Expression of a soybean cytochrome P450 monooxygenase cDNA in yeast and tobacco enhances the metabolism of phenylurea herbicides

• Published in: Proceedings of the National Academy of Sciences - PNAS 1999-02, Vol.96 (4), p.1750-1755
• Main Authors: Siminszky, B. (North Carolina State University, Raleigh.), Corbin, F.T, Ward, E.R, Fleischmann, T.J, Dewey, R.E
• Format: Article
• Language: English
• Subjects: ADN; AMINO ACID SEQUENCES; Biological Sciences; CHEMICAL COMPOSITION; CHLOROTOLURON; CITOCROMO P450; Cloning, Molecular; COMPLEMENTARY DNA; COMPOSICION QUIMICA; COMPOSITION CHIMIQUE; Cytochrome P-450 Enzyme System - genetics; Cytochrome P-450 Enzyme System - metabolism; CYTOCHROME P450; Deoxyribonucleic acid; DETOXIFICACION METABOLICA; DETOXIFICATION METABOLIQUE; DIURON; DNA; Enzymes; FLUOMETURON; GENBANK/AF022157; GENBANK/AF022457; GENBANK/AF022458; GENBANK/AF022459; GENBANK/AF022460; GENBANK/AF022461; GENBANK/AF022462; GENBANK/AF022463; GENBANK/AF022464; GENETIC TRANSFORMATION; GLYCINE MAX; Glycine max - enzymology; Glycine max - genetics; HERBICIDAS; HERBICIDE; HERBICIDE TOLERANCE; HERBICIDES; Herbicides - pharmacokinetics; Inactivation, Metabolic; Insecticides - pharmacokinetics; ISOENZIMAS; ISOENZYME; ISOENZYMES; Kinetics; LINURON; METABOLIC DETOXIFICATION; METABOLISM; METABOLISME; METABOLISMO; Metabolites; Microsomes - enzymology; Mixed Function Oxygenases - genetics; Mixed Function Oxygenases - metabolism; MOLECULAR SEQUENCE DATA; Nicotiana - enzymology; Nicotiana - genetics; NICOTIANA TABACUM; NUCLEOTIDE SEQUENCE; OXIDOREDUCTASES; OXIDORREDUCTASAS; OXYDOREDUCTASE; Phenylurea Compounds - pharmacokinetics; PLANTAS TRANSGENICAS; PLANTE TRANSGENIQUE; Plants; Plants, Genetically Modified; Plants, Toxic; Protein metabolism; RESISTANCE AUX PRODUITS CHIMIQUES; RESISTANCE TO CHEMICALS; RESISTENCIA A PRODUCTOS QUIMICOS; SACCHAROMYCES CEREVISIAE; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae - genetics; SECUENCIA NUCLEOTIDICA; SEQUENCE NUCLEOTIDIQUE; Soybean Proteins; Soybeans; Substrate Specificity; Tobacco; TRANSFORMACION GENETICA; TRANSFORMATION GENETIQUE; TRANSGENIC PLANTS; UNSPECIFIC MONOOXYGENASE; Yeast; Yeasts
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.96.4.1750

A strategy based on the random isolation and screening of soybean cDNAs encoding cytochrome P450 monooxygenases (P450s) was used in an attempt to identify P450 isozymes involved in herbicide metabolism. Nine full-length (or near-full-length) P450 cDNAs representing eight distinct P450 families were isolated by using PCR-based technologies. Five of the soybean P450 cDNAs were expressed successfully in yeast, and microsomal fractions generated from these strains were tested for their potential to catalyze the metabolism of 10 herbicides and 1 insecticide. In vitro enzyme assays showed that the gene product of one heterologously expressed P450 cDNA (CYP71A10) specifically catalyzed the metabolism of phenylurea herbicides, converting four herbicides of this class (fluometuron, linuron, chlortoluron, and diuron) into more polar compounds. Analyses of the metabolites suggest that the CYP71A10 encoded enzyme functions primarily as an N-demethylase with regard to fluometuron, linuron, and diuron, and as a ring-methyl hydroxylase when chlortoluron is the substrate. In vivo assays using excised leaves demonstrated that all four herbicides were more readily metabolized in CYP71A10-transformed tobacco compared with control plants. For linuron and chlortoluron, CYP71A10-mediated herbicide metabolism resulted in significantly enhanced tolerance to these compounds in the transgenic plants