenase is responsible for the initial attack of a range of polycyclic aromatic hydrocarbons (PAHs) composed of up to five rings. The components of this enzyme were separately purified and characterized. The oxygenase component (ht-PhnI) was shown to contain one Rieske-type [2Fe-2S] cluster and one mononuclear Fe center per alpha subunit, based on EPR measurements and iron assay. Steady-state kinetic measurements revealed that the enzyme had a relatively low apparent Michaelis constant for naphthalene (Km= 0.92 ± 0.15 µM), and an apparent specificity constant of 2.0 ± 0.3 µM-1 s-1. Naphthalene was converted to the corresponding 1,2-dihydrodiol with stoichiometric oxidation of NADH. On the other hand, the oxidation of eight other PAHs occurred at slower rates, and with coupling efficiencies that decreased with the enzyme reaction rate. Uncoupling was associated with hydrogen peroxide formation, which is potentially deleterious to cells and might inhibit PAH degradation. In single turnover reactions, ht-PhnI alone catalyzed PAH hydroxylation at a faster rate in the presence of organic solvent, suggesting that the transfer of substrate to the active site is a limiting factor. The four-ring PAHs chrysene and benz[a]anthracene were subjected to a double ring-dihydroxylation, giving rise to the formation of a significant proportion of bis-cis-dihydrodiols. In addition, the dihydroxylation of benz[a]anthracene yielded three dihydrodiols, the enzyme showing a preference for carbons in positions 1,2 and 10,11. This is the first characterization of a dioxygenase able to dihydroxylate PAHs made up of four and five rings

Diversity and catalytic potential of PAH-specific ring-hydroxylating dioxygenases from a hydrocarbon-contaminated soil.

International audienceRing-hydroxylating dioxygenases (RHDs) catalyze the initial oxidation step of a range of aromatic hydrocarbons including polycyclic aromatic hydrocarbons (PAHs). As such, they play a key role in the bacterial degradation of these pollutants in soil. Several polymerase chain reaction (PCR)-based methods have been implemented to assess the diversity of RHDs in soil, allowing limited sequence-based predictions on RHD function. In the present study, we developed a method for the isolation of PAH-specific RHD gene sequences of Gram-negative bacteria, and for analysis of their catalytic function. The genomic DNA of soil PAH degraders was labeled in situ by stable isotope probing, then used to PCR amplify sequences specifying the catalytic domain of RHDs. Sequences obtained fell into five clusters phylogenetically linked to RHDs from either Sphingomonadales or Burkholderiales. However, two clusters comprised sequences distantly related to known RHDs. Some of these sequences were cloned in-frame in place of the corresponding region of the phnAIa gene from Sphingomonas CHY-1 to generate hybrid genes, which were expressed in Escherichia. coli as chimerical enzyme complexes. Some of the RHD chimeras were found to be competent in the oxidation of two- and three-ring PAHs, but other appeared unstable. Our data are interpreted in structural terms based on 3D modeling of the catalytic subunit of hybrid RHDs. The strategy described herein might be useful for exploring the catalytic potential of the soil metagenome and recruit RHDs with new activities from uncultured soil bacteria

\u3ci\u3eParamecium bursaria\u3c/i\u3e Chlorella Virus 1 Encodes a Polyamine Acetyltransferase

Background: PBCV-1 gene a654l encodes a protein with sequence similarity to GCN5 histone acetyltransferases. Results: A crystal structure of A654L bound to coenzyme A reveals how A654L acetylates polyamines, not histone lysines. Conclusion: A654L functions as a polyamine acetyltransferase. Significance: As the first viral polyamine acetyltransferase, A654L has a possible role in host polyamine catabolism in viral replication. Paramecium bursaria chlorella virus 1 (PBCV-1), a large DNA virus that infects green algae, encodes a histone H3 lysine 27-specific methyltransferase that functions in global transcriptional silencing of the host. PBCV-1 has another gene a654l that encodes a protein with sequence similarity to the GCN5 family histone acetyltransferases. In this study, we report a 1.5A˚ crystal structure of PBCV-1 A654L in a complex with coenzyme A. The structure reveals a unique feature of A654L that precludes its acetylation of histone peptide substrates. We demonstrate that A654L, hence named viral polyamine acetyltransferase (vPAT), acetylates polyamines such as putrescine, spermidine, cadaverine, and homospermidine present in both PBCV-1 and its host through a reaction dependent upon a conserved glutamate 27. Our study suggests that as the first virally encoded polyamine acetyltransferase, vPAT plays a possible key role in the regulation of polyamine catabolism in the host during viral replication. Includes Supplemental Material

