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.

Comparison of Earthworm Bioluminescent Systems.

The results of a comparative study of the luciferin-luciferase systems of seven species of bioluminescing oligochaetes-Henlea petushkovi, Henlea rodionovae, Fridericia heliota (Enchytraeidae), Microscolex phosphoreus (Acanthodrilidae), Pontodrilus litoralis (Megascolecidae), Eisenia lucens, and Avelona ligra (Lumbricidae)-are presented.

Low-Molecular-Weight Components of Luminescent Reaction of the Siberian Enchytraeid Henlea sp.

The first results of the study of chromatographic and spectral properties of the detected lowmolecular-weight activators, putative emitters in the luminescent reaction of Siberian enchytraeid Henlea sp., are presented.

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.

Purification and partial spectral characterization of a novel luciferin from the luminous enchytraeid Fridericia heliota.

A homogeneous luciferin preparation has been obtained from the luminous soil enchytraeid Fridericia heliota, which has an ATP-dependent luminescent system. A procedure for luciferin purification without losing fractions of active luciferase has been developed. The luciferin specific activity is 4000 times increased; its UV absorption spectrum maximum is 294 nm with a local minimum at 262 nm. The luciferin of the enchytraeid F. heliota is significantly different from firefly luciferin, whose luminescent reaction also requires ATP, and it also appears to have no similarities to other known luciferins.

Effect of different salts and detergents on luciferin-luciferase luminescence of the enchytraeid Fridericia heliota.

The study addresses the effect produced by different inorganic salts and detergents (SDS, Triton X-100, the Tween series) on the ATP-dependent bioluminescent reaction catalyzed by the luciferase of the new earthworm species Fridericia heliota (Annelida: Clitellata: Oligochaeta: Enchytraeidae). It has been shown that the effect of divalent metal salts on luminescence is determined by the action of cations. Three of them - Mg(2+), Mn(2+) and Ca(2+) - can stimulate luciferase activity at concentrations varying within a wide range, and Mn(2+) can act as a 100%-effective substitute for Mg(2+) in F. heliota luminescence reaction in vitro. The inhibitory effect of monovalent metal salts on luminescence is largely determined by the action of the anion part of the molecule. The effectiveness of the inhibitory effect of anions increases in the following order: Cl(-)

Purification and characterization of flavoproteins and cytochromes from the yellow bioluminescence marine bacterium Vibrio fischeri strain Y1.

Several flavoproteins and cytochromes that occur as major components in extracts of the yellow bioluminescence Y1 strain of the marine bacterium Vibrio fischeri have been purified and characterized with respect to their mass (SDS/PAGE and matrix-assisted laser-desorption/ionization MS), chromatographic properties, N-terminal sequence, and spectroscopy (absorption, fluorescence emission and anisotropy decay). The investigated proteins were as follows: yellow fluorescence protein (YFP) with bound riboflavin, FMN or 6,7-dimethyl-8-ribityllumazine; a blue fluorescence protein (BFP) with bound 6,7-dimethyl-8-ribityllumazine, riboflavin, or 6-methyl-7-oxo-8-ribityllumazine; thioredoxin reductase with FAD as ligand; and two c-type diheme cytochromes, c551 and c554. We present evidence that the riboflavin-bound YFP has an N-terminal sequence corresponding to that published for the dimeric YFP. We show that an equilibrium replacement of the riboflavin can be made with excess lumazine derivative and that lumazine-bound YFP has different bioluminescence properties to those of the lumazine protein from Photobacterium leiognathi. BFP is a different protein again, and in the bacterial lysate it occurs in multiple forms, ligated to either riboflavin, lumazine, or the 7-oxolumazine derivative. The N-terminal sequence for BFP shows similarities to those of the YFP proteins and to lumazine protein and riboflavin synthase from Photobacterium. BFP in any form has no bioluminescence or riboflavin-synthase activity. A 70-kDa fluorescent flavoprotein with FAD as ligand has an N-terminal sequence highly similar to those of thioredoxin reductases from Haemophilus influenzae and Escherichia coli. Cytochrome contaminations in previous preparations of YFP have been removed and are identified as the two c-type cytochromes c551 and c554. Both inhibit the NADH-induced bioluminescence in the reductase/luciferase system with the luciferases from P. leiognathi and V. fischeri. The N-terminal amino acid sequence of the cytochrome (c551) corresponds to a diheme cytochrome c4. The spectral properties of c554 are similar to those of other c5 cytochromes, and both c554 and c551 have absorption spectra similar to those of the respective cytochromes from the gram-negative bacteria Pseudomonas and Azotobacter.

