title: Purification and characteristics of an inducible by polycyclic aromatic hydrocarbons NADP(+)-dependent naphthalenediol dehydrogenase (NDD) in Mucor circinelloides YR-1.
abstract: We detected NADP(+)-dependent dihydrodiol dehydrogenase (DD) activity in a cell-free extract from Mucor circinelloides YR-1, after high-speed centrifugation. We analyzed the enzymatic activity in the cytosolic fraction by zymograms, as described previously, and eight different DD activity bands were revealed. Five constitutive DD activities (DD1-5) were present when glucose was used as carbon source and three inducible activities (NDD, PDD1 and PDD2) when aromatic hydrocarbon compounds were used. NDD activity was induced all of the aromatic hydrocarbon compounds. The highest DD activity inducer was naphthalene and the lowest was pyrene. One of the enzymes showed higher activity with cis-naphthalene-diol rather than with trans-nahthalenediol as a substrate. We purified this particular enzyme to homogeneity and found that it had an isoelectric point of 4.6. The molecular weight for the native protein was 197.4kDa and 49.03±0.5kDa for the monomer that conforms it, suggesting a homotetrameric structure for the complete enzyme. Polyclonal antibodies were raised against it and obtained. NDD activity was almost totally inhibited when antibodies were used at low concentrations, and in native immunoblots only one band, which corresponds to the activity band detected in the zymograms, could be detected. In denaturing PAGE immunoblots only one band was detected. This band corresponds to the purified protein band of 49kDa detected in SDS-PAGE gels. The other two inducible enzymes PDD1 and PDD2 were present only when phenanthrene was used as sole carbon source in the culture media.
authors: Biology Department, Campus Noria Alta, University of Guanajuato, Noria Alta s/n, C.P. 36050 Guanajuato, Gto., Mexico. Electronic address: Lucy-2000@hotmail.com.
authors: Reyna Lucero
authors: RL
authors: Camacho-Morales
authors: Biology Department, Campus Noria Alta, University of Guanajuato, Noria Alta s/n, C.P. 36050 Guanajuato, Gto., Mexico. Electronic address: vanesanovoa@yahoo.com.mx.
authors: Vanesa
authors: V
authors: Zazueta-Novoa
authors: Biology Department, Campus Noria Alta, University of Guanajuato, Noria Alta s/n, C.P. 36050 Guanajuato, Gto., Mexico. Electronic address: lizbeth_g_c_@hotmail.com.
authors: Juana Lizbeth González
authors: JL
authors: Casillas
authors: Departamento de Microbiología y Sistemas Simbióticos, Estación Experimental del Zaidín, Granada, Spain. Electronic address: elizabet.aranda@eez.csic.es.
authors: Elizabeth Aranda
authors: EA
authors: Ballesteros
authors: Departamento de Microbiología y Sistemas Simbióticos, Estación Experimental del Zaidín, Granada, Spain. Electronic address: juanantonio.ocampo@eez.csic.es.
authors: Juan Antonio Ocampo
authors: JA
authors: Bote
authors: Biology Department, Campus Noria Alta, University of Guanajuato, Noria Alta s/n, C.P. 36050 Guanajuato, Gto., Mexico. Electronic address: drbioqu1993@gmail.com.
authors: Roberto
authors: R
authors: Zazueta-Sandoval
mesh_terms: D005656:Fungal Proteins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D009089:Mucor / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism; D009252:NADPH Dehydrogenase / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D009281:Naphthalenes / Q000378:metabolism; D011084:Polycyclic Aromatic Hydrocarbons / Q000378:metabolism*
chemical_list: C518223:1,8-naphthalenediol; D005656:Fungal Proteins; D009281:Naphthalenes; D011084:Polycyclic Aromatic Hydrocarbons; D009252:NADPH Dehydrogenase
keywords: Dihydrodiol dehydrogenases; Mucor circinelloides; Naphthalene