title: Identification of pyrene-induced proteins in Mycobacterium sp. strain 6PY1: evidence for two ring-hydroxylating dioxygenases.
abstract: In this study, the enzymes involved in polycyclic aromatic hydrocarbon (PAH) degradation were investigated in the pyrene-degrading Mycobacterium sp. strain 6PY1. [(14)C]pyrene mineralization experiments showed that bacteria grown with either pyrene or phenanthrene produced high levels of pyrene-catabolic activity but that acetate-grown cells had no activity. As a means of identifying specific catabolic enzymes, protein extracts from bacteria grown on pyrene or on other carbon sources were analyzed by two-dimensional gel electrophoresis. Pyrene-induced proteins were tentatively identified by peptide sequence analysis. Half of them resembled enzymes known to be involved in phenanthrene degradation, with closest similarity to the corresponding enzymes from Nocardioides sp. strain KP7. The genes encoding the terminal components of two distinct ring-hydroxylating dioxygenases were cloned. Sequence analysis revealed that the two enzymes, designated Pdo1 and Pdo2, belong to a subfamily of dioxygenases found exclusively in gram-positive bacteria. When overproduced in Escherichia coli, Pdo1 and Pdo2 showed distinctive selectivities towards PAH substrates, with the former enzyme catalyzing the dihydroxylation of both pyrene and phenanthrene and the latter preferentially oxidizing phenanthrene. The catalytic activity of the Pdo2 enzyme was dramatically enhanced when electron carrier proteins of the phenanthrene dioxygenase from strain KP7 were coexpressed in recombinant cells. The Pdo2 enzyme was purified as a brown protein consisting of two types of subunits with M(r)s of about 52,000 and 20,000. Immunoblot analysis of cell extracts from strain 6PY1 revealed that Pdo1 was present in cells grown on benzoate, phenanthrene, or pyrene and absent in acetate-grown cells. In contrast, Pdo2 could be detected only in PAH-grown cells. These results indicated that the two enzymes were differentially regulated depending on the carbon source used for growth.
authors.affiliation: Laboratoire de Biochimie et Biophysique des Systèmes Intégrés, CNRS UMR 5092, CEA-Grenoble, F-38054 Grenoble Cedex 9, France.
authors.forename: Serge
authors.initials: S
authors.lastname: Krivobok
authors.forename: Sylvain
authors.initials: S
authors.lastname: Kuony
authors.forename: Christine
authors.initials: C
authors.lastname: Meyer
authors.forename: Mathilde
authors.initials: M
authors.lastname: Louwagie
authors.forename: John C
authors.initials: JC
authors.lastname: Willison
authors.forename: Yves
authors.initials: Y
authors.lastname: Jouanneau
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D015180:Electrophoresis, Gel, Two-Dimensional; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D009161:Mycobacterium / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D010105:Oxygenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011084:Polycyclic Aromatic Hydrocarbons / Q000378:metabolism; D011721:Pyrenes / Q000378:metabolism*; D017422:Sequence Analysis, DNA
chemical_list: D001426:Bacterial Proteins; D010455:Peptides; D011084:Polycyclic Aromatic Hydrocarbons; D011721:Pyrenes; C030984:pyrene; D010105:Oxygenases; C406405:phenanthrene dioxygenase