RESUMEN
Multi-enzyme cascades enable the production of valuable chemical compounds, and fusion of the enzymes that catalyze these reactions can improve the reaction outcome. In this work, P450 BM3 from Bacillus megaterium and an alcohol dehydrogenase from Sphingomonas yanoikuyae were fused to bifunctional constructs to enable cofactor regeneration and improve the inâ vitro two-step oxidation of (+)-valencene to (+)-nootkatone. An up to 1.5-fold increased activity of P450 BM3 was achieved with the fusion constructs compared to the individual enzyme. Conversion of (+)-valencene coupled to cofactor regeneration and performed in the presence of the solubilizing agent cyclodextrin resulted in up to 1080â mg L-1 (+)-nootkatone produced by the fusion constructs as opposed to 620â mg L-1 produced by a mixture of the separate enzymes. Thus, a two-step (+)-valencene oxidation was considerably improved through the simple method of enzyme fusion.
Asunto(s)
Alcohol Deshidrogenasa , Bacillus megaterium , Alcohol Deshidrogenasa/genética , Bacillus megaterium/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/genética , NADPH-Ferrihemoproteína Reductasa/química , NADPH-Ferrihemoproteína Reductasa/genética , Sesquiterpenos PolicíclicosRESUMEN
Fusion of multiple enzymes to multifunctional constructs has been recognized as a viable strategy to improve enzymatic properties at various levels such as stability, activity and handling. In this study, the genes coding for cytochrome P450 BM3 from B. megaterium and formate dehydrogenase from Pseudomonas sp. were fused to enable both substrate oxidation catalyzed by P450 BM3 and continuous cofactor regeneration by formate dehydrogenase within one construct. The order of the genes in the fusion as well as the linkers that bridge the enzymes were varied. The resulting constructs were compared to individual enzymes regarding substrate conversion, stability and kinetic parameters to examine whether fusion led to any substantial improvements of enzymatic properties. Most noticeably, an activity increase of up to threefold was observed for the fusion constructs with various substrates which were partly attributed to the increased diflavin reductase activity of the P450 BM3. We suggest that P450 BM3 undergoes conformational changes upon fusion which resulted in altered properties, however, no NADPH channeling was detected for the fusion constructs.
Asunto(s)
Proteínas Bacterianas/genética , Sistema Enzimático del Citocromo P-450/genética , Formiato Deshidrogenasas/genética , NADPH-Ferrihemoproteína Reductasa/genética , Ingeniería de Proteínas/métodos , Bacillus megaterium/enzimología , Bacillus megaterium/genética , Bacillus megaterium/metabolismo , Proteínas Bacterianas/metabolismo , Catálisis , Sistema Enzimático del Citocromo P-450/metabolismo , Enzimas/genética , NADP/metabolismo , NADPH-Ferrihemoproteína Reductasa/metabolismo , Oxidación-Reducción , Pseudomonas/enzimología , Pseudomonas/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/farmacologíaRESUMEN
Ribosome-inactivating proteins (RIPs) are enzymes with N-glycosylase activity that remove adenine bases from the ribosomal RNA. In theory, one single RIP molecule internalized into a cell is sufficient to induce cell death. For this reason, RIPs are of high potential as toxic payload for anti-tumor therapy. A considerable number of RIPs are synthesized by plants that belong to the carnation family (Caryophyllaceae). Prominent examples are the RIPs saporin from Saponaria officinalis L. or dianthin from Dianthus caryophyllus L. In this study, we have isolated and characterized a novel RIP (termed gypsophilin-S) from the tiny seeds of Gypsophila elegans M. Bieb. (Caryophyllaceae). It is noteworthy that this is the first study presenting the complete amino acid sequence of a RIP from a Gypsophila species. Gypsophilin-S was isolated from the defatted seed material following ammonium sulphate precipitation and HPLC-based ion exchange chromatography. Gypsophilin-S-containing fractions were analysed by SDS-PAGE and mass spectrometry. The full amino acid sequence of gypsophilin-S was assembled by MALDI-TOF-MS-MS and PCR. Gypsophilin-S exhibited strong adenine releasing activity and its cytotoxicity in human glioblastoma cells was investigated using an impedance-based real-time assay in comparison with recombinant saporin and dianthin.
