Rational Design and Mutagenesis of Fungal Luciferase from Neonothopanus nambi.

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.

The Recombinant Luciferase of the Fungus Neonothopanus nambi: Obtaining and Properties.

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.

Luciferin-Luciferase System of Marine Polychaete Chaetopterus variopedatus.

This paper presents the preliminary results of the separation of the Chaetopterus variopedatus bioluminescent system into luciferin and luciferase and a brief description of some of their properties.

Isolation and Purification of Fungal Luciferase from Neonothopanus nimbi.

This is the first study to obtain a high-purity luciferase from the fungus Neonothopanus nambi biomass that is suitable for subsequent sequencing.

Why does the bioluminescent fungus Armillaria mellea have luminous mycelium but nonluminous fruiting body?

By determining the components involved in the bioluminescence process in luminous and nonluminous organs of the honey fungus Armillaria mellea, we have established causes of partial luminescence of this fungus. The complete set of enzymes and substrates required for bioluminescence is formed only in the mycelium and only under the conditions of free oxygen access. Since the synthesis of luciferin precursor (hispidin) and 3-hydroxyhispidin hydroxylase in the fruiting bodies is blocked, the formation of luciferin-the key component of fungal bioluminescent system-was not observed. That is why the fruiting body of Armillaria mellea is nonluminous despite the presence of luciferase, the enzyme that catalyzes the oxidation of luciferin with a photon emission.

Structure of fungal oxyluciferin, the product of the bioluminescence reaction.

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.

Nanodiamonds as Carriers for Address Delivery of Biologically Active Substances.

Surface of detonation nanodiamonds was functionalized for the covalent attachment of immunoglobulin, and simultaneously bovine serum albumin and Rabbit Anti-Mouse Antibody. The nanodiamond-IgG(I125) and RAM-nanodiamond-BSA(I125) complexes are stable in blood serum and the immobilized proteins retain their biological activity. It was shown that the RAM-nanodiamond-BSA(I125) complex is able to bind to the target antigen immobilized on the Sepharose 6B matrix through antibody-antigen interaction. The idea can be extended to use nanodiamonds as carriers for delivery of bioactive substances (i.e., drugs) to various targets in vivo.

The interaction of linear and ring forms of DNA molecules with nanodiamonds synthesized by detonation.

Nanodiamonds synthesized by detonation have been found not to immobilize the ring form of pUC19 plasmid DNA. Linear pUC19 molecules with blunt ends, prepared by restriction of the initial ring form of pUC19 DNA, and linear 0.25-10 kb DNA fragments are adsorbed on nanodiamonds. The amount of adsorbed linear DNA molecules depends on the size of the molecules and the size of the nanodiamond clusters.

Role of conservative residue Cys158 in the formation of an active photoprotein complex of obelin.

Using site directed mutagenesis, the conservative residue Cys158 of recombinant apoobelin was substituted for serine (C158S, S-mutant) or alanine (C158A, A-mutant). These point mutations resulted in significant changes in the apoobelin structure accompanied by slowing of photoprotein complex formation, decrease of its stability, and changing of its bioluminescence characteristics. The enzymatic properties of the photoprotein decreased in the series: wild-type protein > S-mutant > A-mutant. This is consistent with rank of nucleophilicity SH > OH > CH(3) of cysteine, serine, and alanine side chain functional groups, respectively. Possible mechanisms of the involvement of the apoobelin Cys158 SH-group in the formation of the enzyme-substrate complex are considered.