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

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

Trends and Challenges in Experimental Macromolecular Crystallography

Macromolecular X-ray crystallography underpins the vigorous field of structural molecular biology having yielded many protein, nucleic acid and virus structures in fine detail. The understanding of the recognition by these macromolecules, as receptors, of their cognate ligands involves the detailed study of the structural chemistry of their molecular interactions. Also these structural details underpin the rational design of novel inhibitors in modern drug discovery in the pharmaceutical industry. Moreover, from such structures the functional details can be inferred, such as the biological chemistry of enzyme reactivity. There is then a vast number and range of types of biological macromolecules that potentially could be studied. The completion of the protein primary sequencing of the yeast genome, and the human genome sequencing project comprising some 105 proteins that is underway, raises expectations for equivalent three dimensional structural database

The catalytic pocket of the ring-hydroxylating dioxygenase from Sphingomonas CHY-1

International audienceRing-hydroxylating dioxygenases are multicomponent bacterial enzymes that catalyze the first step in the oxidative degradation of aromatic hydrocarbons. The dioxygenase from Sphingomonas CHY-1 is unique in that it can oxidize a wide range of polycyclic aromatic hydrocarbons (PAHs). The crystal structure of its catalytic component has been determined to 1.85 Å. The catalytic domain features a large hydrophobic cavity that was occupied in part by an external ligand, possibly an indole molecule. This substrate binding cavity appeared to be much greater than that of seven other dioxygenases so far characterized. Molecular modeling studies indicated that the catalytic cavity is large enough to accommodate the five-ring benzo[a]pyrene molecule. The predicted positions of this and other PAHs in the substrate binding pocket are consistent with the product regio- and stereo-selectivity of the enzyme

Characterization of dislocations in protein crystals by means of synchrotron double-crystal topography

5 pages, 8 figures.Hen egg-white lysozyme (HEWL) crystals have been studied by means of double-crystal synchrotron topography. The crystals reveal a number of features that are quite well known in hydrothermally grown inorganic crystals: dislocations, growth bands and growth sector boundaries. Dislocations in the sectors have been characterized as edge dislocations with Burgers vector parallel to the c axis. They are distinguishable only under weak beam conditions. The presence of edge dislocations shown in this paper is consistent with the spiral growth steps previously reported. This spiral growth on protein crystals has been observed many times by surface techniques.We are very indebted to Dr M. C. Robert who kindly helped us to examine the topographs and to analyse the various features of their contrast.Peer reviewe

Structure of a [2Fe-2S] ferredoxin from Rhodobacter capsulatus likely involved in Fe-S cluster biogenesis and conformational changes observed upon reduction

International audienceFdVI from Rhodobacter capsulatus is structurally related to a group of [2Fe-2S] ferredoxins involved in iron-sulfur cluster biosynthesis. Comparative genomics suggested that FdVI and orthologs found in α−proteobacteria are involved in this process. Here, the crystal structure of FdVI has been determined on both the oxidized and the reduced protein. The [2Fe-2S] cluster lies 6 Å below the protein surface in an hydrophobic pocket without access to the solvent. This particular cluster environment might explain why the FdVI midpoint redox potential (–306 mV at pH 8.0) did not show temperature or ionic strength dependence. Besides the four cysteines that bind the cluster, FdVI features an extra cysteine which is located close to the S1 atom of the cluster and is oriented in a position such that its thiol group points towards the solvent. Upon reduction, the general fold of the polypeptide chain was almost unchanged. The [2Fe-2S] cluster underwent a conformational change from a planar to a distorted lozenge. In the vicinity of the cluster, the side chain of Met24 was rotated by 180° bringing its S atom within H-bonding distance of the S2 atom of the cluster. The reduced molecule also featured a higher content of bound water molecules, and more extensive hydrogen bonding networks compared to the oxidized molecule. The unique conformational changes observed in FdVI upon reduction are discussed in the light of structural studies performed on related ferredoxins