RESUMEN
5-Aminolevulinic acid (5-ALA) has been suggested for improving plant salt tolerance via exogenous application. In this study, we used a transgenic canola (Brassica napus), which contained a constituted gene YHem1 and biosynthesized more 5-ALA, to study salt stress responses. In a long-term pot experiment, the transgenic plants produced higher yield under 200 mmol L(-1) NaCl treatment than the wild type (WT). In a short-term experiment, the YHem1 transformation accelerated endogenous 5-ALA metabolism, leading to more chlorophyll accumulation, higher diurnal photosynthetic rates and upregulated expression of the gene encoding Rubisco small subunit. Furthermore, the activities of antioxidant enzymes, including superoxide dismutase, guaiacol peroxidase, catalase and ascorbate peroxidase, were significantly higher in the transgenic plants than the WT, while the levels of O2 ·(-) and malondialdehyde were lower than the latter. Additionally, the Na(+) content was higher in the transgenic leaves than that in the WT under salinity, but K(+) and Cl(-) were significantly lower. The levels of N, P, Cu, and S in the transgenic plants were also significantly lower than those in the WT, but the Fe content was significantly improved. As the leaf Fe content was decreased by salinity, it was suggested that the stronger salt tolerance of the transgenic plants was related to the higher Fe acquisition. Lastly, YHem1 transformation improved the leaf proline content, but salinity decreased rather than increased it. The content of free amino acids and soluble sugars was similarly decreased as salinity increased, but it was higher in the transgenic plants than that in the WT.
Asunto(s)
Ácido Aminolevulínico/metabolismo , Brassica napus/fisiología , Regulación de la Expresión Génica de las Plantas , Proteínas de Plantas/metabolismo , Cloruro de Sodio/farmacología , Ascorbato Peroxidasas/metabolismo , Brassica napus/efectos de los fármacos , Brassica napus/genética , Catalasa/metabolismo , Clorofila/metabolismo , Fotosíntesis/fisiología , Hojas de la Planta , Proteínas de Plantas/genética , Prolina/metabolismo , Salinidad , Tolerancia a la Sal , Estrés Fisiológico , Superóxido Dismutasa/metabolismoRESUMEN
MPA (mycophenolic acid) is an immunosuppressive drug produced by several fungi in Penicillium subgenus Penicillium. This toxic metabolite is an inhibitor of IMPDH (IMP dehydrogenase). The MPA-biosynthetic cluster of Penicillium brevicompactum contains a gene encoding a B-type IMPDH, IMPDH-B, which confers MPA resistance. Surprisingly, all members of the subgenus Penicillium contain genes encoding IMPDHs of both the A and B types, regardless of their ability to produce MPA. Duplication of the IMPDH gene occurred before and independently of the acquisition of the MPAbiosynthetic cluster. Both P. brevicompactum IMPDHs are MPA-resistant, whereas the IMPDHs from a non-producer are MPA-sensitive. Resistance comes with a catalytic cost: whereas P. brevicompactum IMPDH-B is >1000-fold more resistant to MPA than a typical eukaryotic IMPDH, its kcat/Km value is 0.5% of 'normal'. Curiously, IMPDH-B of Penicillium chrysogenum, which does not produce MPA, is also a very poor enzyme. The MPA-binding site is completely conserved among sensitive and resistant IMPDHs. Mutational analysis shows that the C-terminal segment is a major structural determinant of resistance. These observations suggest that the duplication of the IMPDH gene in the subgenus Penicillium was permissive for MPA production and that MPA production created a selective pressure on IMPDH evolution. Perhaps MPA production rescued IMPDH-B from deleterious genetic drift.
Asunto(s)
Adaptación Fisiológica/genética , Evolución Biológica , IMP Deshidrogenasa/antagonistas & inhibidores , Ácido Micofenólico/metabolismo , Penicillium/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Duplicación de Gen , Regulación Fúngica de la Expresión Génica/fisiología , IMP Deshidrogenasa/genética , IMP Deshidrogenasa/metabolismo , Modelos Moleculares , Familia de Multigenes , Ácido Micofenólico/farmacología , Conformación ProteicaRESUMEN
The filamentous fungus Penicillium brevicompactum produces the immunosuppressive drug mycophenolic acid (MPA), which is a potent inhibitor of eukaryotic IMP dehydrogenases (IMPDHs). IMPDH catalyzes the conversion of IMP to XMP via a covalent enzyme intermediate, E-XMP*; MPA inhibits by trapping E-XMP*. P. brevicompactum (Pb) contains two MPA-resistant IMPDHs, PbIMPDH-A and PbIMPDH-B, which are 17- and 10(3)-fold more resistant to MPA than typically observed. Surprisingly, the active sites of these resistant enzymes are essentially identical to those of MPA-sensitive enzymes, so the mechanistic basis of resistance is not apparent. Here, we show that, unlike MPA-sensitive IMPDHs, formation of E-XMP* is rate-limiting for both PbIMPDH-A and PbIMPDH-B. Therefore, MPA resistance derives from the failure to accumulate the drug-sensitive intermediate.
Asunto(s)
Farmacorresistencia Fúngica , Inhibidores Enzimáticos/farmacología , Ácido Micofenólico/farmacología , Penicillium/efectos de los fármacos , Penicillium/metabolismo , Animales , Cricetinae , IMP Deshidrogenasa/antagonistas & inhibidores , IMP Deshidrogenasa/química , IMP Deshidrogenasa/metabolismo , Cinética , Modelos Moleculares , Penicillium/enzimología , Conformación Proteica , Ribonucleótidos/metabolismo , XantinaRESUMEN
Cryptosporidium spp. cause acute gastrointestinal disease that can be fatal for immunocompromised individuals. These protozoan parasites are resistant to conventional antiparasitic chemotherapies and the currently available drugs to treat these infections are largely ineffective. Genomic studies suggest that, unlike other protozoan parasites, Cryptosporidium is incapable of de novo pyrimidine biosynthesis. Curiously, these parasites possess redundant pathways to produce dTMP, one involving thymidine kinase (TK) and the second via thymidylate synthase-dihydrofolate reductase. Here we report the expression and characterization of TK from C. parvum. Unlike other TKs, CpTK is a stable trimer in the presence and absence of substrates and the activator dCTP. Whereas the values of k(cat) = 0.28 s(-1) and K(m)(,ATP) = 140 microm are similar to those of human TK1, the value of K(m)(thymidine) = 48 microm is 100-fold greater, reflecting the abundance of thymidine in the gastrointestinal tract. Surprisingly, the antiparasitic nucleosides AraT, AraC, and IDC are not substrates for CpTK, indicating that Cryptosporidium possesses another deoxynucleoside kinase. Trifluoromethyl thymidine and 5-fluorodeoxyuridine are good substrates for CpTK, and both compounds inhibit parasite growth in an in vitro model of C. parvum infection. Trifluorothymidine is also effective in a mouse model of acute disease. These observations suggest that CpTK-activated pro-drugs may be an effective strategy for treating cryptosporidiosis.