Assuntos
Antibacterianos/biossíntese , Butiratos/metabolismo , Macrolídeos/metabolismo , Complexos Multienzimáticos/genética , Streptomyces griseus/genética , Isobutiratos , Complexos Multienzimáticos/antagonistas & inibidores , Estereoisomerismo , Streptomyces griseus/enzimologia , Streptomyces griseus/metabolismoRESUMO
[reaction: see text] A recombinant P450-monooxygenase, DoxA, obtained from Streptomyces sp. strain C5, the producer of the anticancer compound daunorubicin, was expressed in S. lividans TK24 and therein used to catalyze the conversion of the anthracycline analogue desacetyladriamycin into the new anthracycline, 10-hydroxydesacetyladriamycin. This work establishes a new function for DoxA and demonstrates the use of a recombinant enzyme to prepare a new anthracycline analogue.
Assuntos
Antibióticos Antineoplásicos/metabolismo , Proteínas de Bactérias , Sistema Enzimático do Citocromo P-450/metabolismo , Doxorrubicina/biossíntese , Doxorrubicina/metabolismo , Oxigenases de Função Mista/metabolismo , Streptomyces/enzimologia , Antibióticos Antineoplásicos/biossíntese , Sistema Enzimático do Citocromo P-450/biossíntese , Sistema Enzimático do Citocromo P-450/genética , Doxorrubicina/análogos & derivados , Hidroxilação , Oxigenases de Função Mista/biossíntese , Oxigenases de Função Mista/genética , Ressonância Magnética Nuclear Biomolecular , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Streptomyces/genética , Streptomyces/metabolismoRESUMO
Nonactin is the parent compound of a group of highly atypical polyketide metabolites produced by Streptomyces griseus subsp. griseus ETH A7796. In this paper we describe the isolation, sequencing, and analysis of 15¿ omitted¿559 bp of chromosomal DNA, containing the potential nonactin biosynthesis gene cluster, from S. griseus subsp. griseus ETH A7796. Fourteen open reading frames were observed in the DNA sequence. Significantly, type II polyketide synthase (PKS) homologues were discovered in an apparent operon structure, which also contained the nonactate synthase gene (nonS), clustered with the tetranactin resistance gene. The deduced products of two of the genes (nonK and nonJ) are quite unusual ketoacyl synthase (KAS) alpha and KASbeta homologues. We speculate that nonactic acid, the polyketide precursor of nonactin, is synthesized by a type II PKS system.
Assuntos
Antibacterianos/biossíntese , Proteínas de Bactérias , Genes Bacterianos , Complexos Multienzimáticos/genética , Streptomyces griseus/genética , Aciltransferases/química , Aciltransferases/genética , Aciltransferases/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Clonagem Molecular , Macrolídeos/metabolismo , Dados de Sequência Molecular , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Família Multigênica , Óperon , Filogenia , Policetídeo Sintases , Análise de Sequência de DNA , Streptomyces griseus/enzimologiaRESUMO
DoxA is a cytochrome P-450 monooxygenase involved in the late stages of daunorubicin and doxorubicin biosynthesis that has a broad substrate specificity for anthracycline glycone substrates. Recombinant DoxA was purified to homogeneity from Streptomyces lividans transformed with a plasmid containing the Streptomyces sp. strain C5 doxA gene under the control of the strong SnpR-activated snpA promoter. The purified enzyme was a monomeric, soluble protein with an apparent Mr of 47,000. Purified DoxA catalyzed the 13-hydroxylation of 13-deoxydaunorubicin, the 13-oxidation of 13-dihydrocarminomycin and 13-dihydrodaunorubicin, and the 14-hydroxylation of daunorubicin. The pH optimum for heme activation was pH 7.5, and the temperature optimum was 30 degreesC. The kcat/Km values for the oxidation of anthracycline substrates by purified DoxA, incubated with appropriate electron-donating components, were as follows: for 13-deoxydaunorubicin, 22,000 M-1 x s-1; for 13-dihydrodaunorubicin, 14,000 M-1 x s-1; for 13-dihydrocarminomycin, 280 M-1 x s-1; and for daunorubicin, 130 M-1 x s-1. Our results indicate that the conversion of daunorubicin to doxorubicin by this enzyme is not a favored reaction and that the main anthracycline flux through the late steps of the daunorubicin biosynthetic pathway catalyzed by DoxA is likely directed through the 4-O-methyl series of anthracyclines.