\u3ci\u3eParamecium bursaria\u3c/i\u3e Chlorella Virus 1 Encodes a Polyamine Acetyltransferase

Background: PBCV-1 gene a654l encodes a protein with sequence similarity to GCN5 histone acetyltransferases. Results: A crystal structure of A654L bound to coenzyme A reveals how A654L acetylates polyamines, not histone lysines. Conclusion: A654L functions as a polyamine acetyltransferase. Significance: As the first viral polyamine acetyltransferase, A654L has a possible role in host polyamine catabolism in viral replication. Paramecium bursaria chlorella virus 1 (PBCV-1), a large DNA virus that infects green algae, encodes a histone H3 lysine 27-specific methyltransferase that functions in global transcriptional silencing of the host. PBCV-1 has another gene a654l that encodes a protein with sequence similarity to the GCN5 family histone acetyltransferases. In this study, we report a 1.5A˚ crystal structure of PBCV-1 A654L in a complex with coenzyme A. The structure reveals a unique feature of A654L that precludes its acetylation of histone peptide substrates. We demonstrate that A654L, hence named viral polyamine acetyltransferase (vPAT), acetylates polyamines such as putrescine, spermidine, cadaverine, and homospermidine present in both PBCV-1 and its host through a reaction dependent upon a conserved glutamate 27. Our study suggests that as the first virally encoded polyamine acetyltransferase, vPAT plays a possible key role in the regulation of polyamine catabolism in the host during viral replication. Includes Supplemental Material

From screen to structure with a harvestable microfluidic device

Microfluidic crystallization using the Crystal Former improves the identification of initial crystallization conditions relative to screening via vapour diffusion

Protein-Protein Interactions in Crystals of the Human Receptor-Type Protein Tyrosine Phosphatase ICA512 Ectodomain

ICA512 (or IA-2) is a transmembrane protein-tyrosine phosphatase located in secretory granules of neuroendocrine cells. Initially, it was identified as one of the main antigens of autoimmune diabetes. Later, it was found that during insulin secretion, the cytoplasmic domain of ICA512 is cleaved and relocated to the nucleus, where it stimulates the transcription of the insulin gene. The role of the other parts of the receptor in insulin secretion is yet to be unveiled. The structures of the intracellular pseudocatalytic and mature extracellular domains are known, but the transmembrane domain and several intracellular and extracellular parts of the receptor are poorly characterized. Moreover the overall structure of the receptor remains to be established. We started to address this issue studying by X-ray crystallography the structure of the mature ectodomain of ICA512 (ME ICA512) and variants thereof. The variants and crystallization conditions were chosen with the purpose of exploring putative association interfaces, metal binding sites and all other structural details that might help, in subsequent works, to build a model of the entire receptor. Several structural features were clarified and three main different association modes of ME ICA512 were identified. The results provide essential pieces of information for the design of new experiments aimed to assess the structure in vivo

Etude structurale de métalloprotéines à centres [2Fe-2S].<br />Cas d'une ferrédoxine et d'une dioxygénase impliquée dans la biodégradation des<br />hydrocarbures aromatiques.<br />Cristallographie des protéines à très haute énergie.<br />Méthodes de phasage d'une protéine modele à 55 keV.

