title: Altered substrate specificity of the Pterygoplichthys sp. (Loricariidae) CYP1A enzyme.
abstract: Ethoxyresorufin is a classical substrate for vertebrate CYP1A enzymes. In Pterygoplichthys sp. (Loricariidae) this enzyme possesses 48 amino acids substitutions compared to CYP1A sequences from other vertebrate species. These substitutions or a certain subset substitution are responsible for the non-detection of the EROD reaction in this species liver microsomes. In the present study, we investigated the catalytic activity of Pterygoplichthys sp. CYP1A toward 15 potential substrates in order to understand the substrate preferences of this modified CYP1A. The fish gene was expressed in yeast and the accumulation of the protein was confirmed by both the characteristic P450-CO absorbance spectra and by detection with monoclonal antibodies. Catalytic activities were assayed with yeast microsomes and four resorufin ethers, six coumarin derivates, three flavones, resveratrol and ethoxyfluoresceinethylester. Results demonstrated that the initial velocity pattern of this enzyme for the resorufin derivatives is different from the one described for most vertebrate CYP1As. The initial velocity for the activity with the coumarin derivatives is several orders of magnitude higher than with the resorufins, i.e. the turnover number (kcat) for ECOD is 400× higher than for EROD. Nonetheless, the specificity constant (kcat/km) for EROD is only slightly higher than for ECOD. EFEE is degraded at a rate comparable to the resorufins. Pterygoplichthys sp. CYP1A also degrades 7-methoxyflavone and β-naphthoflavone but not resveratrol and chrysin. These results indicate a divergent substrate preference for Pterygoplichthys sp. CYP1A, which may be involved in the adaptation of Loricariidae fish to their particular environment and feeding habits.
authors.affiliation: BioMA, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Brasil. Electronic address: parente@biof.ufrj.br.
authors.forename: Thiago E M
authors.initials: TE
authors.lastname: Parente
authors.affiliation: Université de Toulouse; INSA, UPS, INP; LISBP, 135 Avenue de Rangueil, F-31077 Toulouse, France; INRA, UMR792 Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France; CNRS, UMR5504, 135 Avenue de Rangueil, F-31400 Toulouse, France.
authors.forename: Philippe
authors.initials: P
authors.lastname: Urban
authors.affiliation: Université de Toulouse; INSA, UPS, INP; LISBP, 135 Avenue de Rangueil, F-31077 Toulouse, France.
authors.forename: Denis
authors.initials: D
authors.lastname: Pompon
authors.affiliation: BioMA, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Brasil.
authors.forename: Mauro F
authors.initials: MF
authors.lastname: Rebelo
mesh_terms: D000818:Animals; D002397:Catfishes / Q000502:physiology*; D019363:Cytochrome P-450 CYP1A1 / Q000235:genetics / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D008862:Microsomes, Liver / Q000201:enzymology; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013379:Substrate Specificity / Q000502:physiology; D014874:Water Pollutants, Chemical / Q000378:metabolism*
chemical_list: D014874:Water Pollutants, Chemical; D019363:Cytochrome P-450 CYP1A1
keywords: Adaptation; Catfish; Coumarins; Evolution; Kinetics parameters; P450