Interaction of Photobacterium leiognathi and Vibrio fischeri Y1 luciferases with fluorescent (antenna) proteins: bioluminescence effects of the aliphatic additive.

The kinetics of the bacterial bioluminescence reaction is altered in the presence of the fluorescent (antenna) proteins, lumazine protein (LumP) from Photobacterium or the yellow fluorescence proteins (YFP) having FMN or Rf bound, from Vibrio fischeri strain Y1. Depending on reaction conditions, the bioluminescence intensity and its decay rate may be either enhanced or strongly quenched in the presence of the fluorescent proteins. These effects can be simply explained on the basis of the same protein-protein complex model that accounts for the bioluminescence spectral shifts induced by these fluorescent proteins. In such a complex, where the fluorophore evidently is in proximity to the luciferase active site, it is expected that the on-off rate of certain aliphatic components of the reaction should be altered with a consequent shift in the equilibria among the luciferase intermediates, as recently elaborated in a kinetic scheme. These aliphatic components are the bioluminescence reaction substrate, tetradecanal or other long-chain aldehyde, its carboxylic acid product, or dodecanol used as a stabilizer of the luciferase peroxyflavin. No evidence can be found for the protein-protein interaction in the absence of the aliphatic component.

Direct measurement of excitation transfer in the protein complex of bacterial luciferase hydroxyflavin and the associated yellow fluorescence proteins from Vibrio fischeri Y1.

Time-resolved fluorescence was used to directly measure the energy transfer rate constant in the protein-protein complex involved in the yellow bioluminescence of Vibrio fischeri, strain Y1. In this reaction the putative donor is the fluorescent transient intermediate, luciferase hydroxyflavin, which exhibits a major fluorescence lifetime of the bound flavin of 10 ns. On addition of the acceptor, the V. fischeri yellow fluorescence protein containing either FMN or riboflavin as ligand, a rapid decay time, 0.25 ns, becomes predominant. The same results are observed using rec-luciferase from Photobacterium leiognathi to produce the donor. Because of favorable spectral separation in this system, this rapid decay rate of 4 ns-1, can be directly equated to the energy transfer rate. This rate is ten times higher than the rate previously observed in the Photobacterium luciferase hydroxyflavin-lumazine protein, donor-acceptor system, derived from emission anisotropy measurements. This ten-times ratio is close to the ratio of spectral overlaps of the donor fluorescence with the acceptor absorption, between these two systems, so it is concluded that the topology of the protein complexes in both cases, must be very similar. Energy transfer is also monitored by the loss of steady-state fluorescence intensity at 460 nm of the donor, on addition of the acceptor protein. A fluorescence titration indicates that luciferase hydroxyflavin and the yellow protein complex with a 1:1 stoichiometry with a Kd of 0.7 microM (0 degree C). These parameters account for the bioluminescence spectral shifting effects observed in these reactions.

The yellow bioluminescence bacterium, Vibrio fischeri Y1, contains a bioluminescence active riboflavin protein in addition to the yellow fluorescence FMN protein.

The yellow bioluminescence Y1 strain of Vibrio fischeri can produce a 22 kDa protein with either FMN or riboflavin as a bound fluorophore. Both forms are active for shifting the bioluminescence spectral maximum. The fluorescence spectral distribution of the two proteins differs slightly and the in vivo emission appears to be an equal mixture of the two. The bioluminescence activity of the riboflavin Y1 protein contrasts with the inactivity of the related Photobacterium type.