RESUMO
The gene from Streptomyces coelicolor A3(2) encoding CYP102B1, a recently discovered CYP102 subfamily which exists solely as a single P450 heme domain, has been cloned, expressed in Escherichia coli, purified, characterized, and compared to its fusion protein family members. Purified reconstitution metabolism experiments with spinach ferredoxin, ferredoxin reductase, and NADPH revealed differences in the regio- and stereoselective metabolism of arachidonic acid compared to that of CYP102A1, exclusively producing 11,12-epoxyeicosa-5,8,14-trienoic acid in addition to the shared metabolites 18-hydroxy arachidonic acid and 14,15-epoxyeicosa-5,8,11-trienoic acid. Consequently, in order to elucidate the physiological function of CYP102B1, transposon mutagenesis was used to generate an S. coelicolor A3(2) strain lacking CYP102B1 activity and the phenotype was assessed.
Assuntos
Sistema Enzimático do Citocromo P-450/genética , Oxigenases de Função Mista/genética , Streptomyces coelicolor/enzimologia , Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Ácido 8,11,14-Eicosatrienoico/metabolismo , Ácido Araquidônico/metabolismo , Clonagem Molecular , Sistema Enzimático do Citocromo P-450/isolamento & purificação , Sistema Enzimático do Citocromo P-450/metabolismo , Elementos de DNA Transponíveis , Escherichia coli/genética , Ferredoxina-NADP Redutase/metabolismo , Ferredoxinas/metabolismo , Expressão Gênica , Oxigenases de Função Mista/isolamento & purificação , Oxigenases de Função Mista/metabolismo , Mutagênese Insercional , NADP/metabolismo , Streptomyces coelicolor/genética , Especificidade por SubstratoRESUMO
The genus Streptomyces produces about two-thirds of naturally occurring antibiotics and a wide array of other secondary metabolites, including antihelminthic agents, antitumor agents, antifungal agents, and herbicides. The newly completed genome sequence of the avermectin-producing bacterium Streptomyces avermitilis contains 33 cytochromes p450 (CYPs), many more than the 18 observed in Streptomyces coelicolor A3(2). Some of the likely metabolic functions are reported together with their genomic location and bioinformatic analysis. Seven entirely new CYP families were found together with close homologues of some forms observed in S. coelicolor A3(2). The presence of unusual CYP forms associated with conservons is revealed and of these, CYP157 forms in both S. avermitilis and S. coelicolor A3(2) deviate from the previously accepted rule for an EXXR motif within the K-helix of CYPs. Amongst this range of CYPs are forms associated with avermectin, filipin, geosmin, and pentalenolactone biosynthesis as well as unknown pathways of secondary metabolism.
Assuntos
Sistema Enzimático do Citocromo P-450/genética , Ivermectina/análogos & derivados , Streptomyces/enzimologia , Cromossomos Fúngicos , Proteínas do Sistema Complemento , Sistema Enzimático do Citocromo P-450/classificação , Genoma Fúngico , Ivermectina/metabolismo , Filogenia , Streptomyces/genética , Streptomyces/metabolismoRESUMO
In the present study we describe the complete cytochrome P450 complement, the "CYPome," of Streptomyces coelicolor A3(2). Eighteen cytochromes P450 (CYP) are described, in contrast to the absence of CYPs in Escherichia coli, and the twenty observed in Mycobacterium tuberculosis. Here we confirm protein identity as cytochromes P450 by heterologous expression in E. coli and measurement of reduced carbon monoxide difference spectra. We also report on their arrangement in the linear chromosome and relatedness to other CYPs in the superfamily. The future development of manipulation of antibiotic pathways and the use of streptomycetes in bioremediation and biotransformations will involve many of the new CYP forms identified here.