ABSTRACT
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.
Subject(s)
Agaricales/enzymology , Fungal Proteins/chemistry , Luciferases/chemistry , Agaricales/genetics , Amino Acid Sequence , Catalytic Domain , Computational Biology/methods , Fungal Proteins/genetics , Fungal Proteins/metabolism , Luciferases/genetics , Luciferases/metabolism , Luminescence , Models, Molecular , Mutagenesis , Mutation , Sequence Homology, Amino Acid , Structure-Activity RelationshipABSTRACT
A key component of the recently described bioluminescent system of higher fungi is luciferase, a new class of proteins. The properties of fungal luciferase and their relationship with its structure are interesting both for improving autoluminescent systems already created on its basis and for creating new ones. Therefore, it is extremely important to understand the spatial structure of this protein. We have performed heterologous expression and purification of Neonothopanus nambi luciferase, obtained a protein suitable for subsequent crystallization, and also determined some biochemical properties of the recombinant luciferase.
Subject(s)
Agaricales/metabolism , Luciferases/biosynthesis , Luciferases/chemistry , Circular Dichroism , Detergents , Escherichia coli/metabolism , Fungal Proteins/biosynthesis , Fungal Proteins/chemistry , HEK293 Cells , Humans , Hydrogen-Ion Concentration , Industrial Microbiology , Kinetics , Luminescence , Magnetic Resonance Spectroscopy , Protein Conformation , Protein Domains , Recombinant Proteins/chemistry , Saccharomycetales/metabolism , TemperatureABSTRACT
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.
ABSTRACT
This is the first study to obtain a high-purity luciferase from the fungus Neonothopanus nambi biomass that is suitable for subsequent sequencing.
Subject(s)
Basidiomycota/enzymology , Fungal Proteins/chemistry , Fungal Proteins/isolation & purification , Luciferases/chemistry , Luciferases/isolation & purificationABSTRACT
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.
ABSTRACT
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.