Christine Meyer

Characterization of Three XylT-Like [2Fe-2S] Ferredoxins Associated with Catabolism of Cresols or Naphthalene: Evidence for Their Involvement in Catechol Dioxygenase Reactivation

The xylT gene product, a component of the xylene catabolic pathway of Pseudomonas putida mt2, has been recently characterized as a novel [2Fe-2S] ferredoxin which specifically reactivates oxygen-inactivated catechol 2,3-dioxygenase (XylE). In this study, three XylT-like proteins potentially involved in the catabolism of naphthalene (NahT) or cresols (PhhQ and DmpQ) have been overexpressed in Escherichia coli, purified, and compared with respect to their biochemical properties and interaction with XylE. The three XylT analogues show general spectroscopic characteristics common to plant-type [2Fe-2S] ferredoxins as well as distinctive features that appear to be typical for the XylT subgroup of these proteins. The midpoint redox potentials of the PhhQ and DmpQ proteins were -286 mV and -323 mV, respectively. Interestingly, all purified XylT-like proteins promoted in vitro reactivation of XylE almost as efficiently as XylT. The interaction of XylE with XylT and its analogues was studied by cross-linking experiments using the 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide. A polypeptide band with an M(r) of 46,000, which corresponded to the cross-linked product between one XylE subunit and one molecule of ferredoxin, was obtained in all cases. The formation of the complex was affected by ionic strength, indicating that electrostatic forces are involved in the dioxygenase-ferredoxin interaction. In complementation experiments, plasmids expressing xylT or its analogues were introduced into an XylT-null mutant of P. putida which is unable to grow on p-methylbenzoate. All transconjugants regained the wild-type phenotype, indicating that all analogues can substitute for XylT in the in vivo reactivation of XylE. Our results provide evidence for a subgroup of [2Fe-2S] ferredoxins with distinct biochemical properties whose specific function is to reactivate intrinsically labile extradiol ring cleavage dioxygenases involved in the catabolism of various aromatic hydrocarbons.

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

The 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 and Leu356, which contribute to the rather uniform trapezoidal shape 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 towards 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.

Dihydroxylation of four- and five-ring aromatic hydrocarbons by the naphthalene dioxygenase from Sphingomonas CHY-1

The naphthalene dioxygenase from Sphingomonas CHY-1 exhibits extremely broad substrate specificity toward polycyclic aromatic hydrocarbons (PAHs). In a previous study, the catalytic rates of oxidation of nine PAHs were determined using the purified dioxygenase, but the oxidation products formed from four- to five-ring hydrocarbons were incompletely characterized. Here, we reexamined PAH oxygenation reactions using Escherichia coli recombinant cells overproducing strain CHY-1 dioxygenase. Hydroxylated products generated by the dioxygenase were purified and characterized by means of GC-MS, UV absorbance as well as 1H- and 13C-NMR spectroscopy. Fluoranthene was converted to three dihydrodiols, the most abundant of which was identified as cis-7,8-dihydroxy-7,8-dihydrofluoranthene. This diol turned out to be highly unstable, converting to 8-hydroxyfluoranthene by spontaneous dehydration. The dioxygenase also catalyzed dihydroxylations on the C2–C3 and presumably the C1–C2 positions, although at much lower rates. Benz[a]anthracene was converted into three dihydrodiols, hydroxylated in positions C1–C2, C8–C9, and C10–C11, and one bis-cis-dihydrodiol. The latter compound was identified as cis,cis-1,2,10,11-tetrahydroxy-1,2,10,11-tetrahydrobenz[a]anthracene, which resulted from the subsequent dioxygenation of the 1,2- or 10,11-dihydrodiols. Chrysene dioxygenation yielded a single diol identified as cis-3,4-dihydroxy-3,4-dihydrochrysene, which underwent further oxidation to give cis,cis-3,4,9,10 chrysene tetraol. Pyrene was a poor substrate for the CHY-1 dioxygenase and gave a single dihydrodiol hydroxylated on C4 and C5, whereas benzo[a}pyrene was converted to two dihydrodiols, one of which was identified as cis-9,10-dihydrodiol. The selectivity of the dioxygenase is discussed in the light of the known 3D structure of its catalytic component and compared to that of the few enzymes able to attack four- and five-ring PAHs.

Studies on the Electron Transport to Nitrogenase in Rhodobacter capsulatus

Biological nitrogen fixation requires a minimum of 16 ATP molecules and 8 low-potential reducing equivalents per molecule of N2 reduced. Under physiological conditions, a small electron carrier such as a ferredoxin or a flavodoxin is thought to transfer electrons to nitrogenase. However, in most nitrogen fixing bacteria, little is known on the electron transport pathway to nitrogenase. In the photosynthetic bacterium R. capsulatus, a 2[4Fe-4S] ferredoxin called FdI was identified as the major electron donor to nitrogenase (Schatt et al., 1989; Schmehl et al., 1993; Jouanneau et al., 1995). A flavodoxin encoded by nifF, may function as an auxiliary electron carrier when iron is limited (Gennaro et al., 1996). In addition, a group of genes called rnf have been implicated in the electron transport to nitrogenase, based on the characterization of mutants bearing defined deletions within these genes (Schmehl et al., 1993). Here, we report on the identification of two new rnf genes, on the purification of RnfB and RnfC as two membrane-bound iron-sulfur proteins, and on the regulation of rnf gene expression as a function of iron availability. The rnf genes are predicted to code for a membrane-bound complex showing some similarities with a sodium-dependent NADH ubiquinone oxidoreductase.