RESUMO
The recently described bioluminescent system from fungi has great potential for developing highly efficient tools for biomedical research. Luciferase enzyme is one of the most crucial components of this system. The luciferase from Neonothopanus nambi fungus belongs to the novel still undescribed protein family. The structure data for this protein is almost absent. A detailed study of the N. nambi luciferase properties is necessary for the improvement of analytical methods based on the fungal bioluminescent system. Here we present the positions of key amino acid residues and their effect on enzyme function described using bioinformatic and experimental approaches. These results are useful for further fungal luciferase structure determination.
Assuntos
Agaricales/enzimologia , Proteínas Fúngicas/química , Luciferases/química , Agaricales/genética , Sequência de Aminoácidos , Domínio Catalítico , Biologia Computacional/métodos , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Luciferases/genética , Luciferases/metabolismo , Luminescência , Modelos Moleculares , Mutagênese , Mutação , Homologia de Sequência de Aminoácidos , Relação Estrutura-AtividadeRESUMO
Optoanalytical methods based on using genetically encoded bioluminescent enzymes, luciferases, allow one to obtain highly sensitive signals, are non-invasive, and require no external irradiation. Bioluminescence is based on the chemical reaction of oxidation of a low-molecular-weight substrate (luciferin) by atmospheric oxygen, which is catalyzed by an enzyme (luciferase). Relaxation of the luciferin oxidation product from its excited state is accompanied by a release of a quantum of light, which can be detected as an analytical signal. The ability to express luciferase genes in various heterological systems and high quantum yields of luminescence reactions have made these tools rather popular in biology and medicine. Among several naturally available luciferases, a few have been found to be useful for practical application. Luciferase size, the wavelength of its luminescence maximum, enzyme thermostability, optimal pH of the reaction, and the need for cofactors are parameters that may differ for luciferases from different groups of organisms, and this fact directly affects the choice of the application area for each enzyme. It is quite important to overview the whole range of currently available luciferases based on their biochemical properties before choosing one bioluminescent probe suitable for a specific application.
RESUMO
Heterologous pathways are linked series of biochemical reactions occurring in a host organism after the introduction of foreign genes. Incorporation of metabolic pathways into host organisms is a major strategy used to increase the production of valuable secondary metabolites. Unfortunately, simple introduction of the pathway genes into the heterologous host in most cases does not result in successful heterologous expression. Extensive modification of heterologous genes and the corresponding enzymes on many different levels is required to achieve high target metabolite production rates. This review summarizes the essential techniques used to create heterologous biochemical pathways, with a focus on the key challenges arising in the process and the major strategies for overcoming them.
RESUMO
This is the first study to obtain a high-purity luciferase from the fungus Neonothopanus nambi biomass that is suitable for subsequent sequencing.
Assuntos
Basidiomycota/enzimologia , Proteínas Fúngicas/química , Proteínas Fúngicas/isolamento & purificação , Luciferases/química , Luciferases/isolamento & purificaçãoRESUMO
We designed a genetically encoded ratiometric fluorescent probe, SypHer3s, with enhanced brightness and optimized pKa, which responds to pH changes in different cellular compartments. SypHer3s was successfully utilized for imaging the pH dynamics in mitochondria of living neurons and in quantitative pH measurement in zebrafish embryos.
RESUMO
The structure of fungal oxyluciferin was determined, the enzymatic bioluminescence reaction under substrate saturation conditions with discrete monitoring of formed products was conducted, and the structures of the end products of the reaction were established. On the basis of these studies, the scheme of oxyluciferin degradation to the end products was developed. The structure of fungal oxyluciferin was confirmed by counter synthesis.
Assuntos
Fungos/química , Indóis/química , Substâncias Luminescentes/química , Pirazinas/química , Fungos/metabolismo , Medições Luminescentes , Estrutura MolecularAssuntos
Modelos Químicos , ATPase Trocadora de Sódio-Potássio/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Encéfalo/enzimologia , Isoenzimas/química , Isoenzimas/metabolismo , Rim/enzimologia , Cinética , Substâncias Macromoleculares , Miocárdio/enzimologia , ATPase Trocadora de Sódio-Potássio/químicaRESUMO
The basal and stress-induced blood pressure was found to decrease in rats with inherited stress sensitive hypertension (SSHR) followed by androzole acetate administration orally (30 mg/kg body weight) for eight days. The basal levels of arterial blood pressure was decreased by 17.5% and that of stress-induced animals--by 19%.
