Caracterization of arene dioxygenases implied in polycyclic aromatic hydrocarbons biodegradation in Mycobacterium sp. 6PY1 ; Caractérisation d'arène dioxygénases impliquées dans la biodégradation des hydrocarbures aromatiques polycycliques chez Mycobacterium sp. 6PY1

Président :M. D. SCHNEIDER Rapporteurs :Mme F. FAYOLLE-GUICHARD, M. T. VOGEL Examinateur :M. S. KRIVOBOK ; This thesis deals with the bacterial biodegradation of pollutants called polycyclic aromatic hydrocarbons (PAHs). The bacterium Mycobacterium sp. 6PY1 was isolated from a polluted soil for its ability to use pyrene, a 4-ring PAH, as sole source of carbon and energy. To learn about the pyrene metabolic pathway, the identification of the enzymes involved in this process has been undertaken using a proteomic approach. This approach revealed the occurrence of two ring-hydroxylating dioxygenases in strain 6PY1, which could catalyze the initial attack of pyrene. The goal of this study was to clone the genes encoding the dioxygenases identified in Mycobacterium sp. 6PY1, over-express these genes in an heterologous system in order to facilitate the purification of the corresponding enzymes, and determine the biochemical and catalytic properties of these enzymes. The pdoA1B1 genes encoding the terminal component of a dioxygenase were cloned and overexpressed in Escherichia coli. The catalytic properties of this enzyme, called Pdo1, were determined in vivo by measuring the oxidation products of 2- to 4-ring PAHs by gas chromatography coupled to mass spectrometry (GC-MS). Analysis of the selectivity of the enzyme, as determined using GC-MS, showed that Pdo1 preferentially oxidized 3- or 4-ring PAHs, including phenanthrene and pyrene, but was inactive on diaromatic compounds such as naphthalene and biphenyl. Pdo1 was unstable and was therefore purified in inactive form. The genes encoding a second dioxygenase component were found in a locus containing two other catabolic genes. The pdoA2B2 genes encoded an enzyme called Pdo2 showing a narrow specificity towards 2- to 3-ring PAHs, and a high preference for phenanthrene. Pdo2 is an Α3Β3 hexamer, containing [2Fe-2S] Rieske clusters which confer it a characteristic absorbance spectrum. A third set of genes possibly encoding another dioxygenase was discovered in the genome ...