Metalloproteins containing Fe-S clusters play an important role in nature as they areinvolved in essential physiological functions including photosynthesis, respiration, andnitrogen fixation. In this thesis, a [2Fe-2S] ferredoxin involved in Fe-S cluster biogenesis, anda bacterial dioxygenase playing a critical role in aromatic hydrocarbon biodegradation weresubjected to structural analysis by synchrotron X-ray crystallography. The structure of aferredoxin from the photosynthetic bacterium Rhodobacter capsulatus was solved in both itsoxidized and reduced states. Subtle structural changes were observed upon reduction,especially in the vicinity of the [2Fe-2S] cluster. These changes are discussed in comparisonwith those described for ferredoxins with similar structures but different functions.A more complex metalloprotein, belonging to a large family of bacterial dioxygenases,was studied for its ability to oxidize polycyclic aromatic hydrocarbons (PAHs). This multicomponentenzyme, isolated from a PAH-degrading Sphingomonas strain, consists of aNAD(P)H-oxidoreductase, a [2Fe-2S] ferredoxin, and a terminal oxygenase. The terminaloxygenase component, called PhnI, consists of six subunits assembled into an ?3?3 hexamer,and contains one Rieske-type [2Fe-2S] cluster and one Fe(II) ion per ? subunit, which wereidentified by their characteristic EPR signature. The enzyme showed an exceptionally broadsubstrate specificity, as it could hydroxylate a wide range of PAHs made of two to five fusedrings, including the carcinogens, benz[a]anthracene and benzo[a]pyrene. With naphthalene assubstrate, steady-state kinetics showed that the enzyme had a low apparent Km (0.92 WM) anda specificity constant of 2.0 WM-1. s-1. The Phn1 protein was crystallized and its threedimensionalstructure was determined at 1.85 A resolution. In spite of moderate sequencesimilarity with homologous dioxygenases, the 3D polypeptide fold was found to be verysimilar, most of the differences being observed near the substrate binding pocket.Many protein crystals, especially those of Fe-S proteins, have been shown to undergoX-ray radiation damage, leading to artifacts in protein structure determinations. As an attemptto solve the problem, ultra-high energy X-rays (55 keV; 0.22 A), which are only slightlyabsorbed by proteins, were used for the first time to determine the 3D structure of a modelprotein, lysozyme. Beamline specificities as well as optimum energy were determined.Potential applications for structural biology are discussed.Les métalloprotéines contenant des centres Fe-S jouent un rôle important dans lanature car elles sont impliquées dans des fonctions physiologiques essentielles telles que laphotosynthèse, la respiration et la fixation de l'azote.Dans cette thèse, une ferrédoxine impliquée dans la biogenèse des centres Fe-S, et unedioxygenase bactérienne jouant un rôle crucial dans la biodegradation des hydrocarburesaromatiques ont fait l'objet d‘analyses structurales par cristallographie aux rayons X. Lastructure d'une ferrédoxine de la bactérie photosynthétique Rhodobacter capsulatus, a étérésolue dans les états oxyde et réduit. De petits changements structuraux ont été observes lors de la réduction, notamment au voisinage du centre [2Fe-2S]. Ces changements sont compares a ceux décrits pour des ferrédoxines de structure similaire mais de fonction différente.Une métalloprotéine plus complexe, appartenant a une grande famille de dioxygenasesbactériennes, a été étudiée pour son activité d'oxydation des hydrocarbures aromatiquespolycycliques (HAP). Cette enzyme a trois composantes, isolée d'une souche deSphingomonas dégradant les HAP comprend une oxydoréductase a NAD(P)H, uneferrédoxine a centre [2Fe-2S], et composante oxygenase de six sous-unités assemblées en un hexamère de type α3β3. La composante oxygenase, appelée PhnI, contient une centre [2Fe-2S] de type Rieske et un ion ferreux par sous-unité α, qui ont été identifies par leur signature RPE. L'enzyme est douée d'une spécificité du substrat extrêmement large, puisqu'elle est capable d'hydroxyle toute une gamme de HAP fait de 2 a 5 cycles aromatiques, y compris des cancérogènes comme le benz[a]anthracene et le benzo[a]pyrene. Avec le naphtalène comme substrat, des mesures de cinétique ont montre que cette enzyme a un Km bas (0.92 WM) et une constante de spécificité de 2.0 WM-1. s-1. La proteine Phn1 a été cristallisée, et sa structure 3D a été résolue avec une résolution de 1.85 A. En dépit d'une modeste similitude de séquence avec des dioxygenases homologues, le repliement polypeptidique est très semblable.Des différences ont toutefois été observées au niveau de la poche catalytique.Les protéines sous forme cristallisée, notamment les protéines Fe-S, peuvent subir desdommages dus au rayonnement X synchrotron, causant des artefacts lors de la déterminationde la structure. Pour essayer de palier cet inconvénient, des rayons X de très haute énergie (55 keV; 0.22 A), qui sont peu absorbes par les protéines, ont été utilises pour résoudre lastructure d'une proteine modèle, le lysozyme. Une structure a été établie pour la première foispar cette approche, en utilisant les phases expérimentales obtenues par différentes méthodes.Les applications potentielles en biologie structurale sont discutées